InteractiveFly: GeneBrief

Calmodulin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Calmodulin is a protein of primary importance in the regulation of cellular processes dependent on Ca2+. When cellular Ca2+ levels rise, CaM sequesters Ca2+ ions, binding specifically to CaM-binding domains on a variety of proteins. CaM is involved in most of the important signaling pathways in the cell.

CaM targets include:

• cyclic nucleotide metabolism (see Dunce and Rutabaga)
• phosphorylation pathways (see CamKII and zipper, a site that includes information on myosin light chain kinase)
• dephosphorylation (carried out by calcineurin: see Drosophila Calcinerin)
• calcium transport (carried out by the plasma membrane Ca2+ pump)
• nitric oxide pathway (controlling NO synthase)
• regulation of cytoskeletal proteins (Gap-43, Spectrin and NinaC)

CaM in the Ca2+-bound form is a dumbell-shaped molecule: two lobes (globular domains) connected by a long alpha-helix chain, the central portion of which is highly mobile, acting as a flexible tether for the two domains. Each domain binds two Ca2+ ions. The C-terminal lobe binds Ca2+ with high affinity, while the N-terminal shows lower affinity for Ca2+. Binding of Ca2+ ions induces a large conformational change which makes two hydrophobic patches, one in each half of the molecule, available for target interaction (James, 1995 and references).

Most of the characterized Calmodulin binding domains of target proteins are stretches of 16 to 35 amino acids that show a segregation of basic and polar residues on one side of an alpha-helical configuration, and hydrophobic amino acids on the other. Upon target binding of CaM, the extended CaM dumbbell-like structure collapses into a more compact form: the two globular lobes now face each other and the central helical chain twists into a long flexible loop. In many cases, the CaM-binding domain of target proteins constitutes, or is adjacent to, an autoinhibitory domain. Thus, the CaM-binding site interacts with the active site of the enzyme repressing it until inactivated by binding to CaM. In many cases, the CaM-binding domain of target proteins is subject to phosphorylation, generating a CaM independent target (James, 1995 and references). Additional information about these processes can be found at the CaMKII site.

One of CaM's many roles in fly biology is illustrated by the complex interaction of CaM with the unconventional myosin NINAC. CaM localization in the rhabdomere, a microvillar structure in the photoreceptor cell, is disrupted in ninaC mutants. There are two isoforms of NINAC: the p174 isoform is spatially restricted to the rhabdomere and p132 is restricted to the sub-rhabdomeral cytoplasm. Mutant flies lacking the rhabdomere-specific p174 NINAC protein do not concentrate Calmodulin in the rhabdomere, while flies lacking the sub-rhabdomeral p132 isoform have no detectable cytoplasmic Calmodulin (Porter, 1993). Of the two Calmodulin-binding sites in NINAC (C1 and C2), C1 is common to both p132 and p174, while C2 is unique to p174. Spatial location of CaM depends on binding of CaM to both C1 and C2 (Porter, 1995).

Disruption of C1 or C2 disrupts the normal distribution of CaM. In flies carrying a NINAC deleted for C1, Calmodulin staining is detected almost exclusively in the rhabdomeres, and the staining is less intense than in wild-type. In flies carrying a NINAC deleted for C2 (carrying wild-type p132), the relative intensity of staining in the rhabdomeres is reduced, when compared with wild-type. Deletion of C1 or C2, and the consequent disruption of the normal distribution of CaM, results in an abnormal retinal electrophysiology characterized by a prolonged depolarization after-potential (PDA) (Porter, 1995).

The interaction of CaM and NINAC may not be restricted solely to a transport function. Besides being an unconventional myosin, NINAC also possesses a kinase domain. What is the function of this kinase and how is it regulated? Is the myosin function of NINAC regulated by CaM? NinaC myosin activity might be regulated by Calmodulin, since the properties of at least some unconventional myosins are affected by Calmodulin association. As far as the kinase function is concerned, the electrophysiological phenotype (PDA) caused by deletion of C1 and C2 suggests that Calmodulin binding to p174 is required for inactivation of the phototransduction cascade. The only other mutation identified that results in a similar reduction in PDA is arrestin2 . In wild-type organisms, Arrestin2 binds rhodopsin (the photoreceptor of the fly), which is subsequently phosphorylated at multiple serine and threonine residues by rhodopsin kinase. The phosphorylated rhodopsin is then protected by Arrestin2 from dephosphorylation and is no longer able to bind the signal transducing G-protein, causing termination of the photoresponse. Arrestin2 also undergoes serine/threonine phosphorylation, apparently in a Ca2+/CaM dependent fashion. It is intriguing to speculate that NINAC p174 may be the CaM-dependent serine/threonine kinase that phosphorylates Arrestin2 (Porter, 1995).

In addition to the PDA phenotype, the deletion of the C2 CaM interaction site of NINAC, but not C1, results in an abnormal electroretinogram recording, a measurement of the summed response of all retinal cells to light. C2 null flies exhibit an electroretinogram measurement similar to a null allele of ninaC , again suggesting an intimate functional interaction between CaM and NINAC (Porter, 1995).

Calcium levels regulate a multitude of cell functions. Calmodulin is crucial to proper calcium function -- a synergistic relationship both highly evolved and evolutionarily conserved. For example, the following proteins and/or interactions are all conserved in evolution:

• interaction of Calmodulin with CaMKII
• Nitric oxide synthetase
• myosin light chain kinase
• the membrane-associated neuronal phosphoprotein Gap-43
• cAMP phosphodiesterase (Dunce in Drosophila)
• neural adenylate kinase (Rutabaga in Drosophila)

This evolutionary stability suggests that protein interaction in signal transduction pathways may serve as a driving force for conservation of protein structure in evolution.

Ca2+-dependent metarhodopsin inactivation mediated by Calmodulin and NINAC Myosin III

Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. This study investigated the mechanism and role of rhodopsin inactivation. The lifetime of activated rhodopsin (metarhodopsin = M^*) was determined in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M^* by photoreisomerization to rhodopsin could suppress responses to prior illumination. M^* was inactivated rapidly (τ ~20 ms) under control conditions, but ~10-fold more slowly in Ca²⁺-free solutions. This pronounced Ca²⁺ dependence of M^* inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca²⁺ influx acting via CaM and NINAC accelerates the binding of arrestin to M^*. These results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling (Liu, 2008).

This study exploited the bistable nature of invertebrate rhodopsins to measure the lifetime of activated metarhodopsin in Drosophila. The approach measures the time window during which photoreisomerization of M^* can suppress the response to light. The relative lack of overlap of the R and M spectra in UV opsins has been exploited by recording from the UV-sensitive photoreceptors in Limulus median ocelli. This strategy was adapted for Drosophila by using flies engineered to express the UV opsin Rh3; the effective M^* lifetime was found to be very short (τ_dec ≈20 ms) under physiological conditions. Strikingly, M^* lifetime was prolonged ~10-fold in the absence of Ca²⁺ influx, indicating that M-Arr2 binding is Ca²⁺ dependent and that M^* lifetime is the rate-limiting step in response deactivation in Ca²⁺-free solutions. Further experiments led to proposal of a mechanism for Ca²⁺-dependent M^* inactivation by Arr2, mediated by calmodulin (CaM) and myosin III NINAC (Liu, 2008).

Photoisomerization of rhodopsin (R) by short-wavelength light (480 nm for Rh1 or 330 nm for Rh3) generates active metarhodopsin (M^*). M^* continues to activate G_q until it binds arrestin (Arr2) or is reconverted to R by long-wavelength illumination (570/460 nm). M is serially phosphorylated by rhodopsin kinase (RK), but this is not required for M^* inactivation or Arr2 binding. CaMKII-dependent phosphorylation of Arr2 at Ser³⁶⁶ and photoreconversion of M_pp to R_pp is required for the release of Arr_p. Phosphorylation of Arr2 also prevents endocytotic internalization of M-Arr2. In Arr2^S366A or mutants defective in CamKII, photoreconversion fails to release Arr2. Finally, R_pp is dephosphorylated by the Ca-CaM-dependent rhodopsin phosphatase (rdgC) to recreate the ground state, R. The results suggest that under low-Ca²⁺ conditions Arr2 is prevented from rapid binding to M^* because it is sequestered by NINAC or a NINAC-regulated target; however, Ca²⁺ influx acting via CaM rapidly releases Arr2. Each microvillus contains ~70 Arr2 molecules, ensuring rapid quenching of M^* once they are free to diffuse. The role of M^* phosphorylation remains uncertain but may be involved in Rh1 internalization by the minor arrestin, Arr1 (Liu, 2008).

Ca²⁺ dependence of M^* lifetime had not previously been demonstrated in an invertebrate photoreceptor, and the consensus from data in Drosophila suggested no obvious mechanism by which M^* lifetime could be regulated by Ca²⁺. The finding that M^* inactivation is strongly Ca²⁺ dependent prompted a re-examination of possible roles of Rh1 and Arr2 phosphorylation as well as CaM. Although M^* lifetime remained strongly Ca²⁺ dependent in mutants defective in rhodopsin and arrestin phosphorylation, the Ca²⁺ dependence of M^* inactivation was effectively eliminated in hypomorphic cam mutants. This requirement for CaM appeared to be mediated by the myosin III NINAC protein, since the Ca²⁺ dependence of M^* inactivation was effectively abolished in both the null ninaC^P235 mutant and an allele (ninaC^KD) in which CaM levels in the microvilli were unaffected. NINAC, which is the major CaM-binding protein in the photoreceptors, has long been known to be required for normal rapid response deactivation (Porter, 1993b), but the mechanistic basis remained unresolved. These results now strongly suggest that it is specifically required for the Ca²⁺- and CaM-dependent inactivation of M^* by Arr2 (Liu, 2008).

How might NINAC regulate the Ca²⁺-dependent inactivation of M^*? A clue comes from the finding that Arr2 levels were substantially reduced in ninaC mutants. After taking this into account, the lack of Ca²⁺ dependence of M^* inactivation in ninaC mutants was in fact associated with a very pronounced acceleration of response inactivation under Ca²⁺-free conditions. This was most clearly revealed in ninaC^KD, which appears to be specifically defective only in Ca²⁺-dependent M^* inactivation and does not show the additional response defects of the null ninaC phenotype (e.g., Hofstee, 1996). This suggests a disinhibitory mechanism whereby Ca²⁺-dependent inactivation of M^* may be achieved, at least in part, by the NINAC-dependent prevention of Arr2-M^* binding under low-Ca²⁺ conditions. Specifically, it is suggested that in Ca²⁺-free solutions, or in the low-Ca²⁺ conditions prevailing during the latent period of the quantum bump under physiological conditions, Arr2 in the microvilli is predominantly bound to NINAC or a NINAC-regulated target, thus restricting its access to M^*. However, following Ca²⁺ influx, CaCaM would bind to NINAC, causing NINAC to release Arr2, which, as a soluble protein, could then rapidly diffuse to encounter and inactivate M^* (Liu, 2008).

Interestingly, a recent study reported that NINAC can interact with Arr2 in a phosphoinositide-dependent manner (Lee, 2004). This interaction was described in the context of a role of NINAC in light-induced translocation of Arr2, which was reported to be disrupted in ninaC mutants. However, involvement in translocation was challenged by a subsequent study reporting that Arr2 translocation was unaffected in ninaC mutants (Satoh, 2005). It will be interesting to see whether the Arr2-NINAC interactions described by Lee (2004a) reflect a role in the CaCaM- and NINAC-dependent inactivation of M^* reported in this study (Liu, 2008).

It has long been known that responses under Ca²⁺-free conditions decay ~10-fold more slowly than in the presence of Ca²⁺. The current results establish that the inactivation of M^* by Arr2 is the rate-limiting inactivation step in such Ca²⁺-free responses, with a time constant of ~200 ms in wild-type photoreceptors. Following inactivation of M^* by photoreisomerization under Ca²⁺-free conditions, the response decayed with a time constant of ~80 ms. This also provides a unique and direct measure of the time constant(s) of the downstream mechanisms of inactivation, which presumably include GTP-ase activity of the Gq-PLC complex and removal of DAG by DAG kinase. It will be interesting so see whether Ca²⁺ also accelerates these inactivation mechanisms (Liu, 2008).

By contrast, the failure to accelerate response decay by overexpressing Arr2 in the presence of Ca²⁺ indicates that inactivation of M^* is not rate limiting under physiological conditions. This can be understood by recognizing that the macroscopic kinetics are determined by the convolution of the bump latency distribution and bump waveform, the latter probably terminated by Ca²⁺-dependent inactivation of the light-sensitive channels. Until the Ca²⁺ influx associated with the quantum bump, the phototransduction machinery in each microvillus is effectively operating under Ca²⁺-free conditions. The results suggest that it is the Ca²⁺ influx associated with each quantum bump that promotes M^* inactivation, and hence the timing of M^* inactivation will be determined by the bump latency distribution and not vice versa. This leads to the, perhaps counterintuitive, concept that response termination is rate limited, not by any specific inactivation mechanism, but rather by the time course with which the cumulative probability of bump generation approaches 100% (Liu, 2008).

Clearly, rapid quenching of M^* is essential to maintain the fidelity and high temporal resolution of phototransduction. In wild-type cells, an effectively absorbed photon generates only one quantum bump, but never (or extremely rarely) two or more; yet the multiple bump trains observed in arr2, cam, and ninaC mutants show that additional bumps are readily generated within 50–100 ms if M^* fails to be inactivated. To prevent such multiple bumps without Ca²⁺-dependent feedback would require such a high rate of Arr2 binding that many M^* molecules would be inactivated before they had a chance to activate sufficient G proteins to generate a quantum bump. This would result in an effective reduction in sensitivity, as is directly illustrated by the phenotype of p[Arr2] flies overexpressing Arr2. These show not only a 5-fold reduction in quantum efficiency. but also a reduction in bump amplitude and even an increase in bump latency, which is attributed to a decreased rate of second messenger generation. The mechanism proposed in this study provides an elegant solution to this dilemma. The analysis suggests that in the low-Ca²⁺ environment prior to Ca²⁺ influx, much of the Arr2 in the microvillus is bound to NINAC (or NINAC-regulated target), thus allowing M^* to remain active long enough to activate sufficient G proteins to guarantee production of a full-sized quantum bump with high probability. Only after the bump has been initiated does Ca²⁺ influx accelerate the inactivation of M^* by releasing Arr2, thus ensuring that only one bump is generated. This strategy is complemented and enabled by the ultracompartmentalization afforded by the microvillar design, which ensures that the Ca²⁺ rise is both extremely rapid and largely confined to the affected microvillus (Liu, 2008).

An Asp-CaM complex is required for centrosome-pole cohesion and centrosome inheritance in neural stem cells

The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. This study reports that Drosophila melanogaster calmodulin (CaM) regulates the ability of the microcephaly-associated protein, abnormal spindle (Asp), to cross-link spindle microtubules. Both proteins colocalize on spindles and move toward spindle poles, suggesting that they form a complex. Binding and structure-function analysis support this hypothesis. Disruption of the Asp-CaM interaction alone leads to unfocused spindle poles and centrosome detachment. This behavior leads to randomly inherited centrosomes after neuroblast division. It is further shown that spindle polarity is maintained in neuroblasts despite centrosome detachment, with the poles remaining stably associated with the cell cortex. Finally, evidence is provided that CaM is required for Asp's spindle function; however, it is completely dispensable for Asp's role in microcephaly suppression (Schoborg, 2015).

These results provide insight into how Asp, a key protein involved in mitotic spindle function, is regulated by the ubiquitous calcium-sensing protein CaM. CaM was localized near the spindle poles over 35 yr ago; the current data now assign a role for this CaM localization in directly regulating Asp to cross-link spindle MTs. The Asp-CaM interaction is conserved because it has also been biochemically identified in other eukaryotes, such as nematodes and mice, suggesting that this complex performs an essential spindle function. The work presented in this study extends the functional understanding of the Asp-CaM complex in spindle pole focusing and centrosome-pole cohesion, in addition to the cell biology of microcephaly (Schoborg, 2015).

Previous work in Drosophila, C. elegans, and mice has suggested a link between Asp and CaM. Spindle phenotypes are observed after RNAi depletion of either protein in Drosophila S2 cells. In C. elegans, analysis of meiotic spindles in the early embryo showed spindle defects after asp depletion and Asp's dependence on CaM (CMD-1) for pole localization. Furthermore, yeast two-hybrid analysis identified an Asp fragment containing a single IQ motif that could interact with CMD-1. This interaction between CaM and Asp on meiotic spindles was later identified in mouse oocytes using immunoprecipitation. However, in all cases, details of the underlying mechanism of the Asp-CaM association and a direct test of its contribution to spindle architecture remained unexplored (Schoborg, 2015).

The current results demonstrate that CaM functions as the critical factor that dictates Asp's ability to cross-link MTs. This is supported by the fact that Asp transgenes that localize to the spindle in a manner identical to that of the FL protein, yet are defective in CaM binding (Asp^N and Asp^FLΔIQ), fail to maintain pole focusing and centrosome-pole cohesion. Further, transgene analysis also highlighted a second mode of MT binding by Asp, mediated through its C terminus, and is independent of its known N-terminal MT binding domain. This interaction, though clearly weaker and distinct from the punctate signals observed for N-terminal containing transgenes, is supported by previous studies in vitro. It is believed that the stronger spindle pole and punctate localization of WT Asp normally masks this Asp^C localization and possibly contributes to Asp's ability to cross-link MTs (Schoborg, 2015).

Furthermore, a novel mode of Asp-CaM complex behavior on spindles was uncovered, highlighted by dynamic streaming of foci through the spindle lattice toward the pole. Previous work suggested that Asp associates with MT minus ends based on its accumulation at spindle poles where their density is highest. Localization of the Asp-Cam complex in live cells supports this hypothesis. However, it is further suggested that Asp-CaM complexes, seen as discrete puncta that move poleward, reside at MT minus ends distributed throughout the spindle that are collectively transported and organized at poles. These observations are consistent with work showing γ-tubulin-marked minus ends present throughout the spindle that stream toward the poles. Additionally, vertebrate NuMA displays similar streaming behavior, indicating a shared mechanism in which pole focusing is achieved through the concerted movement of protein complexes along the spindle toward the pole. Biochemical analysis will be critical for establishing the relationship between the distribution of minus ends within the spindle, the ability of the Asp-CaM complex to bind MT minus ends, and how the dynamic nature of their movement contribute to pole focusing and centrosome-pole cohesion (Schoborg, 2015).

The complete detachment of centrosomes from the spindle and random movement within the NB could have substantial long-term effects that are not fully appreciated by limited analysis of third-instar larval brains. Although the swapping of mother-daughter centrosome position and improper inheritance is interesting, its significance is unknown. It could be that centrosome position after detachment, rather than detachment, per se, negatively influences mitotic events. One would predict, for example, that centrosomes positioned anywhere in the cell other than the poles could influence the MT architecture within the spindle. In fact, a significant number of aberrantly bent spindles is seen, and live imaging showed that wandering centrosomes transiently interact laterally along the entire length of the spindle. One might also predict that this lateral centrosome position would influence the dynamics and tension across the kinetochores, triggering the spindle assembly checkpoint and an extended metaphase, which was also documentd in asp^t25 mutants. Therefore, the wandering centrosomes and their improper inheritance could have many negative downstream effects. If these results of inheriting too many or too few centrosomes are extrapolated to mammalian cells, one would predict detrimental effects on cilia formation in addition to mitotic defects, as previously documented in other mutant backgrounds (Schoborg, 2015).

This analysis of apical determinants in NBs highlighted a possible role for spindle poles (not centrosomes) in the maintenance of cell polarity. Despite centrosome detachment in the asp^t25/Df NBs and long curving spindles, no misaligned spindles were observed. This was true in fixed tissue using the apical polarity marker aPKC, in which, despite pole splaying and curvature, minus ends of MTs appeared to remain stably associated with the crescent at the cell cortex. Furthermore, significant spindle rotation was never observed after centrosome detachment during the course of live imaging, and NBs divided asymmetrically. These observations support the prevalent model that centrosomes initiate NB polarity but further add that centrosomes are neither necessary nor able to alter polarity once established. This is corroborated by the fact that no significant difference was observed in NB number in the asp^t25/Df mutant, suggesting that cell fate determinants were correctly partitioned during asymmetric division (Schoborg, 2015).

The results also shed light on the role of Asp in microcephaly. Interestingly, this phenotype is not dependent on the Asp-CaM complex. Both Asp^N and Asp^FLΔIQ rescued the brain size defects of the asp^t25/Df despite showing no or reduced binding to CaM. These results are in agreement with previous work demonstrating normal head size in animals expressing an N-terminal Asp fragment in the hypomorphic asp allele background. Importantly, the data using the null allele show that microcephaly is a result of the loss of Asp function and not a dominant-negative effect of the hypomorphic asp alleles. Furthermore, this study showed that the microcephaly phenotype is not a consequence of unfocused spindle poles or detached centrosomes, because the Asp^N and Asp^FLΔIQ rescue fragments displayed both of these defects. Taken collectively, this analysis of the null asp allele uncovered a separation of function that requires both termini of Asp to maintain MT cross-linking and an unknown region of the N terminus to specify proper brain size (Schoborg, 2015).

Two possible models are proposed by which the Asp-CaM complex could function. In both models, CaM exerts its influence on the spindle through directly binding the C terminus of Asp and is required for its stability. The first model proposes that CaM aids Asp oligomerization within the spindle. Putative higher-order Asp assemblies would be analogous to NuMA oligomerization shown to facilitate MT focusing in vertebrate cells. A second model proposes that CaM might regulate the weak association of Asp's C terminus to MTs. In this model, Asp would bind MT minus ends via its N terminus and the MT lattice via its C terminus, effectively bridging and zippering MTs. In both models, CaM might promote a structural conformation that allows for oligomerization or for a single Asp molecule to bind two separate MTs. Both models are not mutually exclusive, because elements of each may cooperate to ensure proper cross-linking between spindle MTs and centrosome MTs for robust pole focusing and centrosome attachment. Future biochemical and structural studies will be required to more fully understand the influence of CaM binding to Asp and the role of this complex in spindle MT cross-linking (Schoborg, 2015).

REGULATION

Transcriptional Regulation

Calmodulin expression was examined in embryos homozygous for mutations in four loci that are known to affect nervous system development: numb, the achaete-scute complex, daughterless, and mastermind. The Calmodulin transcription pattern is altered in embryos mutant for each of these loci, suggesting that regulation by these genes, either directly or indirectly, is taking place (Kovalick, 1992).

Protein Interactions

Interaction of Calmodulin with Calcineurin

Calcineurin is a Ca²⁺-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca²⁺ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca²⁺-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).

Calcineurin is activated by a sustained increase in intracellular Ca²⁺ levels that can result from the opening of intracellular Ca²⁺ channels in response to phosphoinositide (PI) signaling. PI signaling is initiated by the activation of a phosphatidylinositol-specific phospholipase C, either PLCß by G-protein-coupled receptors (GPCR) or PLCgamma by receptor tyrosine kinases (RTK). PI-PLCs cleave phosphatidylinositol 4,5-bisphosphate (PIP₂) to yield inositol 1,4,5-trisphosphate (InsP₃), which then activates the InsP₃ receptor Ca²⁺ channel (Sullivan, 2002).

An activated form of Pp2B-14D, canA^act, was made by deleting the autoinhibitory and calmodulin-binding domains. The canA^act construct was expressed in Drosophila under the control of glass response elements, which induce transcription uniformly in cells posterior to the morphogenetic furrow in the eye imaginal disc (Sullivan, 2002).

Flies carrying one copy of the canA^act.gl transgene have mild rough eyes compared to wild type, and the eyes of flies carrying two copies exhibit a stronger phenotype. Consistent with observations in other systems, neither full-length CanA nor activated canA without a functional CanB-binding domain causes any detectable phenotypes when expressed throughout development (Sullivan, 2002).

Interaction of Calmodulin with CP309 (Pericentrin-like protein)

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. A large coiled-coil centrosome protein identified in Drosophila can bind to calmodulin. Biochemical studies reveal that this novel centrosome protein, centrosome protein of 309 kDa (Cp309), cofractionates with the gamma-tubulin ring complex and the centrosome-complementing activity. CP309 is required for microtubule nucleation mediated by centrosomes and it interacts with the gamma-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309 (Kawaguchi, 2004).

Because CP309 contains a CaM-binding motif, whether CP309 can bind to CaM was examined along with whether this binding is Ca2+ dependent. CaM-agarose beads were incubated with Drosophila embryo extracts in the presence of 2.5 mM Ca2+ or 5 mM EGTA (EGTA was used to chelate the Ca2+ in the extracts). Proteins bound to the CaM agarose beads were analyzed by Western blotting, probing with antibodies against CP309. It was found that significantly more CP309 in the extracts bound to CaM-agarose beads in the presence of Ca2+ than in the presence EGTA. Next, it was asked whether Ca2+ facilitates CaM-free CP309 binding to CaM. CaM-free CP309 was prepared by eluting CP309 from CaM-agarose beads with EGTA and then used in the same binding assay as described above. It was found that the CaM-free CP309 also binds to CaM-agarose beads more efficiently in the presence of Ca2+ than in the presence of EGTA. Therefore, although CP309 can bind to CaM in the absence of Ca2+, Ca2+ significantly enhances the binding (Kawaguchi, 2004).

Interaction of Calmodulin with kinases

For information on the interaction of Calmodulin with Calcium/calmodulin dependent protein kinase II, see CaMKII.

For information on the interaction of Calmodulin with the Regulatory light chain of myosin II (known in Drosophila as Spaghetti squash, or MRLC).

Calcium/calmodulin-dependent protein kinases (CaM kinases) have been reported to be involved in neuroplasticity. A new Drosophila CaM kinase gene has been cloned, named caki. The caki gene is extremely large; comparison of the genomic and cDNA sequences reveals that the caki transcription unit is at least 150 kb. The catalytic domain of this new CaM kinase protein shares homology (41%) with type II CaM kinases, while the C-terminal part is divergent. Constitutively expressed Caki protein is enzymatically active since it causes a 3-fold increase in the level of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter in a co-transfusion assay. In situ hybridization shows that during embryogenesis, larval and pupal life, transcription of caki is restricted almost exclusively to the central nervous system. In the adult head, immunohistochemistry reveals Caki protein in the lamina, the neuropil of the medulla, lobula, lobula plate and in the central brain. Mutant caki flies show reduced walking speed in 'Buridan's paradigm' (Martin, 1996).

Casein kinase II (CKII) is composed of a catalytic subunit (alpha) and a regulatory subunit (beta) that combine to form an alpha 2 beta 2 holoenzyme. The alpha-subunit monomer is enzymatically active, albeit kinetically attenuated relative to the holoenzyme; the addition of purified beta subunit stimulates its activity against casein. A kinetic analysis was performed of the phosphorylation of various protein and peptide substrates by the alpha subunit and the holoenzyme of Drosophila CKII. The alpha subunit, like the holoenzyme, is competent to phosphorylate typical physiological substrates such as the regulatory (RII) subunit of cAMP-dependent protein kinase (cAMPdPK), as well as artificial substrates such as alpha-casein and the synthetic peptide RRREEETEEE. The Km of the alpha subunit in each case is similar to that of the holoenzyme, whereas the Vmax is 5- to 60-fold lower. In contrast, Calmodulin, a protein that is significantly phosphorylated by the holoenzyme only in the presence of polybasic compounds, is readily phosphorylated by the alpha subunit alone. While the Km values of the alpha subunit and the holoenzyme for Calmodulin are similar, the Vmax of the alpha subunit is at least 10-fold higher than that of the holoenzyme. These results suggest that while the alpha subunit contains the necessary determinants for CKII substrate specificity, the beta subunit can either inhibit or activate it, in a substrate-dependent manner. Polybasic compounds stimulate not only the holoenzyme but, to a lesser extent, the alpha subunit as well (Bidwai, 1993).

Calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the CNS and is crucial for cellular and behavioral plasticity. It is thought that the ability of CaMKII to autophosphorylate and become Ca2+ independent allows it to act as a molecular memory switch. Inhibition of Drosophila CaMKII leads to impaired performance in the courtship conditioning associative memory assay, but it was unknown whether the constitutive form of the kinase had a special role in learning. In this study, a tripartite transgenic system combining GAL4/UAS with the tetracycline-off system was used to spatially and temporally manipulate levels of Ca2+-independent CaMKII activity in Drosophila. An enhancement of information processing during the training period was found with Ca2+-independent, but not Ca2+-dependent, CaMKII. During training, control animals have a lag before active suppression of courtship begins. Animals expressing Ca2+-independent CaMKII have no lag, implying that there is a threshold level of Ca2+-independent activity that must be present to suppress courtship. This is the first demonstration, in any organism, of enhanced behavioral plasticity with overexpression of constitutively active CaMKII. Anatomical studies indicate that transgene expression in antennal lobes and extrinsic mushroom body neurons drives this behavioral enhancement. Interestingly, immediate memory was unaffected by expression of T287D CaMKII in mushroom bodies, although previous studies have shown that CaMKII activity is required in this brain region for memory formation. These results suggest that the biochemical mechanisms of CaMKII-dependent memory formation are threshold based in only a subset of neurons (Mehren, 2004; full text or article).

Interaction of Calmodulin with ion channels

Two putative light-sensitive ion channels have been isolated from Drosophila, encoded by the transient-receptor-potential (trp) and transient-receptor-potential-like (trpl ) genes. The cDNA encoding the Trpl protein was initially isolated on the basis that the expressed protein binds Calmodulin. Two Calmodulin-binding sites are present in the C-terminal domain of the Trpl protein: CBS-1 and CBS-2. CBS-1 binds Calmodulin in a Ca2+-dependent fashion, requiring Ca2+ concentrations above 0.3-0.5 microM for Calmodulin binding. In contrast, CBS-2 binds the Ca2+-free form of Calmodulin, with dissociation occurring at Ca2+ concentrations between 5 and 25 microM. Phosphorylation of a serine residue within a peptide encompassing CBS-1 by cyclic AMP-dependent protein kinase (PKA) abolishes Calmodulin binding, and phosphorylation of the adjacent serine by protein kinase C appears to modulate this phosphorylation by PKA. Interpretation of these findings provides a novel model for ion-channel gating and modulation in response to changing levels of intracellular Ca2+ (Warr, 1996).

The effects of expression of the Drosophila Trpl protein, which is thought to encode a putative Ca2+ channel, on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ to oocytes injected with TRPL cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induces a rapid initial and a slower sustained rate of increase in fluorescence, which are respectively designated the initial and sustained rates of Ca2+ inflow. Compared with mock-injected oocytes, TRPL-cRNA-injected oocytes exhibit a higher resting cytoplasmic free Ca2+ concentration, and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in TRPL-cRNA-injected oocytes increases with (1) an increase in the time elapsed between injection of TRPL cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of TRPL cRNA injected and (3) an increase in Ca2+. A GTP antagonist inhibits the trpl cRNA-induced basal rate of Ca2+ inflow. Expression of TRPL cRNA also causes an increase in the basal rate of Mn2+ inflow. The increases in resting Ca2+ and in the basal rate of Ca2+ inflow induced by expression of TRPL cRNA are inhibited by the Calmodulin inhibitors W13, calmodazolium and peptide (amino acids 281-309) of CaM kinase II. A low concentration of exogenous Calmodulin (introduced by microinjection) activates, and a higher concentration inhibits, the TRPL cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous Calmodulin is reversed by W13 and calmodazolium. When rates of Ca2+ inflow in TRPL-cRNA-injected oocytes are compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate is greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes; (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, and is activated by cytoplasmic free Ca2+ (through endogenous Calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum, and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels (Lan, 1996).

The role of the ether a go-go (eag) gene was examined in modulation of K+ currents. The possibility that its protein product Eag is a subunit in the heteromultimeric assembly of K+ channels was examined by voltage-clamp analysis of larval muscle membrane currents. Previous DNA sequence studies indicate that the eag gene codes for a polypeptide homologous to, but distinct from, the Shaker (Sh) K+ channel subunits, and electrophysiological recordings reveal allele-specific effects of eag on four identified K+ currents in Drosophila larval muscles. Further studies of eag alleles indicate that none of the eag mutations, including alleles producing truncated mRNA messages, eliminate any of the four K+ currents, and that the mutational effects exhibit strong temperature dependence. Both W7, an antagonist of Ca2+/Calmodulin, and cGMP analogs modulate K+ currents; their actions are altered or even abolished by eag mutations. These results suggest a role of eag in modulation of K+ currents that may subserve integration of signals at a converging site of the two independent modulatory pathways. The Sh locus is known to encode certain subunits of the IA channel in larval muscle. The existence of multiple eag and Sh alleles enables an independent test of the idea of Eag as a K+ channel subunit by studying IA in different double-mutant combinations. An array of allele-specific interactions between eag and Sh has been observed, reflecting a close association between the Sh and eag subunits within the IA channel. Taken together, these data strengthen the possibility that the eag locus provides a subunit common to different K+ channels. The role of the eag subunit for modulating channels, as opposed to that of Sh subunits required for gating, selectivity, and conductance of the channel, suggest a combinatorial genetic framework for generating diversified K+ channels (Zhong, 1993).

Calmodulin interactions in the Drosophila retina

In the Drosophila retina, Calmodulin is concentrated in the the rhabdomere, a microvillar structure of the photoreceptor cell. Calmodulin is also found in lower amounts in the sub-rhabdomeral cytoplasm. This Calmodulin localization is dependent on the NinaC (neither inactivation nor afterpotential C) unconventional myosins. Mutant flies lacking the rhabdomere-specific p174 NINAC protein do not concentrate Calmodulin in the rhabdomere, whereas flies lacking the sub-rhabdomeral p132 isoform have no detectable cytoplasmic Calmodulin. A defect in vision results when Calmodulin is not concentrated in the rhabdomeres, suggesting a role for Calmodulin in the regulation of fly phototransduction. A general function of unconventional myosins may be to control the subcellular distribution of Calmodulin (Porter, 1993a).

The ninaC locus encodes two unconventional myosins, p132 and p174, both consisting of fused protein kinase and myosin head domains expressed in Drosophila photoreceptor cells. NinaC encodes the major Calmodulin-binding proteins in the retina and the NinaC-Calmodulin interaction is required for the normal subcellular localization of Calmodulin as well as for normal photo-transduction. There are two Calmodulin-binding sites in NinaC, C1 and C2, which have different in vitro binding properties. C1 is common to both p132 and p174 while C2 is unique to p174. To address the requirements for Calmodulin binding at each site in vivo, transgenic flies were generated expressing ninaC genes deleted for either C1 or C2. The spatial localization of Calmodulin depends on binding to both C1 and C2. Mutation of either site results in a defective photoresponse. A prolonged depolarization afterpotential (PDA) is elicited at lower light intensities than necessary to produce a PDA in wild-type flies. These results suggest that Calmodulin binding to both C1 and C2 is required in vivo for termination of phototransduction (Porter, 1995).

Phototransduction in Drosophila occurs through inositol lipid signaling that results in Ca2+ mobilization. The physiological roles of calmodulin (CaM) were studied in light adaptation and in regulation of the inward current that is brought about by depletion of cellular Ca2+ stores. Three resources providing decreased Ca-CaM content in photoreceptors were analysed: (1) transgenic Drosophila P[ninaCDeltaB] flies that have CaM-deficient photoreceptors; (2) the peptide inhibitor M5 that binds to Ca-CaM and prevents its action, and (3) Ca2+-free medium that prevents Ca2+ influx and thereby diminishes the generation of Ca-CaM. Several effects have been noted due to decrease in Ca-CaM level:

1. Fluorescence of Ca2+ indicator reveals an enhanced light-induced Ca2+ release from internal stores.
2. Measurements of the light-induced current in P[ninaCDeltaB] cells show a reduced light adaptation.
3. Internal dialysis of M5 initially enhances excitation and subsequently disrupts the light-induced current.
4. An inward dark current appears after depletion of the Ca2+ stores with ryanodine and caffeine.

Importantly, application of Ca-CaM into the photoreceptor cells prevents all of the above effects. It is proposed that negative feedback of Ca-CaM on Ca2+ release from ryanodine-sensitive stores mediates light adaptation, is essential for light excitation, and keeps the store-operated inward current under a tight control (Arnon, 1997b).

Activation of the Drosophila visual cascade is extremely rapid and results in opening of the cation influx channels transient receptor potential (TRP) and transient receptor potential-like (TRPL) within ~10-20 msec of photostimulation of rhodopsin. The G-protein-signaling cascade that leads to opening of the ion channels has been extensively characterized and is known to involve the inositol phospholipid-signaling system. Termination of the photoresponse, after cessation of the light stimulus, is also rapid and is a Ca2+-regulated process; however, understanding of the mechanism by which Ca2+ contributes to termination of the photoresponse is quite incomplete (Li, 1998 and references).

Several proteins have been identified that seem to mediate Ca2+-dependent termination of phototransduction. These include the Ca2+-binding regulatory protein Calmodulin, which functions in both light adaptation and termination of the light response. The ninaC (neither inactivation nor afterpotential C) locus, which encodes two isoforms, p132 and p174, each of which consists of a protein kinase domain fused to a myosin head domain, also functions in negative feedback regulation of the photoresponse. The two NINAC proteins differ because of unique C-terminal ends. p174 is localized exclusively to the microvillar portion of the photoreceptors, the rhabdomeres, and p132 is restricted to the cell bodies. Null mutations in ninaC cause defects in adaptation and response termination. These functions are caused by p174 because elimination of p174, but not p132, causes each of these phenotypes. Because negative feedback regulation seems to be mediated by Ca2+, it is plausible that p174 is regulated by Ca2+. However, p174 does not contain a known Ca2+-binding motif, such as an EF hand or C2 domain, and there is no evidence that it binds Ca2+ directly. Thus, p174 seems to respond to the light-dependent Ca2+ flux indirectly. One NINAC Ca2+ sensor is Calmodulin because NINAC binds to Calmodulin and the NINAC-Calmodulin interaction is required for both adaptation and termination. NINAC might also be regulated by Ca2+-dependent phosphorylation because p174 contains multiple protein kinase C (PKC) consensus sites including several in its unique C-terminal tail. Moreover, mutation of an eye-specific PKC (ePKC) causes perturbations in adaptation and termination. The role of PKC in negative feedback regulation may be more significant than that indicated by mutation of ePKC because a second PKC, brain PKC (brPKC), is known to be enriched in the Drosophila retina and a third PKC, PKC98F, is highly expressed in adult heads. Two retinal substrates for PKC have been identified. These are the TRP cation influx channel and the PSD95, DLG, and ZO-1 (PDZ)-containing protein inactivation, no afterpotential D (INAD), which binds to most of the proteins that function in phototransduction and organizes a supramolecular signaling complex. However, the consequences of disrupting PKC phosphorylation of any retinal substrate that functions in Drosophila vision have not been determined (Li, 1998 and references).

The current work shows that NINAC p174, which consists of a protein kinase domain joined to the head region of myosin heavy chain, is a phosphoprotein and is phosphorylated in vitro by PKC. Mutation of either of two PKC sites in the p174 tail results in an unusual defect in deactivation that has not been detected previously for other ninaC alleles or other loci. After cessation of the light stimulus, there appeared to be a transient reactivation of the visual cascade. This phenotype suggests that a mechanism exists to prevent reactivation of the visual cascade and that p174 participates in this process. The termination mechanisms controlling Drosophila phototransduction seem to be more complicated than previously envisioned. In addition to a requirement for NINAC in facilitating rapid deactivation after cessation of the light stimulus, there is an additional requirement for this unconventional myosin in preventing transient reactivation of the plasma membrane conductances. Because p174 also functions in adaptation, it seems that NINAC has a central role in many aspects of negative feedback regulation of the visual cascade. Recently, a homolog of NINAC has been identified in the mammalian retina (D. Hillman, A. Dose, and B. Burnside, personal communication to Li, 1998). Thus, it is intriguing to speculate that vertebrate NINAC also functions in negative feedback regulation and that an active mechanism may also exist in mammalian photoreceptor cells to ensure stable termination of phototransduction (Li, 1998).

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). Phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale that is faster than that of any other protein in vivo. A CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. Ser366 is identified as the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI. It has been concluded that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors (Matsumoto, 1994).

Calmodulin regulation of Drosophila light-activated channels and receptor function mediates termination of the light response in vivo

The characterization of Drosophila Calmodulin mutants and the role of CAM in photoreceptor cell function have been described. In Drosophila photoreceptor neurons, light activation of rhodopsin activates a heterotrimeric G protein, which in turn activates phospholipase C (PLC). PLC catalyzes the hydrolysis of the minor membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) into the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG). Activation of PLC then leads to the opening of cation-selective membrane channels encoded by the transient receptor potential (trp) and trp-like (trpl) genes. It has been hypothesized that calcium release from internal stores is required for activation of the phototransduction cascade and that the TRP channel functions as a store-operated channel gated by the light-induced emptying of the internal stores (Scott, 1997 and references).

Contrary to current models of excitation and TRP channel function, the transient phenotype of trp mutants can be explained by CAM regulation of the TRPL channel rather than by the loss of a store-operated conductance leading to depletion of the internal stores. In fact, introduction of calcium intracellularly in trp mutants does not restore responsiveness. The finding that trp mutants can maintain responsiveness in the absence of calcium suggests that there is calcium-dependent inactivation of light-induced currents in the trp mutant. Light responses were analyzed in a variety of mutant and transgenic backgrounds. The transient respone of trp mutants reflects TRPL channel function. Deletion of either of the two CAM binding sites of TRPL results in a prolonged current suggesting that CAM binding functions to inactivate TRPL. Thus, Calmodulin is essential for calcium-dependent negative regulation of phototransduction. Mutants for cam display dramatic defects in deactivation kinetics, displaying greatly prolonged deactivation times. In the absence of extracellular calcium, mutant and wild-type responses are not significantly different from each other, demonstrating that calium entry is required to reveal the cam mutant phenotype and highlighting the absolute requirement for calcium for the rapid deactivation of the phototransduction cascade. CAM ilso regulates the catalytic lifetime of activated rhodopsin by regulating the binding of arrestin to rhodopsin. Thus CAM coordinates termination of the light response by modulating receptor and ion channel activity (Scott, 1997).

Retinal targets for calmodulin include proteins implicated in synaptic transmission

To identify Calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, a screen was conducted for retinal Calmodulin-binding proteins. Twelve Calmodulin-binding proteins were found that are expressed in the Drosophila retina. The functions of Calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the Trp and Trp-like ion channels, NinaC and InaD. Eight calmodulin-binding proteins have been identified that have not been previously reported to be expressed in the Drosophila retina. The full-length sequences corresponding to three of the calmodulin-binding proteins have been described. These corresponded to two Calmodulin-dependent protein kinases, MLCK and CaM kinase II, as well as to one of two previously described Calcineurin proteins. A third calmodulin-dependent protein kinase is expressed in the Drosophila retina, CaM kinase I. No CaM kinase I has previously been reported from any invertebrate, raising the possibility that this protein kinase is specific to vertebrates. Nevertheless, the Drosophila CaM kinase I is highly related to vertebrate CaM kinase I: ~60% identical over 349 amino acids. The remaining four calmodulin-binding proteins have not been known to bind calmodulin prior to the current work. Six targets have been found that are related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein (UNC13) and a protein (CRAG) related to Rab3 GTPase exchange proteins. Two other calmodulin-binding proteins found are Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that Calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission (Xu, 1998).

At least two of the four novel calmodulin-binding proteins share similarities to components implicated in synaptic transmission. One of these proteins (1441 residues), is referred to as CRAG (calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein; due to its similarity to a domain in the recently identified rat Rab3 GDP/GTP exchange protein (rRab3 GEP) and the C. elegans homolog, AEX-3, which has been implicated in synaptic vesicle release. The sequences of AEX-3 and the rRab3 GEP were published contemporaneously and have therefore not been directly compared. AEX-3 and the rRab3 GEP (1409 and 1602 amino acids, respectively) contain three regions of homology, the first of which (~500 residues) is conserved in CRAG, AEX-3, rRab3 GEP (CAR domain) and the human homolog, MADD (death domain MAP kinase activator). The latter two regions, are conserved in AEX-3 and rRab3 GEP (AR1 and AR2), but not CRAG, and are shorter (~100 and 300 residues, respectively) than the CAR homology. The CAR domain in CRAG is ~36 identical over 321 amino acids (residues 95-415) to either rRab3 GEP or AEX-3. In addition, there is weak homology (16%) in the flanking sequences that extend the CAR domain in CRAG to residues 73-490. The C-terminal ~800 residues of CRAG do not share significant primary amino acid sequence homology with the rRab3 GEP, AEX-3, or any other protein in the data banks (Xu, 1998 and references).

While it remains to be determined if CRAG is also a Rab3 GEP, such a finding would have interesting implications regarding the mechanism by which GTP exchange on Rab3 is regulated. Rab3 binds to synaptic vesicles; however, this association only occurs in resting nerve terminals and requires Rab3 in the GTP bound state. Mutation of the C. elegans Rab3 GEP, known as AEX-3, causes accumulation of Rab3 in neuronal cell bodies and an impairment in the release of neurotransmitter. Thus, the Rab3 GEP appears to play a critical role in association of Rab3 with synaptic vesicles and in synaptic transmission. The observation that CRAG binds calmodulin implies that the putative GEP activity of this protein could be regulated by changes in Ca²⁺ levels, which are spatially restricted to microdomains near the active zones in presynaptic terminals. A variety of evidence suggests that the absolute level of Rab3-GTP bound to synaptic vesicles regulates the rate of exocytosis by limiting the number of vesicles that can be fused with the plasma membrane. Thus, formation of Rab3-GTP appears to be a crucial step in synaptic transmission. The mechanisms controlling the GDP-GTP exchange are not known but one possibility is that CRAG is a Rab3 GEP and the associated calmodulin provides a sensor to differentiate between the lower Ca²⁺ levels in resting nerve terminals and higher levels resulting from Ca²⁺ influx. While it remains to be determined if CRAG is a Rab3 GEP and whether the exchange activity is regulated through the associated calmodulin, an exchange factor for another small GTPase, RAS, binds to and is regulated by Ca²⁺/calmodulin (Xu, 1998 and references).

Pollux (Plx) is a protein previously reported to be 732 amino acids in length and required for viability (S. D. Zhang, 1996). The protein is predicted to have a transmembrane domain and a leucine zipper (S. D. Zhang, 1996). Plx has now been found to be 1379 amino acids in length and the formerly assigned initiator methionine corresponds to residue 648. A protein related to Plx is TBC1 (Richardson, 1995), a mouse protein which had homology to the majority of Plx. The region in Plx that contains the greatest similarity to TBC1 is a 337-amino acid segment (51% identity, residues 676-1012) that includes the putative transmembrane domain. Of particular interest, the region most highly conserved between Plx and TBC1 includes a 153-amino acid domain (residues 811-963) that displays moderate homology to the yeast Rab family GTPase-activating proteins, GYP6 or GYP7 (Strom, 1994). GYP7 is ~29% identical to this domain in either Plx or TBC1; however, if two gaps of 18 and 36 amino acids have been introduced in Plx and TBC1, the 29% homology extends to over 222 amino acids (742-963). This ~200 amino acid sequence corresponds to the domain previously referred to as a TBC domain due to its similarity to segments in the TRE-2 oncogene and the yeast regulators of mitosis, BUB2 and CDC16. TBC1 is 1141 residues and is found to be a nuclear protein. Thus, TBC1 and Plx have very disparate spatial distributions. (Xu, 1998).

The portion of the Plx protein that was isolated in the screen extends from residues 180-1379. Using a series of overlapping GST fusion proteins and the gel overlay assay, the calmodulin-binding site(s) contained in the original fusion protein was further mapped to residues 657-680. The sequence of the calmodulin-binding site is not conserved in the mouse homolog, TBC1, but is in several human ESTs. A bovine homolog of Plx (Lyncein), which was isolated from a bovine retinal library, is highly conserved in the calmodulin-binding domain despite having no higher overall sequence conservation to Plx than TBC1. Moreover, a fusion protein containing the conserved sequence in Lyncein binds calmodulin. Plx also bind to calmodulin in a pull-down assay; although this interaction is Ca²⁺ independent (Xu, 1998).

Thus it has been found that Plx is 1379 residues rather than 732 amino acids as previously reported (S. D. Zhang, 1996). The additional sequence is not due to a chimeric cDNA since multiple plx cDNAs were obtained and TBC1 shares similarity to Plx both N- and C-terminal to the formerly assigned initiating methionine at residue 648. Plx has been shown to bind calmodulin and does so in a Ca²⁺-independent manner. Although the sequence of the calmodulin-binding site is not conserved in TBC1, the region is very similar in Lyncein, a homolog isolated from a bovine retinal library. Furthermore, the Lyncein sequence also binds calmodulin. Thus, it appears that a Plx homolog is expressed in the vertebrate retina. A possible clue as to the function of Plx in the retina is that it shares some similarity to two yeast Rab GAP proteins, although no homology was found to the Rab3 GAP expressed in the rat brain. Nevertheless, the observation that Plx contains a domain related to Rab GAPs combined with the finding that it appears to be localized to the plasma membrane and lumen of the trachael system raises the possibility that Plx may be involved in exocytosis. In the Drosophila visual system, exocytosis is important not only in synaptic transmission but in turn-over of the microvillar membrane of the photoreceptor cells. Shedding of membrane does not occur uniformly during the diurnal cycle, but occurs maximally soon after dawn. Thus, an increase in the exocytotic process is correlated with the light dependent rise in Ca²⁺ and therefore might be regulated in part by a Ca²⁺ sensing component in a Rab cycle. Alternative potential functions for Plx in photoreceptor cells include other processes that involve vesicular trafficking such as insertion of new membrane in the microvilli and the budding, targeting, and fusion of rhodopsin carrier vesicles with the plasma membrane. These latter events involve a variety of Rab proteins and also appear to be regulated during the daily light cycle (Xu, 1998 and references).

A third protein, not previously known to bind calmodulin, is a Drosophila homolog of UNC13 (dUNC13), a diacylglycerol-binding protein that may be required for release of neurotransmitter from the presynaptic terminal. dUNC13 is expressed as at least two alternatively spliced forms encoding proteins of >1304 (dUNC13A) and >1724 (dUNC13B) amino acids. dUNC13A and dUNC13B shared a common C-terminal region of >1216 amino acids and differ due to unique N-terminal sequences (>88 and >508 residues, respectively). dUNC13 contains extensive homology (>68%) with the C. elegans UNC13 and rat homologs (mUNC13), beginning in the unique region of dUNC13A and extending over the entire region common between both isoforms (residues 72-1304). UNC13 and mUNC13-1 share a similar level of homology over the same region and are only weakly related over the N-terminal ~500 amino acids. The 508 amino acids specific to dUNC13B are not homologous to the UNC13 proteins or any proteins in the data banks. Features common between dUNC13 and other members of the UNC13 family include strong homology to two conserved sequence motifs, C1 and C2, originally recognized in various protein kinase C isoforms. A large variety of other signaling proteins, such as RAF, diacylglycerol kinase, RAS GTPase-activating protein, synaptotagmin, and phoshopholipase C contain these domains. C1 domains typically bind diacylglycerol, while many C2 domains are Ca²⁺-binding regulatory domains. Some C2 domains also bind phospholipids and do so in a Ca²⁺-dependent manner. Other C2 domains confer Ca²⁺ dependence to functions, such as protein kinase activity, mediated by domains distinct from C2. Biochemical analyses of UNC13 demonstrate that it is a bona fide Ca²⁺-dependent phorbol ester-binding protein. The putative C1 domain in dUNC13 (residues 182-232) include six invariant cysteines as well as a seventh cysteine conserved among all UNC13 proteins. Overall, the C1 domain is 92% identical to the corresponding region in mUNC13. The two C2 domains present in each of the three other UNC13 proteins (C2-1 and C2-2) are also found in dUNC13. C2-1 (residues 299-393) and C2-2 (residues 1170-1264) are 76 and 67% identical with the same motifs in mUNC13-1 (Xu, 1998 and references).

In addition to aex-3, several other mutations have been identified in C. elegans that appear to disrupt exocytosis of synaptic vesicles and release of neurotransmitter. One such mutation is in the gene encoding the diacylglycerol-binding protein, UNC13. Although the specific function of UNC13 remains unclear, it may operate in docking and/or fusion of synaptic vesicles since the rat brain-specific mUNC13-1 protein binds directly to two proteins, syntaxin and Doc2alpha, which function in Ca²⁺-dependent exocytosis. The C2 domains present in UNC13 homologs could potentially serve as a Ca²⁺ sensor that responds to the Ca²⁺ influx required for exocytosis. Therefore, the question arises as to the function of a potential second type of Ca²⁺ sensor provided by the binding of calmodulin to dUNC13. One possibility is that each UNC13 protein really has only one Ca²⁺ sensor and that it is supplied in some isoforms by the C2 domain and in others through Ca²⁺/calmodulin. Consistent with this proposal, the calmodulin-binding domain is not conserved in UNC13 suggesting that the C2 domain provides the only Ca²⁺ detector in this protein. The reverse may be the case in mUNC13-1 since this protein does not appear to contain Ca²⁺-binding C2 domains but does show sequence similarity to the dUNC13 calmodulin-binding site. An alternative proposal, which is favored by the authors, is that some UNC13 proteins may be regulated by Ca²⁺ via both C2 domains and calmodulin. Such dual regulation may provide a mechanism for extremely rapid as well as sustained responses to highly transient increases in Ca²⁺. The rise in Ca²⁺, resulting from opening of the voltage-gated channels in synaptic terminals, occurs in microdomains and collapses within microseconds after closing of the ion channels. C2 domains comprise an unusual Ca²⁺ binding motif in that Ca²⁺ appears to regulate this domain through a shift in electrostatic potential rather than a conformational change. As such, C2 domains have the potential to respond very quickly, but transiently, to the rapid Ca²⁺ flux in the active zones of the presynaptic terminal. Although fusion and release of neurotransmitter is extremely rapid (submilliseconds to milliseconds), there is some latency between the opening and closing of the ion channels and these latter events. Ca²⁺ binding to calmodulin, which induces a conformational change, may induce a more delayed but sustained response to Ca²⁺ than that provided by the C2 domain. Thus, dual binding of Ca²⁺ to calmodulin and C2 domains may enable UNC13 proteins to sense the Ca²⁺ rise within a few microseconds and sustain the response for several hundred microseconds to several milliseconds (Xu, 1998 and references).

The fourth novel calmodulin-binding protein is referred to as Calossin (CALO) due to its interaction with calmodulin and colossal molecular mass (predicted >450 kDa). Several overlapping cDNAs have been obtained resulting in the identification of a single open reading frame encoding >4118 amino acids. Several hydrophobic regions are predicted according to a computer algorithm; however, it is unclear if any is sufficiently long to span a lipid bilayer. CALO is related to a predicted C. elegans protein (cCalossin) of similar size (3864 residues) that was identified as part of the C. elegans Genome Sequencing Consortium. The homology between CALO and cCALO was not uniform but concentrated in several domains. The longest continuous region of identity begins at amino acid 2460 and extends ~1650 residues to near the C terminus. In addition, there are two shorter stretches of similarity between residues 604 and 1150. The highest levels of identity (each ~70%) are in three ~50-100 amino acid regions: (1) residues 604-649; ( 2) residues 2587-2638, and (3) residues 3276-3380. The first two of these conserved regions are cysteine-rich domains, CRD1 and CRD2, respectively, that resemble different classes of zinc finger domains. CRD1 is most similar to the zinc finger family defined by Requiem, a protein required for apoptosis, while CRD2 shares features equally well with several families of zinc family proteins and can not be included within a single group (Xu, 1998 and references).

Regulation of the Rhodopsin protein phosphatase Rdgc by Calmodulin

Hundreds of G protein-coupled receptors (GPCRs) and at least six GPCR kinases have been identified, but the only GPCR phosphatase that has been definitively demonstrated is the rhodopsin phosphatase encoded by the rdgC locus of Drosophila. Mutations in rdgC result in defects in termination of the light response and cause severe retinal degeneration. RDGC is shown to bind to Calmodulin, and a mutation in an IQ motif that eliminates the Calmodulin/RDGC interaction prevents dephosphorylation of rhodopsin in vivo and disrupts termination of the photoresponse. These data indicate that RDGC is a novel calmodulin-dependent protein phosphatase and raise the possibility that regulation of other GPCRs through dephosphorylation may be controlled by calmodulin-dependent protein phosphatases related to RDGC (Lee, 2001).

Stimulation of G protein-coupled receptors (GPCRs) by hormones, growth factors, neurotransmitters, sensory stimuli, and other agonists frequently results in an increase in intracellular Ca²⁺. Such changes in Ca²⁺ concentration regulate a variety of effects ranging from apoptosis to differentiation, cell movement, the modulation of synaptic plasticity, and visual transduction. A primary mechanism through which alterations in Ca²⁺ levels lead to discrete physiological consequences involves the control of protein phosphorylation by the Ca²⁺ sensor calmodulin. Several calmodulin-dependent serine/threonine kinases have been described, such as myosin light chain kinase and Ca²⁺/calmodulin-dependent protein kinases I, II, and IV. However, the only known calmodulin-dependent protein phosphatase is calcineurin, despite the ~20 years that have elapsed since its discovery. Calcineurin is conserved from yeast to humans and is a heterodimer consisting of a catalytic subunit, CnA, and a regulatory subunit, CnB, comprised of four Ca²⁺ binding motifs referred to as EF hands (Lee, 2001).

Given the central role of Ca²⁺ in regulating a vast array of essential processes, it seems likely that there exist additional calmodulin-dependent protein phosphatases. Candidates include phosphatases known to function in Ca²⁺-regulated signaling cascades. One such protein is the rhodopsin phosphatase, RDGC, which participates in Drosophila phototransduction (Steele, 1990; Steele, 1992; Byk, 1993; Vinós, 1997). Drosophila phototransduction culminates with Ca²⁺ and Na⁺ influx via the TRP, TRPL, and TRPgamma channels (Lee, 2001).

A key mediator of the Ca²⁺-mediated feedback regulation is calmodulin; however, there are only a few signaling proteins known to function in Drosophila phototransduction that bind to calmodulin. These include the NINAC myosin III, TRP, TRPL, and INAD. Some calmodulin binding proteins, such as TRPL, interact with calmodulin via the positive face of an amphiphilic alpha helix, while others, such as NINAC, associate through IQ motifs. IQ motifs, which contain the core consensus IQxxxRGxxxR (x denotes any amino acid), may associate with calmodulin in either a Ca²⁺-dependent or independent manner. IQ motifs are present in a wide diversity of proteins ranging from myosins to neuromodulin (GAP-43, voltage-gated Ca²⁺ channels, and the Ras guanine nucleotide exchange factor, RAS-GRF). Moreover, the IQ/calmodulin interactions regulate the activities of each of these classes of proteins (Lee, 2001).

The rhodopsin phosphatase, RDGC, is a potential target for regulation by calmodulin, since mutations in rdgC result in severe defects in the Ca²⁺-dependent termination of the photoresponse (Steele, 1990; Vinós, 1997). Disruption of rdgC function also results in age- and light-dependent retinal degeneration (Steele, 1990). Homologs of RDGC are conserved from C. elegans to humans and are collectively referred to as the PPEF family due to the protein phosphatase domain and the presence of multiple C-terminal Ca²⁺ binding motifs, EF hands (Lee, 2001).

The present work shows that RDGC is a calmodulin-regulated protein phosphatase. RDGC binds directly to calmodulin, and this interaction disrupts an association between the N-terminal domain of RDGC and the catalytic domain. Furthermore, the calmodulin/RDGC interaction is required to potentiate dephosphorylation of rhodopsin in vivo and for rapid termination of the photoresponse (Lee, 2001).

To test whether RDGC binds to calmodulin, calmodulin-agarose pull-down assays were performed. Agarose beads conjugated to calmodulin were incubated with full-length RDGC labeled in vitro with ³⁵S. As a negative control, a segment of the PDZ protein, INAD (PDZ domains 3 and 4; amino acids 346-581), which is devoid of calmodulin binding activity, was used. RDGC binds to calmodulin-agarose, although the INAD-PDZ3-4 segment does not. Binding of RDGC to calmodulin-agarose is not strictly Ca²⁺ dependent. However, a greater proportion of RDGC binds to calmodulin in the presence of Ca²⁺ (Lee, 2001).

To determine whether RDGC and calmodulin interact in vivo, whether the two proteins coimmunoprecipitate from fly heads was tested. Anti-RDGC antibodies were generated that recognize three bands (84, 78, and 76 kDa) in wild-type but not rdgC head extracts. The 78 and 76 bands appear to be eye specific, since they are not detected in the eyeless mutant, sine oculis (so). The 84 kDa isoform, which is not eye specific, may be responsible for the previously reported RDGC expression in the mushroom bodies of the central brain (Steele, 1992). To assess whether RDGC and calmodulin associate in vivo, RDGC was immunoprecipitated from wild-type or null rdgC head extracts, and Western blots of the immune complexes were probed with anti-calmodulin antibodies. Calmodulin is detected in the immune complexes from wild-type but not from null rdgC head extracts. Furthermore, calmodulin coimmunoprecipitates with RDGC in the presence or absence of Ca²⁺, although more calmodulin immunoprecipitates in the presence of Ca²⁺ (Lee, 2001).

To map the sites of interaction between RDGC and calmodulin, calmodulin overlay assays were used. Various fragments of RDGC were expressed in E. coli as GST fusion proteins. Total bacterial extracts were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with [¹²⁵I]calmodulin. All the fusion proteins that bind to calmodulin contain the N-terminal 32 residues, while those proteins that lack these residues fail to associate with calmodulin. Thus, the N-terminal 32 residues contain the calmodulin binding site (Lee, 2001).

RDGC contains a sequence similar to the IQ-calmodulin binding motif, and this sequence maps to the N terminus of RDGC (residues 12-22). To address whether RDGC binds to calmodulin through the IQ motif, most of the sequence (deltaIQ, amino acids 12-22) was deleted and the effects on calmodulin binding were assessed using calmodulin overlay assays. GST-fusion proteins containing full-length RDGC or the N-terminal 73 residues of RDGC bind calmodulin (RDGC and 73^WT). However, derivatives of these fusion proteins that lacked residues 12-22 fail to associate with calmodulin (RDGC^deltaIQ and 73^deltaIQ) (Lee, 2001).

To obtain additional evidence that RDGC binds to calmodulin through the IQ motif, the effects of a variety of conservative and nonconservative substitutions of the most invariant residues within the IQ sequence were assessed. Among the point mutations generated, the only one that almost completely abolishes interaction with calmodulin is a glutamic acid substitution of the isoleucine (residue 12) that begins the motif (73^I12E). A relatively conservative alanine substitution of the same residue (73^I12A) does not eliminate calmodulin binding, but appears to result in an increase in the level of calmodulin bound to RDGC. Thus, it appears that the IQ sequence is the only calmodulin binding site in RDGC (Lee, 2001).

To confirm the effects of the single amino acid substitutions in residue 12 and to test whether these alterations influence the effect of Ca²⁺ on the RDGC/calmodulin interaction, calmodulin-agarose pull-down assays were performed. Consistent with the results of the overlay assay, 73^deltaIQ and 73^I12E virtually abolished binding whereas 73^I12A displays an increase in calmodulin binding relative to 73^WT. In addition, the I12A mutation alters the Ca²⁺ dependence for calmodulin binding. While wild-type RDGC binds calmodulin in the presence or absence of Ca²⁺, the interaction between 73^I12A and calmodulin is strictly Ca²⁺ dependent (Lee, 2001).

Other members of the RDGC/PPEF family also contain an N-terminal IQ consensus sequence, suggesting that other RDGC-related proteins may interact with calmodulin. To test whether the IQ sequence in human PPEF2 is a calmodulin binding site, calmodulin-agarose pull-down assays were performed. The core region of the IQ motif (amino acids 21-42) of human PPEF2 was fused to GST (GST-HsIQ), purified using a glutathione-Sepharose column, and incubated with calmodulin-agarose beads. GST-HsIQ binds to calmodulin-agarose, but GST alone does not. In addition, GST-HsIQ binds to calmodulin-agarose in the presence or absence of Ca²⁺. Thus, the IQ-type motif domain in human PPEF-2 is sufficient to bind calmodulin (Lee, 2001).

To determine the physiological role of the RDGC/calmodulin interaction, transgenic flies were generated that express full-length derivatives of RDGC that incorporate the I12A mutation, the I12E mutation, and the IQ deletion of amino acids 12-22. All of the mutations were introduced into the 7.1 kb genomic DNA shown to restore wild-type visual function to rdgC-mutant flies (Steele, 1992). The wild-type (P[rdgC⁺]) and mutant transgenes (P[rdgC^I12A], P[rdgC^I12E], and P[rdgC^deltaIQ]) were expressed in a null rdgC (rdgC^co6) background so that the only RDGC proteins originated from the transgenes. The wild-type transgene restored normal levels of both eye-specific isoforms of RDGC. In addition, the point mutations (I12A and I12E) do not affect the stability of RDGC, since the level of the proteins was similar to that observed in wild-type (y,w) and P[rdgC⁺] flies. However, the deletion of the IQ motif appears to render the protein unstable, since the RDGC^deltaIQ is undetectable (Lee, 2001).

To test whether calmodulin binding is critical for RDGC function in vivo, the retinal morphology of the transgenic flies was examined. Drosophila compound eyes consist of ~800 ommatidia, each of which contains eight photoreceptor cells, though only seven are present in any given plane of section. The photoreceptor cells include a microvillar structure, the rhabdomere, which contains the proteins critical for visual transduction. The morphology of the wild-type and P[rdgC⁺] rhabdomeres do not change with age or depending on the light conditions. However, rdgC flies are characterized by age- and light-dependent retinal degeneration (Steele, 1990). One-day-old rdgC flies maintained under a 12 hr light/12 hr dark cycle show little if any decrease in the size of the rhabdomeres. After rearing rdgC flies for 7 days under a light-dark cycle, the rhabdomeres of the R1-6 cells are almost completely degenerated. The rhabdomeres of rdgC^I12E flies show a pattern of degeneration similar to rdgC null flies. By contrast, no degeneration is observed in rdgC^I12A flies aged for 7 days under the light-dark cycle. These data indicated that the I12E but not the I12A mutation disrupted RDGC function (Lee, 2001).

To test whether association of calmodulin with RDGC is critical for rapid inactivation, the light response was examined using electroretinograms (ERGs). ERGs are extracellular recordings that measure the summed responses of the retinal cells to light. Exposure of rdgC⁺ flies to a light stimulus results in a corneal negative deflection in the ERG. Upon termination of the light response, there was a rapid return to the baseline. The deactivation of the light response is significantly delayed in rdgC null mutant flies. There was no delay in termination in rdgC^I12A; rather, the deactivation kinetics in these flies is slightly faster than rdgC⁺. However, the deactivation rate of rdgC^I12E decreases significantly compared with rdgC⁺, although the delay is slightly less severe than in the null mutant (Lee, 2001).

When a substantial amount of rhodopsin is photoconverted from rhodopsin to the light activated metarhodopsin state, a sustained photoresponse or prolonged depolarization afterpotential (PDA) persists after cessation of the light stimulus. In wild-type, intense light is required to produce a PDA. Blue rather than orange or white light is most effective in producing a PDA, since rhodopsin, but not metarhodopsin, is maximally activated by blue light. A second photon of light, white or orange, is required to convert metarhodopsin to rhodopsin and terminate the PDA (Lee, 2001).

A PDA can be generated in rdgC with considerably less light than in rdgC⁺. A PDA results from an excess of metarhodopsin relative to arrestin. Therefore, mutants that express less arrestin than wild-type also exhibit a PDA with less intense light. It has been suggested that rdgC flies display a PDA with less light than wild-type, since mutations in rdgC cause hyperphosphorylation of rhodopsin, which in turn impairs arrestin function (Lee, 2001).

To address whether the rdgC transgenic flies display defective PDAs, the relative intensity of light needed to produce a PDA was assayed. Like rdgC null mutants, rdgC^I12E enters a PDA with <10% the light intensity required in rdgC⁺. In contrast, rdgC^I12A shows a PDA similar to rdgC⁺. The observations that rdgC^I12E flies display retinal degeneration, a delay in deactivation kinetics, and a low light PDA indicates that calmodulin binding is necessary for normal function of RDGC in vivo (Lee, 2001).

The simplest hypothesis to account for the requirement of the RDGC/calmodulin interaction is that calmodulin regulates the phosphatase activity of RDGC in vivo. To test this proposal, the relative levels of phosphorylated rhodopsin (Rh1) were measured in rdgC⁺ and rdgC^I12E eyes. Dark-reared flies were fed ³²P and then exposed to light. As expected, rhodopsin is hyperphosphorylated in rdgC relative to rdgC⁺. Of primary importance here, rhodopsin is also hyperphosphorylated in the rdgC^I12E eyes. However, the level of rhodopsin phosphorylation in rdgC^I12A was similar to control flies (rdgC⁺). ninaE^P332 flies, which express only 0.1% of rhodopsin, were used as a negative control. Thus, association of calmodulin with RDGC appears to be required for RDGC phosphatase activity in vivo (Lee, 2001).

Whether calmodulin potentiates RDGC activity in vitro was tested using purified recombinant RDGC and p-nitrophenyl phosphate as a pseudosubstrate. Consistent with studies of a human homolog of RDGC, the activity of RDGC is increased by Ca²⁺, possibly through interaction of Ca²⁺ with the EF hands. Similar results were obtained with RDGC^I12E. Significantly, the addition of calmodulin in the presence of 100 µM Ca²⁺ further increases the phosphatase activity of wild-type RDGC but not that of RDGC^I12E. Thus, the enzymatic activity of RDGC seems to be augmented by calmodulin in vitro, although the level of calmodulin-dependent potentiation is much greater in vivo than in vitro (Lee, 2001).

Calmodulin might augment RDGC phosphatase activity by relieving a putative intramolecular interaction that inhibits the phosphatase activity. As a first test of this model, an examination was carried out to see whether the N-terminal region of RDGC, which includes the calmodulin binding site, associates with the catalytic domain. ³⁵S-labeled N-terminal RDGC (amino acids 1-253) binds to a GST-catalytic domain fusion protein (amino acids 153-423) but not to GST alone. Moreover, neither the middle nor the C-terminal portions of RDGC associate with the catalytic domain (Lee, 2001).

To test whether calmodulin binding to RDGC affects the interaction between the N-terminal portion of RDGC and the catalytic domain, pull-down assays were performed. ³⁵S-labeled N-terminal RDGC was incubated with the GST-catalytic domain fusion in the presence or absence of Ca²⁺/calmodulin. The addition of Ca²⁺/calmodulin decreases the interaction between N-terminal RDGC and the GST-catalytic domain fusion nearly 5-fold. However, the interaction between the catalytic domain and the N-terminal fragment of RDGC containing the I12E is not decreased by Ca²⁺/calmodulin. The addition of only BSA or Ca²⁺ does not change the binding of N-terminal RDGC and the catalytic domain (Lee, 2001).

Thus, several lines of evidence support the conclusion that RDGC is a calmodulin-regulated protein phosphatase. RDGC binds to calmodulin in vitro and in vivo, and the interaction is through an established calmodulin binding sequence, the IQ motif. An I to E substitution in this motif disrupts calmodulin binding and interfers with dephosphorylation of rhodopsin in vivo. Calmodulin also potentiates dephosphorylation of a pseudosubstrate in vitro, although the effect is smaller than that observed in vivo, possibly due to a contribution of one or more cofactors that remain to be identified. Finally, addition of calmodulin interfers with an intramolecular interaction between the N-terminal region and the RDGC catalytic domain (Lee, 2001).

It had been assumed previously that RDGC is not regulated by calmodulin since an inhibitor of calmodulin, M5, does not perturb the RDGC-dependent phosphatase activity in fly head extracts (Byk, 1993). However, in addition to RDGC, the activities of several other calmodulin-regulated proteins, such as a Ca²⁺-activated K⁺ channel and an L-type Ca²⁺ channel, are unaffected by application of calmodulin inhibitors. Thus, a lack of effect of calmodulin inhibitors does not rule out the possibility that a given protein interacts with and is regulated by calmodulin (Lee, 2001).

The identification of RDGC as a calmodulin-regulated protein phosphatase addresses a key question in Drosophila phototransduction, the identity of in vivo targets for negative feedback regulation by Ca²⁺/calmodulin. Calmodulin is present at ~0.5 mM concentration in the rhabdomeres and a decrease in rhabdomeral calmodulin has a profound effect on termination of the photoresponse. However, the only rhabdomeral proteins previously shown to be regulated in vivo by calmodulin are the NINAC myosin III, the TRPL cation channel, and Arrestin 2. As is the case for the major rhodopsin, Rh1, Arrestin 2 also undergoes rapid light-dependent phosphorylation. The phosphorylation of Arrestin 2, which is mediated by Ca²⁺/calmodulin-dependent protein kinase II, appears to regulate the release of Arrestin 2 from Rh1 (Lee, 2001).

Despite the observation that Rh1 undergoes light-dependent phosphorylation, the role of this phosphorylation event is controversial. In mammals, desensitization or inactivation of rhodopsin and other GPCRs is initiated by phosphorylation by GPCR kinases, which increases the affinity for arrestin. Arrestin binding disrupts signaling by interfering with engagement of the receptor with the G protein. The GPCR must be subsequently dephosphorylated before the inactivation and recycling is complete (Lee, 2001).

Evidence that phosphorylation of Rh1 is required for the photoresponse is that mutations in rdgC result in hyperphosphorylation of the receptor and a defect in response termination (Steele, 1990; Byk, 1993; Vinós, 1997). However, a C-terminal truncation of Rh1 (Rh1^delta356), or a combination of mutations that eliminate the phosphorylation sites, has no apparent effect on the photoresponse or on Arrestin 2 binding. Nevertheless, the defect in termination in rdgC flies appears to be due to hyperphosphorylation of the receptor, since the rdgC phenotype is suppressed in rdgC/Rh1^delta356 double-mutants. To reconcile these findings, it has been proposed that the C terminus of Rh1 is an autoinhibitory domain for Arrestin 2 binding and phosphorylation relieves the intramolecular interaction. The mutations that prevent phosphorylation of Rh1 may also eliminate the putative autoinhibitory interaction, thereby abrogating the requirement for phosphorylation (Lee, 2001).

Rhodopsin cannot be the only substrate for RDGC, since it is also expressed in a region of the brain, the mushroom bodies, implicated in learning and memory. Furthermore, while one of the two human RDGC homologs, PPEF-2, is highly enriched in the retina, the other, PPEF-1, is expressed primarily in a variety of sensory neurons of neural crest origin. Thus, PPEF-1 must also engage substrates other than rhodopsin. Likely candidates are other GPCRs that initiate signaling cascades that culminate in a rise in intracellular Ca²⁺. The identification of such substrates should provide valuable insights into additional roles for this new class of calmodulin-regulated protein phosphatases (Lee, 2001).

Ca2+-dependent metarhodopsin inactivation mediated by Calmodulin and NINAC Myosin III

Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. This study investigated the mechanism and role of rhodopsin inactivation. The lifetime of activated rhodopsin (metarhodopsin = M^*) was determined in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M^* by photoreisomerization to rhodopsin could suppress responses to prior illumination. M^* was inactivated rapidly (τ ~20 ms) under control conditions, but ~10-fold more slowly in Ca²⁺-free solutions. This pronounced Ca²⁺ dependence of M^* inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca²⁺ influx acting via CaM and NINAC accelerates the binding of arrestin to M^*. These results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling (Liu, 2008).

This study exploited the bistable nature of invertebrate rhodopsins to measure the lifetime of activated metarhodopsin in Drosophila. The approach measures the time window during which photoreisomerization of M^* can suppress the response to light. The relative lack of overlap of the R and M spectra in UV opsins has been exploited by recording from the UV-sensitive photoreceptors in Limulus median ocelli. This strategy was adapted for Drosophila by using flies engineered to express the UV opsin Rh3; the effective M^* lifetime was found to be very short (τ_dec ≈20 ms) under physiological conditions. Strikingly, M^* lifetime was prolonged ~10-fold in the absence of Ca²⁺ influx, indicating that M-Arr2 binding is Ca²⁺ dependent and that M^* lifetime is the rate-limiting step in response deactivation in Ca²⁺-free solutions. Further experiments led to proposal of a mechanism for Ca²⁺-dependent M^* inactivation by Arr2, mediated by calmodulin (CaM) and myosin III NINAC (Liu, 2008).

Photoisomerization of rhodopsin (R) by short-wavelength light (480 nm for Rh1 or 330 nm for Rh3) generates active metarhodopsin (M^*). M^* continues to activate G_q until it binds arrestin (Arr2) or is reconverted to R by long-wavelength illumination (570/460 nm). M is serially phosphorylated by rhodopsin kinase (RK), but this is not required for M^* inactivation or Arr2 binding. CaMKII-dependent phosphorylation of Arr2 at Ser³⁶⁶ and photoreconversion of M_pp to R_pp is required for the release of Arr_p. Phosphorylation of Arr2 also prevents endocytotic internalization of M-Arr2. In Arr2^S366A or mutants defective in CamKII, photoreconversion fails to release Arr2. Finally, R_pp is dephosphorylated by the Ca-CaM-dependent rhodopsin phosphatase (rdgC) to recreate the ground state, R. The results suggest that under low-Ca²⁺ conditions Arr2 is prevented from rapid binding to M^* because it is sequestered by NINAC or a NINAC-regulated target; however, Ca²⁺ influx acting via CaM rapidly releases Arr2. Each microvillus contains ~70 Arr2 molecules, ensuring rapid quenching of M^* once they are free to diffuse. The role of M^* phosphorylation remains uncertain but may be involved in Rh1 internalization by the minor arrestin, Arr1 (Liu, 2008).

Ca²⁺ dependence of M^* lifetime had not previously been demonstrated in an invertebrate photoreceptor, and the consensus from data in Drosophila suggested no obvious mechanism by which M^* lifetime could be regulated by Ca²⁺. The finding that M^* inactivation is strongly Ca²⁺ dependent prompted a re-examination of possible roles of Rh1 and Arr2 phosphorylation as well as CaM. Although M^* lifetime remained strongly Ca²⁺ dependent in mutants defective in rhodopsin and arrestin phosphorylation, the Ca²⁺ dependence of M^* inactivation was effectively eliminated in hypomorphic cam mutants. This requirement for CaM appeared to be mediated by the myosin III NINAC protein, since the Ca²⁺ dependence of M^* inactivation was effectively abolished in both the null ninaC^P235 mutant and an allele (ninaC^KD) in which CaM levels in the microvilli were unaffected. NINAC, which is the major CaM-binding protein in the photoreceptors, has long been known to be required for normal rapid response deactivation (Porter, 1993b), but the mechanistic basis remained unresolved. These results now strongly suggest that it is specifically required for the Ca²⁺- and CaM-dependent inactivation of M^* by Arr2 (Liu, 2008).

How might NINAC regulate the Ca²⁺-dependent inactivation of M^*? A clue comes from the finding that Arr2 levels were substantially reduced in ninaC mutants. After taking this into account, the lack of Ca²⁺ dependence of M^* inactivation in ninaC mutants was in fact associated with a very pronounced acceleration of response inactivation under Ca²⁺-free conditions. This was most clearly revealed in ninaC^KD, which appears to be specifically defective only in Ca²⁺-dependent M^* inactivation and does not show the additional response defects of the null ninaC phenotype (e.g., Hofstee, 1996). This suggests a disinhibitory mechanism whereby Ca²⁺-dependent inactivation of M^* may be achieved, at least in part, by the NINAC-dependent prevention of Arr2-M^* binding under low-Ca²⁺ conditions. Specifically, it is suggested that in Ca²⁺-free solutions, or in the low-Ca²⁺ conditions prevailing during the latent period of the quantum bump under physiological conditions, Arr2 in the microvilli is predominantly bound to NINAC or a NINAC-regulated target, thus restricting its access to M^*. However, following Ca²⁺ influx, CaCaM would bind to NINAC, causing NINAC to release Arr2, which, as a soluble protein, could then rapidly diffuse to encounter and inactivate M^* (Liu, 2008).

Interestingly, a recent study reported that NINAC can interact with Arr2 in a phosphoinositide-dependent manner (Lee, 2004). This interaction was described in the context of a role of NINAC in light-induced translocation of Arr2, which was reported to be disrupted in ninaC mutants. However, involvement in translocation was challenged by a subsequent study reporting that Arr2 translocation was unaffected in ninaC mutants (Satoh, 2005). It will be interesting to see whether the Arr2-NINAC interactions described by Lee (2004a) reflect a role in the CaCaM- and NINAC-dependent inactivation of M^* reported in this study (Liu, 2008).

It has long been known that responses under Ca²⁺-free conditions decay ~10-fold more slowly than in the presence of Ca²⁺. The current results establish that the inactivation of M^* by Arr2 is the rate-limiting inactivation step in such Ca²⁺-free responses, with a time constant of ~200 ms in wild-type photoreceptors. Following inactivation of M^* by photoreisomerization under Ca²⁺-free conditions, the response decayed with a time constant of ~80 ms. This also provides a unique and direct measure of the time constant(s) of the downstream mechanisms of inactivation, which presumably include GTP-ase activity of the Gq-PLC complex and removal of DAG by DAG kinase. It will be interesting so see whether Ca²⁺ also accelerates these inactivation mechanisms (Liu, 2008).

By contrast, the failure to accelerate response decay by overexpressing Arr2 in the presence of Ca²⁺ indicates that inactivation of M^* is not rate limiting under physiological conditions. This can be understood by recognizing that the macroscopic kinetics are determined by the convolution of the bump latency distribution and bump waveform, the latter probably terminated by Ca²⁺-dependent inactivation of the light-sensitive channels. Until the Ca²⁺ influx associated with the quantum bump, the phototransduction machinery in each microvillus is effectively operating under Ca²⁺-free conditions. The results suggest that it is the Ca²⁺ influx associated with each quantum bump that promotes M^* inactivation, and hence the timing of M^* inactivation will be determined by the bump latency distribution and not vice versa. This leads to the, perhaps counterintuitive, concept that response termination is rate limited, not by any specific inactivation mechanism, but rather by the time course with which the cumulative probability of bump generation approaches 100% (Liu, 2008).

Clearly, rapid quenching of M^* is essential to maintain the fidelity and high temporal resolution of phototransduction. In wild-type cells, an effectively absorbed photon generates only one quantum bump, but never (or extremely rarely) two or more; yet the multiple bump trains observed in arr2, cam, and ninaC mutants show that additional bumps are readily generated within 50–100 ms if M^* fails to be inactivated. To prevent such multiple bumps without Ca²⁺-dependent feedback would require such a high rate of Arr2 binding that many M^* molecules would be inactivated before they had a chance to activate sufficient G proteins to generate a quantum bump. This would result in an effective reduction in sensitivity, as is directly illustrated by the phenotype of p[Arr2] flies overexpressing Arr2. These show not only a 5-fold reduction in quantum efficiency. but also a reduction in bump amplitude and even an increase in bump latency, which is attributed to a decreased rate of second messenger generation. The mechanism proposed in this study provides an elegant solution to this dilemma. The analysis suggests that in the low-Ca²⁺ environment prior to Ca²⁺ influx, much of the Arr2 in the microvillus is bound to NINAC (or NINAC-regulated target), thus allowing M^* to remain active long enough to activate sufficient G proteins to guarantee production of a full-sized quantum bump with high probability. Only after the bump has been initiated does Ca²⁺ influx accelerate the inactivation of M^* by releasing Arr2, thus ensuring that only one bump is generated. This strategy is complemented and enabled by the ultracompartmentalization afforded by the microvillar design, which ensures that the Ca²⁺ rise is both extremely rapid and largely confined to the affected microvillus (Liu, 2008).

Miscellaneous interactions

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain has been quantitified with purified species-specific calmodulins. In all three species, adenylyl cyclase is activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity is dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raises the threshold for adenylyl cyclase activation by Ca2+ but also acts synergistically with Ca2+ to activate maximally adenylyl cyclase (Yovell, 1992).

For more information on the interaction of Calmodulin with adenylyl cyclase, see rutabaga.

Nitric oxide (NO) is an intercellular messenger involved in various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). A Drosophila NOS gene, dNOS, located at cytological position 32B encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, dNOS protein contains putative binding sites for Calmodulin, FMN, FAD, and NADPH. dNOS activity is Ca2+/Calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates (Regulski, 1995).

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/Calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is (respectively) 67% and 65% identical to human calcineurin A alpha and beta cDNAs. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The Calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurin As, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man (Guerini, 1992).

The Drosophila Cactus and Dorsal proteins are required for the development of embryonic dorso-ventral polarity and most likelyfor the innate immune response of the insect as well. Like their mammalian counterparts (the cytoplasmic anchor protein I kappa B and the rel/NF kappa B transcription factors) cactus and dorsal are regulated at the level of nuclear localization. Increased intra-cellular calcium levels induced by the ionophore ionomycin can activate dorsal/cactus complexes in the Drosophila cell line SL2. In a cell line (SLDL) in which dorsal is expressed constitutively, ionomycin induces a rapid destruction of Cactus and dephosphorylation of Dorsal. These results suggest a role for the protein phosphatase calcineurin in calcium mediated activation of dorsal/cactus complexes. They also indicate that in the resting cell, constitutive phosphorylation of Dorsal is in equilibrium with calcium dependent dephosphorylation (Kubota, 1995).

Partial and total loss of function mutant alleles of a putative Drosophila homolog (DPhK-gamma) of the vertebrate phosphorylase kinase gamma-subunit gene have been isolated. DPhK-gamma is required in early embryonic processes, such as gastrulation and mesoderm formation; however, defects in these processes are seen only when both the maternal and zygotic components of DPhK-gamma expression are eliminated. Loss of zygotic expression alone does not appear to affect normal embryonic and larval development; some pupal lethality is observed but the majority of mutant animals eclose as adults. Many of these adults show defects in their leg musculature (e.g. missing and degenerating muscles), in addition to exhibiting melanised "tumours" on their leg joints. Loss of only the maternal component has no obvious phenotypic consequences. The DPhK-gamma gene has been cloned and sequenced. It has an open reading frame (ORF) of 1680 bp encoding a 560 amino acid protein. The predicted amino acid sequence of DPhK-gamma has two conserved domains, the catalytic kinase and Calmodulin-binding domains, separated by a linker sequence. The amino acid sequence of DPhK-gamma is homologous to that of mammalian PhK-gamma proteins but differs in the length and amino acid composition of its linker sequence. The expression of DPhK-gamma mRNA is developmentally regulated (Bahri, 1994).

GAP-43 (growth-associated protein, 43 x 10(3) M[r]) is an essential, membrane-associated, neuronal phosphoprotein in vertebrates. The protein is abundantly produced in the growth cones of developing and regenerating neurons, and it is phosphorylated upon induction of long-term potentiation (LTP). Prior work has identified GAP-43-like proteins only in chordates. In this paper, a nervous system-specific gene from Drosophila melanogaster is described that encodes two proteins sharing biochemical activities and sequence homology with GAP-43. The region of homology encompasses the Calmodulin-binding domain and protein kinase C (PKC) phosphorylation site of GAP-43. The fly proteins are shown to bind Drosophila Calmodulin (CaM), and are phosphorylated by purified PKC after a fashion predicted from prior work with vertebrate GAP-43. GAP-43 is modified by palmitoylation. An amino-terminal myristoylation site is described for the Drosophila protein, which may play a similar role in membrane association in the fly. While a small family of GAP-43-related genes has been recognized in vertebrates, only a single gene appears to be present in the fly. Since the Drosophila gene encodes two proteins, each with multiple Calmodulin-binding domains and repeated sites for PKC phosphorylation, it may illuminate functions carried out by the family of vertebrate genes (Neel, 1994).

A 3.3 kb cDNA encoding the complete amino acid sequence of a calcium/Calmodulin regulated protein phosphatase has been isolated from a Drosophila eye disc cDNA library. The predicted protein of 560 amino acids (molecular mass 62 kDa) is 73-78% identical to human PP2B isoforms. The cDNA hybridizes to the X-chromosome at cytological position 14D1-4. Two transcripts of 3.5 kb and 3.0 kb are expressed during embryonic development, their levels being highest in the early embryo. The larger transcript was also clearly present in adult females. This pattern of expression indicates a role for calcium/Calmodulin regulated protein phosphatase in embryonic development (Brown, 1994).

Drosophila A kinase anchor protein 200 (Akap200), is predicted to be involved in routing, mediating, and integrating signals carried by cAMP, Ca²⁺, and diacylglycerol. Experiments designed to assess this hypothesis establish (1) the function, boundaries and identity of critical amino acids of the protein kinase AII (PKAII) tethering site of Akap200; (2) demonstrate that residues 119-148 mediate binding with Ca²⁺-calmodulin and F-actin; (3) show that a polybasic region of Akap200 is a substrate for protein kinase C; (4) reveal that phosphorylation of the polybasic domain regulates affinity for F-actin and Ca²⁺-calmodulin, and (5) indicate that Akap200 is myristoylated and that this modification promotes targeting of Akap200 to plasma membrane. DAkap200, a second product of the Akap200 gene, cannot tether PKAII. However, DAkap200 is myristoylated and contains a phosphorylation site domain that binds Ca²⁺-calmodulin and F-actin. An atypical amino acid composition, a high level of negative charge, exceptional thermostability, unusual hydrodynamic properties, properties of the phosphorylation site domain, and a calculated M_r of 38,000 suggest that DAkap200 is a new member of the myristoylated alanine-rich C kinase substrate protein family. Akap200 is a potentially mobile, chimeric A kinase anchor protein-myristoylated alanine-rich C kinase substrate protein that may facilitate localized reception and targeted transmission of signals carried by cAMP, Ca²⁺, and diacylglycerol (Rossi, 1999).

Calmodulin interactions in the Drosophila retina

In the Drosophila retina, Calmodulin is concentrated in the the rhabdomere, a microvillar structure of the photoreceptor cell. Calmodulin is also found in lower amounts in the sub-rhabdomeral cytoplasm. This Calmodulin localization is dependent on the NinaC (neither inactivation nor afterpotential C) unconventional myosins. Mutant flies lacking the rhabdomere-specific p174 NINAC protein do not concentrate Calmodulin in the rhabdomere, whereas flies lacking the sub-rhabdomeral p132 isoform have no detectable cytoplasmic Calmodulin. A defect in vision results when Calmodulin is not concentrated in the rhabdomeres, suggesting a role for Calmodulin in the regulation of fly phototransduction. A general function of unconventional myosins may be to control the subcellular distribution of Calmodulin (Porter, 1993a).

The ninaC locus encodes two unconventional myosins, p132 and p174, both consisting of fused protein kinase and myosin head domains expressed in Drosophila photoreceptor cells. NinaC encodes the major Calmodulin-binding proteins in the retina and the NinaC-Calmodulin interaction is required for the normal subcellular localization of Calmodulin as well as for normal photo-transduction. There are two Calmodulin-binding sites in NinaC, C1 and C2, which have different in vitro binding properties. C1 is common to both p132 and p174 while C2 is unique to p174. To address the requirements for Calmodulin binding at each site in vivo, transgenic flies were generated expressing ninaC genes deleted for either C1 or C2. The spatial localization of Calmodulin depends on binding to both C1 and C2. Mutation of either site results in a defective photoresponse. A prolonged depolarization afterpotential (PDA) is elicited at lower light intensities than necessary to produce a PDA in wild-type flies. These results suggest that Calmodulin binding to both C1 and C2 is required in vivo for termination of phototransduction (Porter, 1995).

Phototransduction in Drosophila occurs through inositol lipid signaling that results in Ca2+ mobilization. The physiological roles of calmodulin (CaM) were studied in light adaptation and in regulation of the inward current that is brought about by depletion of cellular Ca2+ stores. Three resources providing decreased Ca-CaM content in photoreceptors were analysed: (1) transgenic Drosophila P[ninaCDeltaB] flies that have CaM-deficient photoreceptors; (2) the peptide inhibitor M5 that binds to Ca-CaM and prevents its action, and (3) Ca2+-free medium that prevents Ca2+ influx and thereby diminishes the generation of Ca-CaM. Several effects have been noted due to decrease in Ca-CaM level:

1. Fluorescence of Ca2+ indicator reveals an enhanced light-induced Ca2+ release from internal stores.
2. Measurements of the light-induced current in P[ninaCDeltaB] cells show a reduced light adaptation.
3. Internal dialysis of M5 initially enhances excitation and subsequently disrupts the light-induced current.
4. An inward dark current appears after depletion of the Ca2+ stores with ryanodine and caffeine.

Importantly, application of Ca-CaM into the photoreceptor cells prevents all of the above effects. It is proposed that negative feedback of Ca-CaM on Ca2+ release from ryanodine-sensitive stores mediates light adaptation, is essential for light excitation, and keeps the store-operated inward current under a tight control (Arnon, 1997b).

Activation of the Drosophila visual cascade is extremely rapid and results in opening of the cation influx channels transient receptor potential (TRP) and transient receptor potential-like (TRPL) within ~10-20 msec of photostimulation of rhodopsin. The G-protein-signaling cascade that leads to opening of the ion channels has been extensively characterized and is known to involve the inositol phospholipid-signaling system. Termination of the photoresponse, after cessation of the light stimulus, is also rapid and is a Ca2+-regulated process; however, understanding of the mechanism by which Ca2+ contributes to termination of the photoresponse is quite incomplete (Li, 1998 and references).

Several proteins have been identified that seem to mediate Ca2+-dependent termination of phototransduction. These include the Ca2+-binding regulatory protein Calmodulin, which functions in both light adaptation and termination of the light response. The ninaC (neither inactivation nor afterpotential C) locus, which encodes two isoforms, p132 and p174, each of which consists of a protein kinase domain fused to a myosin head domain, also functions in negative feedback regulation of the photoresponse. The two NINAC proteins differ because of unique C-terminal ends. p174 is localized exclusively to the microvillar portion of the photoreceptors, the rhabdomeres, and p132 is restricted to the cell bodies. Null mutations in ninaC cause defects in adaptation and response termination. These functions are caused by p174 because elimination of p174, but not p132, causes each of these phenotypes. Because negative feedback regulation seems to be mediated by Ca2+, it is plausible that p174 is regulated by Ca2+. However, p174 does not contain a known Ca2+-binding motif, such as an EF hand or C2 domain, and there is no evidence that it binds Ca2+ directly. Thus, p174 seems to respond to the light-dependent Ca2+ flux indirectly. One NINAC Ca2+ sensor is Calmodulin because NINAC binds to Calmodulin and the NINAC-Calmodulin interaction is required for both adaptation and termination. NINAC might also be regulated by Ca2+-dependent phosphorylation because p174 contains multiple protein kinase C (PKC) consensus sites including several in its unique C-terminal tail. Moreover, mutation of an eye-specific PKC (ePKC) causes perturbations in adaptation and termination. The role of PKC in negative feedback regulation may be more significant than that indicated by mutation of ePKC because a second PKC, brain PKC (brPKC), is known to be enriched in the Drosophila retina and a third PKC, PKC98F, is highly expressed in adult heads. Two retinal substrates for PKC have been identified. These are the TRP cation influx channel and the PSD95, DLG, and ZO-1 (PDZ)-containing protein inactivation, no afterpotential D (INAD), which binds to most of the proteins that function in phototransduction and organizes a supramolecular signaling complex. However, the consequences of disrupting PKC phosphorylation of any retinal substrate that functions in Drosophila vision have not been determined (Li, 1998 and references).

The current work shows that NINAC p174, which consists of a protein kinase domain joined to the head region of myosin heavy chain, is a phosphoprotein and is phosphorylated in vitro by PKC. Mutation of either of two PKC sites in the p174 tail results in an unusual defect in deactivation that has not been detected previously for other ninaC alleles or other loci. After cessation of the light stimulus, there appeared to be a transient reactivation of the visual cascade. This phenotype suggests that a mechanism exists to prevent reactivation of the visual cascade and that p174 participates in this process. The termination mechanisms controlling Drosophila phototransduction seem to be more complicated than previously envisioned. In addition to a requirement for NINAC in facilitating rapid deactivation after cessation of the light stimulus, there is an additional requirement for this unconventional myosin in preventing transient reactivation of the plasma membrane conductances. Because p174 also functions in adaptation, it seems that NINAC has a central role in many aspects of negative feedback regulation of the visual cascade. Recently, a homolog of NINAC has been identified in the mammalian retina (D. Hillman, A. Dose, and B. Burnside, personal communication to Li, 1998). Thus, it is intriguing to speculate that vertebrate NINAC also functions in negative feedback regulation and that an active mechanism may also exist in mammalian photoreceptor cells to ensure stable termination of phototransduction (Li, 1998).

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). Phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale that is faster than that of any other protein in vivo. A CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. Ser366 is identified as the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI. It has been concluded that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors (Matsumoto, 1994).

The characterization of Drosophila Calmodulin mutants and the role of CAM in photoreceptor cell function have been described. In Drosophila photoreceptor neurons, light activation of rhodopsin activates a heterotrimeric G protein, which in turn activates phospholipase C (PLC). PLC catalyzes the hydrolysis of the minor membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) into the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG). Activation of PLC then leads to the opening of cation-selective membrane channels encoded by the transient receptor potential (trp) and trp-like (trpl) genes. It has been hypothesized that calcium release from internal stores is required for activation of the phototransduction cascade and that the TRP channel functions as a store-operated channel gated by the light-induced emptying of the internal stores (Scott, 1997 and references).

Contrary to current models of excitation and TRP channel function, the transient phenotype of trp mutants can be explained by CAM regulation of the TRPL channel rather than by the loss of a store-operated conductance leading to depletion of the internal stores. In fact, introduction of calcium intracellularly in trp mutants does not restore responsiveness. The finding that trp mutants can maintain responsiveness in the absence of calcium suggests that there is calcium-dependent inactivation of light-induced currents in the trp mutant. Light responses were analyzed in a variety of mutant and transgenic backgrounds. The transient respone of trp mutants reflects TRPL channel function. Deletion of either of the two CAM binding sites of TRPL results in a prolonged current suggesting that CAM binding functions to inactivate TRPL. Thus, Calmodulin is essential for calcium-dependent negative regulation of phototransduction. Mutants for cam display dramatic defects in deactivation kinetics, displaying greatly prolonged deactivation times. In the absence of extracellular calcium, mutant and wild-type responses are not significantly different from each other, demonstrating that calium entry is required to reveal the cam mutant phenotype and highlighting the absolute requirement for calcium for the rapid deactivation of the phototransduction cascade. CAM ilso regulates the catalytic lifetime of activated rhodopsin by regulating the binding of arrestin to rhodopsin. Thus CAM coordinates termination of the light response by modulating receptor and ion channel activity (Scott, 1997).

To identify Calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, a screen was conducted for retinal Calmodulin-binding proteins. Twelve Calmodulin-binding proteins were found that are expressed in the Drosophila retina. The functions of Calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the Trp and Trp-like ion channels, NinaC and InaD. Eight calmodulin-binding proteins have been identified that have not been previously reported to be expressed in the Drosophila retina. The full-length sequences corresponding to three of the calmodulin-binding proteins have been described. These corresponded to two Calmodulin-dependent protein kinases, MLCK and CaM kinase II, as well as to one of two previously described Calcineurin proteins. A third calmodulin-dependent protein kinase is expressed in the Drosophila retina, CaM kinase I. No CaM kinase I has previously been reported from any invertebrate, raising the possibility that this protein kinase is specific to vertebrates. Nevertheless, the Drosophila CaM kinase I is highly related to vertebrate CaM kinase I: ~60% identical over 349 amino acids. The remaining four calmodulin-binding proteins have not been known to bind calmodulin prior to the current work. Six targets have been found that are related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein (UNC13) and a protein (CRAG) related to Rab3 GTPase exchange proteins. Two other calmodulin-binding proteins found are Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that Calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission (Xu, 1998).

At least two of the four novel calmodulin-binding proteins share similarities to components implicated in synaptic transmission. One of these proteins (1441 residues), is referred to as CRAG (calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein; due to its similarity to a domain in the recently identified rat Rab3 GDP/GTP exchange protein (rRab3 GEP) and the C. elegans homolog, AEX-3, which has been implicated in synaptic vesicle release. The sequences of AEX-3 and the rRab3 GEP were published contemporaneously and have therefore not been directly compared. AEX-3 and the rRab3 GEP (1409 and 1602 amino acids, respectively) contain three regions of homology, the first of which (~500 residues) is conserved in CRAG, AEX-3, rRab3 GEP (CAR domain) and the human homolog, MADD (death domain MAP kinase activator). The latter two regions, are conserved in AEX-3 and rRab3 GEP (AR1 and AR2), but not CRAG, and are shorter (~100 and 300 residues, respectively) than the CAR homology. The CAR domain in CRAG is ~36 identical over 321 amino acids (residues 95-415) to either rRab3 GEP or AEX-3. In addition, there is weak homology (16%) in the flanking sequences that extend the CAR domain in CRAG to residues 73-490. The C-terminal ~800 residues of CRAG do not share significant primary amino acid sequence homology with the rRab3 GEP, AEX-3, or any other protein in the data banks (Xu, 1998 and references).

While it remains to be determined if CRAG is also a Rab3 GEP, such a finding would have interesting implications regarding the mechanism by which GTP exchange on Rab3 is regulated. Rab3 binds to synaptic vesicles; however, this association only occurs in resting nerve terminals and requires Rab3 in the GTP bound state. Mutation of the C. elegans Rab3 GEP, known as AEX-3, causes accumulation of Rab3 in neuronal cell bodies and an impairment in the release of neurotransmitter. Thus, the Rab3 GEP appears to play a critical role in association of Rab3 with synaptic vesicles and in synaptic transmission. The observation that CRAG binds calmodulin implies that the putative GEP activity of this protein could be regulated by changes in Ca²⁺ levels, which are spatially restricted to microdomains near the active zones in presynaptic terminals. A variety of evidence suggests that the absolute level of Rab3-GTP bound to synaptic vesicles regulates the rate of exocytosis by limiting the number of vesicles that can be fused with the plasma membrane. Thus, formation of Rab3-GTP appears to be a crucial step in synaptic transmission. The mechanisms controlling the GDP-GTP exchange are not known but one possibility is that CRAG is a Rab3 GEP and the associated calmodulin provides a sensor to differentiate between the lower Ca²⁺ levels in resting nerve terminals and higher levels resulting from Ca²⁺ influx. While it remains to be determined if CRAG is a Rab3 GEP and whether the exchange activity is regulated through the associated calmodulin, an exchange factor for another small GTPase, RAS, binds to and is regulated by Ca²⁺/calmodulin (Xu, 1998 and references).

Pollux (Plx) is a protein previously reported to be 732 amino acids in length and required for viability (S. D. Zhang, 1996). The protein is predicted to have a transmembrane domain and a leucine zipper (S. D. Zhang, 1996). Plx has now been found to be 1379 amino acids in length and the formerly assigned initiator methionine corresponds to residue 648. A protein related to Plx is TBC1 (Richardson, 1995), a mouse protein which had homology to the majority of Plx. The region in Plx that contains the greatest similarity to TBC1 is a 337-amino acid segment (51% identity, residues 676-1012) that includes the putative transmembrane domain. Of particular interest, the region most highly conserved between Plx and TBC1 includes a 153-amino acid domain (residues 811-963) that displays moderate homology to the yeast Rab family GTPase-activating proteins, GYP6 or GYP7 (Strom, 1994). GYP7 is ~29% identical to this domain in either Plx or TBC1; however, if two gaps of 18 and 36 amino acids have been introduced in Plx and TBC1, the 29% homology extends to over 222 amino acids (742-963). This ~200 amino acid sequence corresponds to the domain previously referred to as a TBC domain due to its similarity to segments in the TRE-2 oncogene and the yeast regulators of mitosis, BUB2 and CDC16. TBC1 is 1141 residues and is found to be a nuclear protein. Thus, TBC1 and Plx have very disparate spatial distributions. (Xu, 1998).

The portion of the Plx protein that was isolated in the screen extends from residues 180-1379. Using a series of overlapping GST fusion proteins and the gel overlay assay, the calmodulin-binding site(s) contained in the original fusion protein was further mapped to residues 657-680. The sequence of the calmodulin-binding site is not conserved in the mouse homolog, TBC1, but is in several human ESTs. A bovine homolog of Plx (Lyncein), which was isolated from a bovine retinal library, is highly conserved in the calmodulin-binding domain despite having no higher overall sequence conservation to Plx than TBC1. Moreover, a fusion protein containing the conserved sequence in Lyncein binds calmodulin. Plx also bind to calmodulin in a pull-down assay; although this interaction is Ca²⁺ independent (Xu, 1998).

Thus it has been found that Plx is 1379 residues rather than 732 amino acids as previously reported (S. D. Zhang, 1996). The additional sequence is not due to a chimeric cDNA since multiple plx cDNAs were obtained and TBC1 shares similarity to Plx both N- and C-terminal to the formerly assigned initiating methionine at residue 648. Plx has been shown to bind calmodulin and does so in a Ca²⁺-independent manner. Although the sequence of the calmodulin-binding site is not conserved in TBC1, the region is very similar in Lyncein, a homolog isolated from a bovine retinal library. Furthermore, the Lyncein sequence also binds calmodulin. Thus, it appears that a Plx homolog is expressed in the vertebrate retina. A possible clue as to the function of Plx in the retina is that it shares some similarity to two yeast Rab GAP proteins, although no homology was found to the Rab3 GAP expressed in the rat brain. Nevertheless, the observation that Plx contains a domain related to Rab GAPs combined with the finding that it appears to be localized to the plasma membrane and lumen of the trachael system raises the possibility that Plx may be involved in exocytosis. In the Drosophila visual system, exocytosis is important not only in synaptic transmission but in turn-over of the microvillar membrane of the photoreceptor cells. Shedding of membrane does not occur uniformly during the diurnal cycle, but occurs maximally soon after dawn. Thus, an increase in the exocytotic process is correlated with the light dependent rise in Ca²⁺ and therefore might be regulated in part by a Ca²⁺ sensing component in a Rab cycle. Alternative potential functions for Plx in photoreceptor cells include other processes that involve vesicular trafficking such as insertion of new membrane in the microvilli and the budding, targeting, and fusion of rhodopsin carrier vesicles with the plasma membrane. These latter events involve a variety of Rab proteins and also appear to be regulated during the daily light cycle (Xu, 1998 and references).

A third protein, not previously known to bind calmodulin, is a Drosophila homolog of UNC13 (dUNC13), a diacylglycerol-binding protein that may be required for release of neurotransmitter from the presynaptic terminal. dUNC13 is expressed as at least two alternatively spliced forms encoding proteins of >1304 (dUNC13A) and >1724 (dUNC13B) amino acids. dUNC13A and dUNC13B shared a common C-terminal region of >1216 amino acids and differ due to unique N-terminal sequences (>88 and >508 residues, respectively). dUNC13 contains extensive homology (>68%) with the C. elegans UNC13 and rat homologs (mUNC13), beginning in the unique region of dUNC13A and extending over the entire region common between both isoforms (residues 72-1304). UNC13 and mUNC13-1 share a similar level of homology over the same region and are only weakly related over the N-terminal ~500 amino acids. The 508 amino acids specific to dUNC13B are not homologous to the UNC13 proteins or any proteins in the data banks. Features common between dUNC13 and other members of the UNC13 family include strong homology to two conserved sequence motifs, C1 and C2, originally recognized in various protein kinase C isoforms. A large variety of other signaling proteins, such as RAF, diacylglycerol kinase, RAS GTPase-activating protein, synaptotagmin, and phoshopholipase C contain these domains. C1 domains typically bind diacylglycerol, while many C2 domains are Ca²⁺-binding regulatory domains. Some C2 domains also bind phospholipids and do so in a Ca²⁺-dependent manner. Other C2 domains confer Ca²⁺ dependence to functions, such as protein kinase activity, mediated by domains distinct from C2. Biochemical analyses of UNC13 demonstrate that it is a bona fide Ca²⁺-dependent phorbol ester-binding protein. The putative C1 domain in dUNC13 (residues 182-232) include six invariant cysteines as well as a seventh cysteine conserved among all UNC13 proteins. Overall, the C1 domain is 92% identical to the corresponding region in mUNC13. The two C2 domains present in each of the three other UNC13 proteins (C2-1 and C2-2) are also found in dUNC13. C2-1 (residues 299-393) and C2-2 (residues 1170-1264) are 76 and 67% identical with the same motifs in mUNC13-1 (Xu, 1998 and references).

In addition to aex-3, several other mutations have been identified in C. elegans that appear to disrupt exocytosis of synaptic vesicles and release of neurotransmitter. One such mutation is in the gene encoding the diacylglycerol-binding protein, UNC13. Although the specific function of UNC13 remains unclear, it may operate in docking and/or fusion of synaptic vesicles since the rat brain-specific mUNC13-1 protein binds directly to two proteins, syntaxin and Doc2alpha, which function in Ca²⁺-dependent exocytosis. The C2 domains present in UNC13 homologs could potentially serve as a Ca²⁺ sensor that responds to the Ca²⁺ influx required for exocytosis. Therefore, the question arises as to the function of a potential second type of Ca²⁺ sensor provided by the binding of calmodulin to dUNC13. One possibility is that each UNC13 protein really has only one Ca²⁺ sensor and that it is supplied in some isoforms by the C2 domain and in others through Ca²⁺/calmodulin. Consistent with this proposal, the calmodulin-binding domain is not conserved in UNC13 suggesting that the C2 domain provides the only Ca²⁺ detector in this protein. The reverse may be the case in mUNC13-1 since this protein does not appear to contain Ca²⁺-binding C2 domains but does show sequence similarity to the dUNC13 calmodulin-binding site. An alternative proposal, which is favored by the authors, is that some UNC13 proteins may be regulated by Ca²⁺ via both C2 domains and calmodulin. Such dual regulation may provide a mechanism for extremely rapid as well as sustained responses to highly transient increases in Ca²⁺. The rise in Ca²⁺, resulting from opening of the voltage-gated channels in synaptic terminals, occurs in microdomains and collapses within microseconds after closing of the ion channels. C2 domains comprise an unusual Ca²⁺ binding motif in that Ca²⁺ appears to regulate this domain through a shift in electrostatic potential rather than a conformational change. As such, C2 domains have the potential to respond very quickly, but transiently, to the rapid Ca²⁺ flux in the active zones of the presynaptic terminal. Although fusion and release of neurotransmitter is extremely rapid (submilliseconds to milliseconds), there is some latency between the opening and closing of the ion channels and these latter events. Ca²⁺ binding to calmodulin, which induces a conformational change, may induce a more delayed but sustained response to Ca²⁺ than that provided by the C2 domain. Thus, dual binding of Ca²⁺ to calmodulin and C2 domains may enable UNC13 proteins to sense the Ca²⁺ rise within a few microseconds and sustain the response for several hundred microseconds to several milliseconds (Xu, 1998 and references).

The fourth novel calmodulin-binding protein is referred to as Calossin (CALO) due to its interaction with calmodulin and colossal molecular mass (predicted >450 kDa). Several overlapping cDNAs have been obtained resulting in the identification of a single open reading frame encoding >4118 amino acids. Several hydrophobic regions are predicted according to a computer algorithm; however, it is unclear if any is sufficiently long to span a lipid bilayer. CALO is related to a predicted C. elegans protein (cCalossin) of similar size (3864 residues) that was identified as part of the C. elegans Genome Sequencing Consortium. The homology between CALO and cCALO was not uniform but concentrated in several domains. The longest continuous region of identity begins at amino acid 2460 and extends ~1650 residues to near the C terminus. In addition, there are two shorter stretches of similarity between residues 604 and 1150. The highest levels of identity (each ~70%) are in three ~50-100 amino acid regions: (1) residues 604-649; ( 2) residues 2587-2638, and (3) residues 3276-3380. The first two of these conserved regions are cysteine-rich domains, CRD1 and CRD2, respectively, that resemble different classes of zinc finger domains. CRD1 is most similar to the zinc finger family defined by Requiem, a protein required for apoptosis, while CRD2 shares features equally well with several families of zinc family proteins and can not be included within a single group (Xu, 1998 and references).

Regulation of the Rhodopsin protein phosphatase Rdgc by Calmodulin

Hundreds of G protein-coupled receptors (GPCRs) and at least six GPCR kinases have been identified, but the only GPCR phosphatase that has been definitively demonstrated is the rhodopsin phosphatase encoded by the rdgC locus of Drosophila. Mutations in rdgC result in defects in termination of the light response and cause severe retinal degeneration. RDGC is shown to bind to Calmodulin, and a mutation in an IQ motif that eliminates the Calmodulin/RDGC interaction prevents dephosphorylation of rhodopsin in vivo and disrupts termination of the photoresponse. These data indicate that RDGC is a novel calmodulin-dependent protein phosphatase and raise the possibility that regulation of other GPCRs through dephosphorylation may be controlled by calmodulin-dependent protein phosphatases related to RDGC (Lee, 2001).

Stimulation of G protein-coupled receptors (GPCRs) by hormones, growth factors, neurotransmitters, sensory stimuli, and other agonists frequently results in an increase in intracellular Ca²⁺. Such changes in Ca²⁺ concentration regulate a variety of effects ranging from apoptosis to differentiation, cell movement, the modulation of synaptic plasticity, and visual transduction. A primary mechanism through which alterations in Ca²⁺ levels lead to discrete physiological consequences involves the control of protein phosphorylation by the Ca²⁺ sensor calmodulin. Several calmodulin-dependent serine/threonine kinases have been described, such as myosin light chain kinase and Ca²⁺/calmodulin-dependent protein kinases I, II, and IV. However, the only known calmodulin-dependent protein phosphatase is calcineurin, despite the ~20 years that have elapsed since its discovery. Calcineurin is conserved from yeast to humans and is a heterodimer consisting of a catalytic subunit, CnA, and a regulatory subunit, CnB, comprised of four Ca²⁺ binding motifs referred to as EF hands (Lee, 2001).

Given the central role of Ca²⁺ in regulating a vast array of essential processes, it seems likely that there exist additional calmodulin-dependent protein phosphatases. Candidates include phosphatases known to function in Ca²⁺-regulated signaling cascades. One such protein is the rhodopsin phosphatase, RDGC, which participates in Drosophila phototransduction (Steele, 1990; Steele, 1992; Byk, 1993; Vinós, 1997). Drosophila phototransduction culminates with Ca²⁺ and Na⁺ influx via the TRP, TRPL, and TRPgamma channels (Lee, 2001).

A key mediator of the Ca²⁺-mediated feedback regulation is calmodulin; however, there are only a few signaling proteins known to function in Drosophila phototransduction that bind to calmodulin. These include the NINAC myosin III, TRP, TRPL, and INAD. Some calmodulin binding proteins, such as TRPL, interact with calmodulin via the positive face of an amphiphilic alpha helix, while others, such as NINAC, associate through IQ motifs. IQ motifs, which contain the core consensus IQxxxRGxxxR (x denotes any amino acid), may associate with calmodulin in either a Ca²⁺-dependent or independent manner. IQ motifs are present in a wide diversity of proteins ranging from myosins to neuromodulin (GAP-43, voltage-gated Ca²⁺ channels, and the Ras guanine nucleotide exchange factor, RAS-GRF). Moreover, the IQ/calmodulin interactions regulate the activities of each of these classes of proteins (Lee, 2001).

The rhodopsin phosphatase, RDGC, is a potential target for regulation by calmodulin, since mutations in rdgC result in severe defects in the Ca²⁺-dependent termination of the photoresponse (Steele, 1990; Vinós, 1997). Disruption of rdgC function also results in age- and light-dependent retinal degeneration (Steele, 1990). Homologs of RDGC are conserved from C. elegans to humans and are collectively referred to as the PPEF family due to the protein phosphatase domain and the presence of multiple C-terminal Ca²⁺ binding motifs, EF hands (Lee, 2001).

The present work shows that RDGC is a calmodulin-regulated protein phosphatase. RDGC binds directly to calmodulin, and this interaction disrupts an association between the N-terminal domain of RDGC and the catalytic domain. Furthermore, the calmodulin/RDGC interaction is required to potentiate dephosphorylation of rhodopsin in vivo and for rapid termination of the photoresponse (Lee, 2001).

To test whether RDGC binds to calmodulin, calmodulin-agarose pull-down assays were performed. Agarose beads conjugated to calmodulin were incubated with full-length RDGC labeled in vitro with ³⁵S. As a negative control, a segment of the PDZ protein, INAD (PDZ domains 3 and 4; amino acids 346-581), which is devoid of calmodulin binding activity, was used. RDGC binds to calmodulin-agarose, although the INAD-PDZ3-4 segment does not. Binding of RDGC to calmodulin-agarose is not strictly Ca²⁺ dependent. However, a greater proportion of RDGC binds to calmodulin in the presence of Ca²⁺ (Lee, 2001).

To determine whether RDGC and calmodulin interact in vivo, whether the two proteins coimmunoprecipitate from fly heads was tested. Anti-RDGC antibodies were generated that recognize three bands (84, 78, and 76 kDa) in wild-type but not rdgC head extracts. The 78 and 76 bands appear to be eye specific, since they are not detected in the eyeless mutant, sine oculis (so). The 84 kDa isoform, which is not eye specific, may be responsible for the previously reported RDGC expression in the mushroom bodies of the central brain (Steele, 1992). To assess whether RDGC and calmodulin associate in vivo, RDGC was immunoprecipitated from wild-type or null rdgC head extracts, and Western blots of the immune complexes were probed with anti-calmodulin antibodies. Calmodulin is detected in the immune complexes from wild-type but not from null rdgC head extracts. Furthermore, calmodulin coimmunoprecipitates with RDGC in the presence or absence of Ca²⁺, although more calmodulin immunoprecipitates in the presence of Ca²⁺ (Lee, 2001).

To map the sites of interaction between RDGC and calmodulin, calmodulin overlay assays were used. Various fragments of RDGC were expressed in E. coli as GST fusion proteins. Total bacterial extracts were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with [¹²⁵I]calmodulin. All the fusion proteins that bind to calmodulin contain the N-terminal 32 residues, while those proteins that lack these residues fail to associate with calmodulin. Thus, the N-terminal 32 residues contain the calmodulin binding site (Lee, 2001).

RDGC contains a sequence similar to the IQ-calmodulin binding motif, and this sequence maps to the N terminus of RDGC (residues 12-22). To address whether RDGC binds to calmodulin through the IQ motif, most of the sequence (deltaIQ, amino acids 12-22) was deleted and the effects on calmodulin binding were assessed using calmodulin overlay assays. GST-fusion proteins containing full-length RDGC or the N-terminal 73 residues of RDGC bind calmodulin (RDGC and 73^WT). However, derivatives of these fusion proteins that lacked residues 12-22 fail to associate with calmodulin (RDGC^deltaIQ and 73^deltaIQ) (Lee, 2001).

To obtain additional evidence that RDGC binds to calmodulin through the IQ motif, the effects of a variety of conservative and nonconservative substitutions of the most invariant residues within the IQ sequence were assessed. Among the point mutations generated, the only one that almost completely abolishes interaction with calmodulin is a glutamic acid substitution of the isoleucine (residue 12) that begins the motif (73^I12E). A relatively conservative alanine substitution of the same residue (73^I12A) does not eliminate calmodulin binding, but appears to result in an increase in the level of calmodulin bound to RDGC. Thus, it appears that the IQ sequence is the only calmodulin binding site in RDGC (Lee, 2001).

To confirm the effects of the single amino acid substitutions in residue 12 and to test whether these alterations influence the effect of Ca²⁺ on the RDGC/calmodulin interaction, calmodulin-agarose pull-down assays were performed. Consistent with the results of the overlay assay, 73^deltaIQ and 73^I12E virtually abolished binding whereas 73^I12A displays an increase in calmodulin binding relative to 73^WT. In addition, the I12A mutation alters the Ca²⁺ dependence for calmodulin binding. While wild-type RDGC binds calmodulin in the presence or absence of Ca²⁺, the interaction between 73^I12A and calmodulin is strictly Ca²⁺ dependent (Lee, 2001).

Other members of the RDGC/PPEF family also contain an N-terminal IQ consensus sequence, suggesting that other RDGC-related proteins may interact with calmodulin. To test whether the IQ sequence in human PPEF2 is a calmodulin binding site, calmodulin-agarose pull-down assays were performed. The core region of the IQ motif (amino acids 21-42) of human PPEF2 was fused to GST (GST-HsIQ), purified using a glutathione-Sepharose column, and incubated with calmodulin-agarose beads. GST-HsIQ binds to calmodulin-agarose, but GST alone does not. In addition, GST-HsIQ binds to calmodulin-agarose in the presence or absence of Ca²⁺. Thus, the IQ-type motif domain in human PPEF-2 is sufficient to bind calmodulin (Lee, 2001).

To determine the physiological role of the RDGC/calmodulin interaction, transgenic flies were generated that express full-length derivatives of RDGC that incorporate the I12A mutation, the I12E mutation, and the IQ deletion of amino acids 12-22. All of the mutations were introduced into the 7.1 kb genomic DNA shown to restore wild-type visual function to rdgC-mutant flies (Steele, 1992). The wild-type (P[rdgC⁺]) and mutant transgenes (P[rdgC^I12A], P[rdgC^I12E], and P[rdgC^deltaIQ]) were expressed in a null rdgC (rdgC^co6) background so that the only RDGC proteins originated from the transgenes. The wild-type transgene restored normal levels of both eye-specific isoforms of RDGC. In addition, the point mutations (I12A and I12E) do not affect the stability of RDGC, since the level of the proteins was similar to that observed in wild-type (y,w) and P[rdgC⁺] flies. However, the deletion of the IQ motif appears to render the protein unstable, since the RDGC^deltaIQ is undetectable (Lee, 2001).

To test whether calmodulin binding is critical for RDGC function in vivo, the retinal morphology of the transgenic flies was examined. Drosophila compound eyes consist of ~800 ommatidia, each of which contains eight photoreceptor cells, though only seven are present in any given plane of section. The photoreceptor cells include a microvillar structure, the rhabdomere, which contains the proteins critical for visual transduction. The morphology of the wild-type and P[rdgC⁺] rhabdomeres do not change with age or depending on the light conditions. However, rdgC flies are characterized by age- and light-dependent retinal degeneration (Steele, 1990). One-day-old rdgC flies maintained under a 12 hr light/12 hr dark cycle show little if any decrease in the size of the rhabdomeres. After rearing rdgC flies for 7 days under a light-dark cycle, the rhabdomeres of the R1-6 cells are almost completely degenerated. The rhabdomeres of rdgC^I12E flies show a pattern of degeneration similar to rdgC null flies. By contrast, no degeneration is observed in rdgC^I12A flies aged for 7 days under the light-dark cycle. These data indicated that the I12E but not the I12A mutation disrupted RDGC function (Lee, 2001).

To test whether association of calmodulin with RDGC is critical for rapid inactivation, the light response was examined using electroretinograms (ERGs). ERGs are extracellular recordings that measure the summed responses of the retinal cells to light. Exposure of rdgC⁺ flies to a light stimulus results in a corneal negative deflection in the ERG. Upon termination of the light response, there was a rapid return to the baseline. The deactivation of the light response is significantly delayed in rdgC null mutant flies. There was no delay in termination in rdgC^I12A; rather, the deactivation kinetics in these flies is slightly faster than rdgC⁺. However, the deactivation rate of rdgC^I12E decreases significantly compared with rdgC⁺, although the delay is slightly less severe than in the null mutant (Lee, 2001).

When a substantial amount of rhodopsin is photoconverted from rhodopsin to the light activated metarhodopsin state, a sustained photoresponse or prolonged depolarization afterpotential (PDA) persists after cessation of the light stimulus. In wild-type, intense light is required to produce a PDA. Blue rather than orange or white light is most effective in producing a PDA, since rhodopsin, but not metarhodopsin, is maximally activated by blue light. A second photon of light, white or orange, is required to convert metarhodopsin to rhodopsin and terminate the PDA (Lee, 2001).

A PDA can be generated in rdgC with considerably less light than in rdgC⁺. A PDA results from an excess of metarhodopsin relative to arrestin. Therefore, mutants that express less arrestin than wild-type also exhibit a PDA with less intense light. It has been suggested that rdgC flies display a PDA with less light than wild-type, since mutations in rdgC cause hyperphosphorylation of rhodopsin, which in turn impairs arrestin function (Lee, 2001).

To address whether the rdgC transgenic flies display defective PDAs, the relative intensity of light needed to produce a PDA was assayed. Like rdgC null mutants, rdgC^I12E enters a PDA with <10% the light intensity required in rdgC⁺. In contrast, rdgC^I12A shows a PDA similar to rdgC⁺. The observations that rdgC^I12E flies display retinal degeneration, a delay in deactivation kinetics, and a low light PDA indicates that calmodulin binding is necessary for normal function of RDGC in vivo (Lee, 2001).

The simplest hypothesis to account for the requirement of the RDGC/calmodulin interaction is that calmodulin regulates the phosphatase activity of RDGC in vivo. To test this proposal, the relative levels of phosphorylated rhodopsin (Rh1) were measured in rdgC⁺ and rdgC^I12E eyes. Dark-reared flies were fed ³²P and then exposed to light. As expected, rhodopsin is hyperphosphorylated in rdgC relative to rdgC⁺. Of primary importance here, rhodopsin is also hyperphosphorylated in the rdgC^I12E eyes. However, the level of rhodopsin phosphorylation in rdgC^I12A was similar to control flies (rdgC⁺). ninaE^P332 flies, which express only 0.1% of rhodopsin, were used as a negative control. Thus, association of calmodulin with RDGC appears to be required for RDGC phosphatase activity in vivo (Lee, 2001).

Whether calmodulin potentiates RDGC activity in vitro was tested using purified recombinant RDGC and p-nitrophenyl phosphate as a pseudosubstrate. Consistent with studies of a human homolog of RDGC, the activity of RDGC is increased by Ca²⁺, possibly through interaction of Ca²⁺ with the EF hands. Similar results were obtained with RDGC^I12E. Significantly, the addition of calmodulin in the presence of 100 µM Ca²⁺ further increases the phosphatase activity of wild-type RDGC but not that of RDGC^I12E. Thus, the enzymatic activity of RDGC seems to be augmented by calmodulin in vitro, although the level of calmodulin-dependent potentiation is much greater in vivo than in vitro (Lee, 2001).

Calmodulin might augment RDGC phosphatase activity by relieving a putative intramolecular interaction that inhibits the phosphatase activity. As a first test of this model, an examination was carried out to see whether the N-terminal region of RDGC, which includes the calmodulin binding site, associates with the catalytic domain. ³⁵S-labeled N-terminal RDGC (amino acids 1-253) binds to a GST-catalytic domain fusion protein (amino acids 153-423) but not to GST alone. Moreover, neither the middle nor the C-terminal portions of RDGC associate with the catalytic domain (Lee, 2001).

To test whether calmodulin binding to RDGC affects the interaction between the N-terminal portion of RDGC and the catalytic domain, pull-down assays were performed. ³⁵S-labeled N-terminal RDGC was incubated with the GST-catalytic domain fusion in the presence or absence of Ca²⁺/calmodulin. The addition of Ca²⁺/calmodulin decreases the interaction between N-terminal RDGC and the GST-catalytic domain fusion nearly 5-fold. However, the interaction between the catalytic domain and the N-terminal fragment of RDGC containing the I12E is not decreased by Ca²⁺/calmodulin. The addition of only BSA or Ca²⁺ does not change the binding of N-terminal RDGC and the catalytic domain (Lee, 2001).

Thus, several lines of evidence support the conclusion that RDGC is a calmodulin-regulated protein phosphatase. RDGC binds to calmodulin in vitro and in vivo, and the interaction is through an established calmodulin binding sequence, the IQ motif. An I to E substitution in this motif disrupts calmodulin binding and interfers with dephosphorylation of rhodopsin in vivo. Calmodulin also potentiates dephosphorylation of a pseudosubstrate in vitro, although the effect is smaller than that observed in vivo, possibly due to a contribution of one or more cofactors that remain to be identified. Finally, addition of calmodulin interfers with an intramolecular interaction between the N-terminal region and the RDGC catalytic domain (Lee, 2001).

It had been assumed previously that RDGC is not regulated by calmodulin since an inhibitor of calmodulin, M5, does not perturb the RDGC-dependent phosphatase activity in fly head extracts (Byk, 1993). However, in addition to RDGC, the activities of several other calmodulin-regulated proteins, such as a Ca²⁺-activated K⁺ channel and an L-type Ca²⁺ channel, are unaffected by application of calmodulin inhibitors. Thus, a lack of effect of calmodulin inhibitors does not rule out the possibility that a given protein interacts with and is regulated by calmodulin (Lee, 2001).

The identification of RDGC as a calmodulin-regulated protein phosphatase addresses a key question in Drosophila phototransduction, the identity of in vivo targets for negative feedback regulation by Ca²⁺/calmodulin. Calmodulin is present at ~0.5 mM concentration in the rhabdomeres and a decrease in rhabdomeral calmodulin has a profound effect on termination of the photoresponse. However, the only rhabdomeral proteins previously shown to be regulated in vivo by calmodulin are the NINAC myosin III, the TRPL cation channel, and Arrestin 2. As is the case for the major rhodopsin, Rh1, Arrestin 2 also undergoes rapid light-dependent phosphorylation. The phosphorylation of Arrestin 2, which is mediated by Ca²⁺/calmodulin-dependent protein kinase II, appears to regulate the release of Arrestin 2 from Rh1 (Lee, 2001).

Despite the observation that Rh1 undergoes light-dependent phosphorylation, the role of this phosphorylation event is controversial. In mammals, desensitization or inactivation of rhodopsin and other GPCRs is initiated by phosphorylation by GPCR kinases, which increases the affinity for arrestin. Arrestin binding disrupts signaling by interfering with engagement of the receptor with the G protein. The GPCR must be subsequently dephosphorylated before the inactivation and recycling is complete (Lee, 2001).

Evidence that phosphorylation of Rh1 is required for the photoresponse is that mutations in rdgC result in hyperphosphorylation of the receptor and a defect in response termination (Steele, 1990; Byk, 1993; Vinós, 1997). However, a C-terminal truncation of Rh1 (Rh1^delta356), or a combination of mutations that eliminate the phosphorylation sites, has no apparent effect on the photoresponse or on Arrestin 2 binding. Nevertheless, the defect in termination in rdgC flies appears to be due to hyperphosphorylation of the receptor, since the rdgC phenotype is suppressed in rdgC/Rh1^delta356 double-mutants. To reconcile these findings, it has been proposed that the C terminus of Rh1 is an autoinhibitory domain for Arrestin 2 binding and phosphorylation relieves the intramolecular interaction. The mutations that prevent phosphorylation of Rh1 may also eliminate the putative autoinhibitory interaction, thereby abrogating the requirement for phosphorylation (Lee, 2001).

Rhodopsin cannot be the only substrate for RDGC, since it is also expressed in a region of the brain, the mushroom bodies, implicated in learning and memory. Furthermore, while one of the two human RDGC homologs, PPEF-2, is highly enriched in the retina, the other, PPEF-1, is expressed primarily in a variety of sensory neurons of neural crest origin. Thus, PPEF-1 must also engage substrates other than rhodopsin. Likely candidates are other GPCRs that initiate signaling cascades that culminate in a rise in intracellular Ca²⁺. The identification of such substrates should provide valuable insights into additional roles for this new class of calmodulin-regulated protein phosphatases (Lee, 2001).

Ca2+-dependent metarhodopsin inactivation mediated by Calmodulin and NINAC Myosin III

Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. This study investigated the mechanism and role of rhodopsin inactivation. The lifetime of activated rhodopsin (metarhodopsin = M^*) was determined in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M^* by photoreisomerization to rhodopsin could suppress responses to prior illumination. M^* was inactivated rapidly (τ ~20 ms) under control conditions, but ~10-fold more slowly in Ca²⁺-free solutions. This pronounced Ca²⁺ dependence of M^* inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca²⁺ influx acting via CaM and NINAC accelerates the binding of arrestin to M^*. These results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling (Liu, 2008).

This study exploited the bistable nature of invertebrate rhodopsins to measure the lifetime of activated metarhodopsin in Drosophila. The approach measures the time window during which photoreisomerization of M^* can suppress the response to light. The relative lack of overlap of the R and M spectra in UV opsins has been exploited by recording from the UV-sensitive photoreceptors in Limulus median ocelli. This strategy was adapted for Drosophila by using flies engineered to express the UV opsin Rh3; the effective M^* lifetime was found to be very short (τ_dec ≈20 ms) under physiological conditions. Strikingly, M^* lifetime was prolonged ~10-fold in the absence of Ca²⁺ influx, indicating that M-Arr2 binding is Ca²⁺ dependent and that M^* lifetime is the rate-limiting step in response deactivation in Ca²⁺-free solutions. Further experiments led to proposal of a mechanism for Ca²⁺-dependent M^* inactivation by Arr2, mediated by calmodulin (CaM) and myosin III NINAC (Liu, 2008).

Photoisomerization of rhodopsin (R) by short-wavelength light (480 nm for Rh1 or 330 nm for Rh3) generates active metarhodopsin (M^*). M^* continues to activate G_q until it binds arrestin (Arr2) or is reconverted to R by long-wavelength illumination (570/460 nm). M is serially phosphorylated by rhodopsin kinase (RK), but this is not required for M^* inactivation or Arr2 binding. CaMKII-dependent phosphorylation of Arr2 at Ser³⁶⁶ and photoreconversion of M_pp to R_pp is required for the release of Arr_p. Phosphorylation of Arr2 also prevents endocytotic internalization of M-Arr2. In Arr2^S366A or mutants defective in CamKII, photoreconversion fails to release Arr2. Finally, R_pp is dephosphorylated by the Ca-CaM-dependent rhodopsin phosphatase (rdgC) to recreate the ground state, R. The results suggest that under low-Ca²⁺ conditions Arr2 is prevented from rapid binding to M^* because it is sequestered by NINAC or a NINAC-regulated target; however, Ca²⁺ influx acting via CaM rapidly releases Arr2. Each microvillus contains ~70 Arr2 molecules, ensuring rapid quenching of M^* once they are free to diffuse. The role of M^* phosphorylation remains uncertain but may be involved in Rh1 internalization by the minor arrestin, Arr1 (Liu, 2008).

Ca²⁺ dependence of M^* lifetime had not previously been demonstrated in an invertebrate photoreceptor, and the consensus from data in Drosophila suggested no obvious mechanism by which M^* lifetime could be regulated by Ca²⁺. The finding that M^* inactivation is strongly Ca²⁺ dependent prompted a re-examination of possible roles of Rh1 and Arr2 phosphorylation as well as CaM. Although M^* lifetime remained strongly Ca²⁺ dependent in mutants defective in rhodopsin and arrestin phosphorylation, the Ca²⁺ dependence of M^* inactivation was effectively eliminated in hypomorphic cam mutants. This requirement for CaM appeared to be mediated by the myosin III NINAC protein, since the Ca²⁺ dependence of M^* inactivation was effectively abolished in both the null ninaC^P235 mutant and an allele (ninaC^KD) in which CaM levels in the microvilli were unaffected. NINAC, which is the major CaM-binding protein in the photoreceptors, has long been known to be required for normal rapid response deactivation (Porter, 1993b), but the mechanistic basis remained unresolved. These results now strongly suggest that it is specifically required for the Ca²⁺- and CaM-dependent inactivation of M^* by Arr2 (Liu, 2008).

How might NINAC regulate the Ca²⁺-dependent inactivation of M^*? A clue comes from the finding that Arr2 levels were substantially reduced in ninaC mutants. After taking this into account, the lack of Ca²⁺ dependence of M^* inactivation in ninaC mutants was in fact associated with a very pronounced acceleration of response inactivation under Ca²⁺-free conditions. This was most clearly revealed in ninaC^KD, which appears to be specifically defective only in Ca²⁺-dependent M^* inactivation and does not show the additional response defects of the null ninaC phenotype (e.g., Hofstee, 1996). This suggests a disinhibitory mechanism whereby Ca²⁺-dependent inactivation of M^* may be achieved, at least in part, by the NINAC-dependent prevention of Arr2-M^* binding under low-Ca²⁺ conditions. Specifically, it is suggested that in Ca²⁺-free solutions, or in the low-Ca²⁺ conditions prevailing during the latent period of the quantum bump under physiological conditions, Arr2 in the microvilli is predominantly bound to NINAC or a NINAC-regulated target, thus restricting its access to M^*. However, following Ca²⁺ influx, CaCaM would bind to NINAC, causing NINAC to release Arr2, which, as a soluble protein, could then rapidly diffuse to encounter and inactivate M^* (Liu, 2008).

Interestingly, a recent study reported that NINAC can interact with Arr2 in a phosphoinositide-dependent manner (Lee, 2004). This interaction was described in the context of a role of NINAC in light-induced translocation of Arr2, which was reported to be disrupted in ninaC mutants. However, involvement in translocation was challenged by a subsequent study reporting that Arr2 translocation was unaffected in ninaC mutants (Satoh, 2005). It will be interesting to see whether the Arr2-NINAC interactions described by Lee (2004a) reflect a role in the CaCaM- and NINAC-dependent inactivation of M^* reported in this study (Liu, 2008).

It has long been known that responses under Ca²⁺-free conditions decay ~10-fold more slowly than in the presence of Ca²⁺. The current results establish that the inactivation of M^* by Arr2 is the rate-limiting inactivation step in such Ca²⁺-free responses, with a time constant of ~200 ms in wild-type photoreceptors. Following inactivation of M^* by photoreisomerization under Ca²⁺-free conditions, the response decayed with a time constant of ~80 ms. This also provides a unique and direct measure of the time constant(s) of the downstream mechanisms of inactivation, which presumably include GTP-ase activity of the Gq-PLC complex and removal of DAG by DAG kinase. It will be interesting so see whether Ca²⁺ also accelerates these inactivation mechanisms (Liu, 2008).

By contrast, the failure to accelerate response decay by overexpressing Arr2 in the presence of Ca²⁺ indicates that inactivation of M^* is not rate limiting under physiological conditions. This can be understood by recognizing that the macroscopic kinetics are determined by the convolution of the bump latency distribution and bump waveform, the latter probably terminated by Ca²⁺-dependent inactivation of the light-sensitive channels. Until the Ca²⁺ influx associated with the quantum bump, the phototransduction machinery in each microvillus is effectively operating under Ca²⁺-free conditions. The results suggest that it is the Ca²⁺ influx associated with each quantum bump that promotes M^* inactivation, and hence the timing of M^* inactivation will be determined by the bump latency distribution and not vice versa. This leads to the, perhaps counterintuitive, concept that response termination is rate limited, not by any specific inactivation mechanism, but rather by the time course with which the cumulative probability of bump generation approaches 100% (Liu, 2008).

Clearly, rapid quenching of M^* is essential to maintain the fidelity and high temporal resolution of phototransduction. In wild-type cells, an effectively absorbed photon generates only one quantum bump, but never (or extremely rarely) two or more; yet the multiple bump trains observed in arr2, cam, and ninaC mutants show that additional bumps are readily generated within 50–100 ms if M^* fails to be inactivated. To prevent such multiple bumps without Ca²⁺-dependent feedback would require such a high rate of Arr2 binding that many M^* molecules would be inactivated before they had a chance to activate sufficient G proteins to generate a quantum bump. This would result in an effective reduction in sensitivity, as is directly illustrated by the phenotype of p[Arr2] flies overexpressing Arr2. These show not only a 5-fold reduction in quantum efficiency. but also a reduction in bump amplitude and even an increase in bump latency, which is attributed to a decreased rate of second messenger generation. The mechanism proposed in this study provides an elegant solution to this dilemma. The analysis suggests that in the low-Ca²⁺ environment prior to Ca²⁺ influx, much of the Arr2 in the microvillus is bound to NINAC (or NINAC-regulated target), thus allowing M^* to remain active long enough to activate sufficient G proteins to guarantee production of a full-sized quantum bump with high probability. Only after the bump has been initiated does Ca²⁺ influx accelerate the inactivation of M^* by releasing Arr2, thus ensuring that only one bump is generated. This strategy is complemented and enabled by the ultracompartmentalization afforded by the microvillar design, which ensures that the Ca²⁺ rise is both extremely rapid and largely confined to the affected microvillus (Liu, 2008).

Miscellaneous interactions

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain has been quantitified with purified species-specific calmodulins. In all three species, adenylyl cyclase is activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity is dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raises the threshold for adenylyl cyclase activation by Ca2+ but also acts synergistically with Ca2+ to activate maximally adenylyl cyclase (Yovell, 1992).

For more information on the interaction of Calmodulin with adenylyl cyclase, see rutabaga.

Nitric oxide (NO) is an intercellular messenger involved in various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). A Drosophila NOS gene, dNOS, located at cytological position 32B encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, dNOS protein contains putative binding sites for Calmodulin, FMN, FAD, and NADPH. dNOS activity is Ca2+/Calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates (Regulski, 1995).

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/Calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is (respectively) 67% and 65% identical to human calcineurin A alpha and beta cDNAs. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The Calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurin As, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man (Guerini, 1992).

The Drosophila Cactus and Dorsal proteins are required for the development of embryonic dorso-ventral polarity and most likelyfor the innate immune response of the insect as well. Like their mammalian counterparts (the cytoplasmic anchor protein I kappa B and the rel/NF kappa B transcription factors) cactus and dorsal are regulated at the level of nuclear localization. Increased intra-cellular calcium levels induced by the ionophore ionomycin can activate dorsal/cactus complexes in the Drosophila cell line SL2. In a cell line (SLDL) in which dorsal is expressed constitutively, ionomycin induces a rapid destruction of Cactus and dephosphorylation of Dorsal. These results suggest a role for the protein phosphatase calcineurin in calcium mediated activation of dorsal/cactus complexes. They also indicate that in the resting cell, constitutive phosphorylation of Dorsal is in equilibrium with calcium dependent dephosphorylation (Kubota, 1995).

Partial and total loss of function mutant alleles of a putative Drosophila homolog (DPhK-gamma) of the vertebrate phosphorylase kinase gamma-subunit gene have been isolated. DPhK-gamma is required in early embryonic processes, such as gastrulation and mesoderm formation; however, defects in these processes are seen only when both the maternal and zygotic components of DPhK-gamma expression are eliminated. Loss of zygotic expression alone does not appear to affect normal embryonic and larval development; some pupal lethality is observed but the majority of mutant animals eclose as adults. Many of these adults show defects in their leg musculature (e.g. missing and degenerating muscles), in addition to exhibiting melanised "tumours" on their leg joints. Loss of only the maternal component has no obvious phenotypic consequences. The DPhK-gamma gene has been cloned and sequenced. It has an open reading frame (ORF) of 1680 bp encoding a 560 amino acid protein. The predicted amino acid sequence of DPhK-gamma has two conserved domains, the catalytic kinase and Calmodulin-binding domains, separated by a linker sequence. The amino acid sequence of DPhK-gamma is homologous to that of mammalian PhK-gamma proteins but differs in the length and amino acid composition of its linker sequence. The expression of DPhK-gamma mRNA is developmentally regulated (Bahri, 1994).

GAP-43 (growth-associated protein, 43 x 10(3) M[r]) is an essential, membrane-associated, neuronal phosphoprotein in vertebrates. The protein is abundantly produced in the growth cones of developing and regenerating neurons, and it is phosphorylated upon induction of long-term potentiation (LTP). Prior work has identified GAP-43-like proteins only in chordates. In this paper, a nervous system-specific gene from Drosophila melanogaster is described that encodes two proteins sharing biochemical activities and sequence homology with GAP-43. The region of homology encompasses the Calmodulin-binding domain and protein kinase C (PKC) phosphorylation site of GAP-43. The fly proteins are shown to bind Drosophila Calmodulin (CaM), and are phosphorylated by purified PKC after a fashion predicted from prior work with vertebrate GAP-43. GAP-43 is modified by palmitoylation. An amino-terminal myristoylation site is described for the Drosophila protein, which may play a similar role in membrane association in the fly. While a small family of GAP-43-related genes has been recognized in vertebrates, only a single gene appears to be present in the fly. Since the Drosophila gene encodes two proteins, each with multiple Calmodulin-binding domains and repeated sites for PKC phosphorylation, it may illuminate functions carried out by the family of vertebrate genes (Neel, 1994).

A 3.3 kb cDNA encoding the complete amino acid sequence of a calcium/Calmodulin regulated protein phosphatase has been isolated from a Drosophila eye disc cDNA library. The predicted protein of 560 amino acids (molecular mass 62 kDa) is 73-78% identical to human PP2B isoforms. The cDNA hybridizes to the X-chromosome at cytological position 14D1-4. Two transcripts of 3.5 kb and 3.0 kb are expressed during embryonic development, their levels being highest in the early embryo. The larger transcript was also clearly present in adult females. This pattern of expression indicates a role for calcium/Calmodulin regulated protein phosphatase in embryonic development (Brown, 1994).

Drosophila A kinase anchor protein 200 (Akap200), is predicted to be involved in routing, mediating, and integrating signals carried by cAMP, Ca²⁺, and diacylglycerol. Experiments designed to assess this hypothesis establish (1) the function, boundaries and identity of critical amino acids of the protein kinase AII (PKAII) tethering site of Akap200; (2) demonstrate that residues 119-148 mediate binding with Ca²⁺-calmodulin and F-actin; (3) show that a polybasic region of Akap200 is a substrate for protein kinase C; (4) reveal that phosphorylation of the polybasic domain regulates affinity for F-actin and Ca²⁺-calmodulin, and (5) indicate that Akap200 is myristoylated and that this modification promotes targeting of Akap200 to plasma membrane. DAkap200, a second product of the Akap200 gene, cannot tether PKAII. However, DAkap200 is myristoylated and contains a phosphorylation site domain that binds Ca²⁺-calmodulin and F-actin. An atypical amino acid composition, a high level of negative charge, exceptional thermostability, unusual hydrodynamic properties, properties of the phosphorylation site domain, and a calculated M_r of 38,000 suggest that DAkap200 is a new member of the myristoylated alanine-rich C kinase substrate protein family. Akap200 is a potentially mobile, chimeric A kinase anchor protein-myristoylated alanine-rich C kinase substrate protein that may facilitate localized reception and targeted transmission of signals carried by cAMP, Ca²⁺, and diacylglycerol (Rossi, 1999).

An Asp-CaM complex is required for centrosome-pole cohesion and centrosome inheritance in neural stem cells

The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. This study reports that Drosophila melanogaster calmodulin (CaM) regulates the ability of the microcephaly-associated protein, abnormal spindle (Asp), to cross-link spindle microtubules. Both proteins colocalize on spindles and move toward spindle poles, suggesting that they form a complex. Binding and structure-function analysis support this hypothesis. Disruption of the Asp-CaM interaction alone leads to unfocused spindle poles and centrosome detachment. This behavior leads to randomly inherited centrosomes after neuroblast division. It is further shown that spindle polarity is maintained in neuroblasts despite centrosome detachment, with the poles remaining stably associated with the cell cortex. Finally, evidence is provided that CaM is required for Asp's spindle function; however, it is completely dispensable for Asp's role in microcephaly suppression (Schoborg, 2015).

These results provide insight into how Asp, a key protein involved in mitotic spindle function, is regulated by the ubiquitous calcium-sensing protein CaM. CaM was localized near the spindle poles over 35 yr ago; the current data now assign a role for this CaM localization in directly regulating Asp to cross-link spindle MTs. The Asp-CaM interaction is conserved because it has also been biochemically identified in other eukaryotes, such as nematodes and mice, suggesting that this complex performs an essential spindle function. The work presented in this study extends the functional understanding of the Asp-CaM complex in spindle pole focusing and centrosome-pole cohesion, in addition to the cell biology of microcephaly (Schoborg, 2015).

Previous work in Drosophila, C. elegans, and mice has suggested a link between Asp and CaM. Spindle phenotypes are observed after RNAi depletion of either protein in Drosophila S2 cells. In C. elegans, analysis of meiotic spindles in the early embryo showed spindle defects after asp depletion and Asp's dependence on CaM (CMD-1) for pole localization. Furthermore, yeast two-hybrid analysis identified an Asp fragment containing a single IQ motif that could interact with CMD-1. This interaction between CaM and Asp on meiotic spindles was later identified in mouse oocytes using immunoprecipitation. However, in all cases, details of the underlying mechanism of the Asp-CaM association and a direct test of its contribution to spindle architecture remained unexplored (Schoborg, 2015).

The current results demonstrate that CaM functions as the critical factor that dictates Asp's ability to cross-link MTs. This is supported by the fact that Asp transgenes that localize to the spindle in a manner identical to that of the FL protein, yet are defective in CaM binding (Asp^N and Asp^FLΔIQ), fail to maintain pole focusing and centrosome-pole cohesion. Further, transgene analysis also highlighted a second mode of MT binding by Asp, mediated through its C terminus, and is independent of its known N-terminal MT binding domain. This interaction, though clearly weaker and distinct from the punctate signals observed for N-terminal containing transgenes, is supported by previous studies in vitro. It is believed that the stronger spindle pole and punctate localization of WT Asp normally masks this Asp^C localization and possibly contributes to Asp's ability to cross-link MTs (Schoborg, 2015).

Furthermore, a novel mode of Asp-CaM complex behavior on spindles was uncovered, highlighted by dynamic streaming of foci through the spindle lattice toward the pole. Previous work suggested that Asp associates with MT minus ends based on its accumulation at spindle poles where their density is highest. Localization of the Asp-Cam complex in live cells supports this hypothesis. However, it is further suggested that Asp-CaM complexes, seen as discrete puncta that move poleward, reside at MT minus ends distributed throughout the spindle that are collectively transported and organized at poles. These observations are consistent with work showing γ-tubulin-marked minus ends present throughout the spindle that stream toward the poles. Additionally, vertebrate NuMA displays similar streaming behavior, indicating a shared mechanism in which pole focusing is achieved through the concerted movement of protein complexes along the spindle toward the pole. Biochemical analysis will be critical for establishing the relationship between the distribution of minus ends within the spindle, the ability of the Asp-CaM complex to bind MT minus ends, and how the dynamic nature of their movement contribute to pole focusing and centrosome-pole cohesion (Schoborg, 2015).

The complete detachment of centrosomes from the spindle and random movement within the NB could have substantial long-term effects that are not fully appreciated by limited analysis of third-instar larval brains. Although the swapping of mother-daughter centrosome position and improper inheritance is interesting, its significance is unknown. It could be that centrosome position after detachment, rather than detachment, per se, negatively influences mitotic events. One would predict, for example, that centrosomes positioned anywhere in the cell other than the poles could influence the MT architecture within the spindle. In fact, a significant number of aberrantly bent spindles is seen, and live imaging showed that wandering centrosomes transiently interact laterally along the entire length of the spindle. One might also predict that this lateral centrosome position would influence the dynamics and tension across the kinetochores, triggering the spindle assembly checkpoint and an extended metaphase, which was also documentd in asp^t25 mutants. Therefore, the wandering centrosomes and their improper inheritance could have many negative downstream effects. If these results of inheriting too many or too few centrosomes are extrapolated to mammalian cells, one would predict detrimental effects on cilia formation in addition to mitotic defects, as previously documented in other mutant backgrounds (Schoborg, 2015).

This analysis of apical determinants in NBs highlighted a possible role for spindle poles (not centrosomes) in the maintenance of cell polarity. Despite centrosome detachment in the asp^t25/Df NBs and long curving spindles, no misaligned spindles were observed. This was true in fixed tissue using the apical polarity marker aPKC, in which, despite pole splaying and curvature, minus ends of MTs appeared to remain stably associated with the crescent at the cell cortex. Furthermore, significant spindle rotation was never observed after centrosome detachment during the course of live imaging, and NBs divided asymmetrically. These observations support the prevalent model that centrosomes initiate NB polarity but further add that centrosomes are neither necessary nor able to alter polarity once established. This is corroborated by the fact that no significant difference was observed in NB number in the asp^t25/Df mutant, suggesting that cell fate determinants were correctly partitioned during asymmetric division (Schoborg, 2015).

The results also shed light on the role of Asp in microcephaly. Interestingly, this phenotype is not dependent on the Asp-CaM complex. Both Asp^N and Asp^FLΔIQ rescued the brain size defects of the asp^t25/Df despite showing no or reduced binding to CaM. These results are in agreement with previous work demonstrating normal head size in animals expressing an N-terminal Asp fragment in the hypomorphic asp allele background. Importantly, the data using the null allele show that microcephaly is a result of the loss of Asp function and not a dominant-negative effect of the hypomorphic asp alleles. Furthermore, this study showed that the microcephaly phenotype is not a consequence of unfocused spindle poles or detached centrosomes, because the Asp^N and Asp^FLΔIQ rescue fragments displayed both of these defects. Taken collectively, this analysis of the null asp allele uncovered a separation of function that requires both termini of Asp to maintain MT cross-linking and an unknown region of the N terminus to specify proper brain size (Schoborg, 2015).

Two possible models are proposed by which the Asp-CaM complex could function. In both models, CaM exerts its influence on the spindle through directly binding the C terminus of Asp and is required for its stability. The first model proposes that CaM aids Asp oligomerization within the spindle. Putative higher-order Asp assemblies would be analogous to NuMA oligomerization shown to facilitate MT focusing in vertebrate cells. A second model proposes that CaM might regulate the weak association of Asp's C terminus to MTs. In this model, Asp would bind MT minus ends via its N terminus and the MT lattice via its C terminus, effectively bridging and zippering MTs. In both models, CaM might promote a structural conformation that allows for oligomerization or for a single Asp molecule to bind two separate MTs. Both models are not mutually exclusive, because elements of each may cooperate to ensure proper cross-linking between spindle MTs and centrosome MTs for robust pole focusing and centrosome attachment. Future biochemical and structural studies will be required to more fully understand the influence of CaM binding to Asp and the role of this complex in spindle MT cross-linking (Schoborg, 2015).

Amplification of Drosophila olfactory responses by a DEG/ENaC Channel

Insect olfactory receptors operate as ligand-gated ion channels that directly transduce odor stimuli into electrical signals. However, in the absence of any known intermediate transduction steps, it remains unclear whether and how these ionotropic inputs are amplified in olfactory receptor neurons (ORNs). This study finds that amplification occurs in the Drosophila courtship-promoting ORNs through Pickpocket (PPK25), a member of the degenerin/epithelial sodium channel family (DEG/ENaC). Pharmacological and genetic manipulations indicate that, in Or47b and Ir84a ORNs, PPK mediates Ca(2+)-dependent signal amplification via an intracellular calmodulin-binding motif. Additionally, hormonal signaling upregulates PPK expression to determine the degree of amplification, with striking effects on male courtship. Together, these findings advance understanding of sensory neurobiology by identifying an amplification mechanism compatible with ionotropic signaling. Moreover, this study offers new insights into DEG/ENaC activation by highlighting a novel means of regulation that is likely conserved across species (Ng, 2019).

Vertebrates detect odorants with G-protein-coupled receptors (GPCRs), the activation of which triggers subsequent metabotropic signaling cascades in the olfactory receptor neurons (ORNs) to transduce chemical stimuli into electrical signals. These series of transduction events also provide opportunities to amplify input signals. In contrast, insect olfaction is initiated by ligand-gated receptor channels that lack canonical G protein interacting domains. Although various G proteins and effectors have been implicated in the function of insect ORNs, it remains an open question whether those molecules play a specific role in olfactory transduction or a regulatory role in neuronal development, maintenance, and neuromodulation. Given that ligand-gated receptor channels can directly convert sensory stimuli to neuronal depolarization, it is unclear whether and how ionotropic inputs can be amplified in the absence of any known intermediate transduction steps (Ng, 2019).

In earlier work, it was found that the responses of Or47b ORNs, which detect aphrodisiac fly odors in D. melanogaster, increase with age in male flies (Lin, 2016), pointing to the possibility of signal amplification downstream of insect olfactory receptors. This age-dependent plasticity therefore presents an opportunity to investigate the mechanisms by which ionotropic sensory inputs can be amplified. Intriguingly, Or47b ORNs express a degenerin/epithelial sodium channel (DEG/ENaC) subunit named Pickpocket (PPK25). Within invertebrate genomes, DEG/ENaCs constitute one of the largest ion channel families. In mechanosensory and gustatory neurons, PPK subunits are involved in touch, proprioception, nociception, salt taste, water sensation, and recognition of contact pheromones. However, the functional role of PPK in olfaction remains unknown (Ng, 2019).

If PPK amplifies olfactory signals, how then is its activity regulated? Can it function as a transduction channel activated by intracellular second messengers downstream of receptor activation? Members of the DEG/ENaC superfamily, including the mammalian nonvoltage-gated sodium channels (SCNNs) and acid-sensing sodium ion channels (ASICs), are known to open in response to mechanical stimuli, extracellular ligands, or are otherwise constitutively active. In cultured cell lines, various intracellular signaling mechanisms can influence DEG/ENaC currents by regulating channel transcription, endocytosis, degradation, or translocation. Post-translational modification is also known to modulate DEG/ENaC function; for example, constitutive channel activity can be regulated by CaMKII-mediated phosphorylation or by protease-mediated cleavage of the extracellular domain. However, the possibility of DEG/ENaC activation by direct interaction with an intracellular ligand has not been explored (Ng, 2019).

This study shows that signal amplification can occur downstream of ligand-gated receptor channels. The age-dependent response plasticity of Or47b ORNs arises through PPK25-mediated amplification. Additionally, this mechanism is employed in another type of courtship-promoting ORN expressing the Ir84a receptor. Interestingly, the degree of amplification is determined by PPK expression levels, which are in turn upregulated by a reproductive hormone. Thus, a common hormone regulates these two parallel olfactory pathways to coordinate courtship behavior. Mechanistically, PPK operates as a transduction channel: its activation requires odor-induced Ca2+ influx and a calmodulin binding motif (CBM) in the N-terminal intracellular domain. This result therefore highlights a novel mechanism whereby DEG/ENaCs can be activated by second messengers, a critical feature common to all transduction channels. Moreover, similar intracellular CBMs are predicted in multiple DEG/ENaCs across animal species, suggesting an evolutionarily conserved regulatory mechanism for channels in this superfamily (Ng, 2019).

This study has demonstrated that ionotropic sensory inputs can be amplified in select Drosophila ORNs whose receptors are ligand-gated cation channels. Pharmacological and genetic experiments reveal a simple and elegant mechanism for this amplification. Upon odor stimulation, receptor excitation allows for direct influx of Ca2+, which serves as a second messenger to activate a DEG/ENaC channel, PPK25, and thereby amplify ORN responses (Ng, 2019).

Ionotropic signal amplification, as described, affords remarkable versatility in sensory signaling when compared against G-protein-mediated metabotropic mechanisms. In vertebrate olfaction, separate families of metabotropic receptors typically couple to different G proteins, each engaging a unique downstream signaling cascade. In contrast, activation of specific G proteins is not required for Ca2+-mediated amplification, making it compatible with a wide variety of ionotropic receptor channels, so long as these receptors are permeable to Ca2+. As evidenced in this study, PPK can function downstream of Or47b, Ir84a, and ChR2, despite their low sequence similarity and distinct topologies because these receptors can flux Ca2+ (Ng, 2019).

These findings highlight striking differences and commonalities between insect and vertebrate olfactory transduction. This study observed surprising heterogeneity within insect olfactory transduction: PPK is neither expressed nor functional in another ORN type expressing Or22a, which belongs to the same receptor family as Or47b. It is unclear whether input signals in Or22a ORNs are amplified. If so, it is likely through a different mechanism. This finding indicates that insect ORNs expressing the same family of receptors do not necessarily employ the same mechanism for amplification, in contrast with vertebrate olfaction where receptors from the same family share a common signaling pathway. Despite this difference, the activation mechanism and function of PPK are remarkably similar to those of Anoctamin (ANO2), a key transduction channel in vertebrate ORNs. Specifically, both PPK and ANO are activated by calcium to amplify olfactory inputs. Although the sources of Ca2+may differ-ligand-gated ion channels in insects or cyclic nucleotide-gated cation channels in vertebrates-Ca2+-mediated amplification may represent a shared signaling motif between the two olfactory systems (Ng, 2019).

Interestingly, the impact of signal amplification on spike output differs between insect and vertebrate ORNs. Consistent with its role as a transduction channel, Ano mutation markedly reduces odor-evoked currents. However, the spike output of Ano knockout ORNs is higher than that of wild-type neurons. Vertebrate ORN spike number peaks when the transduction current is largely carried by ANO at intermediate odor concentrations, suggesting a strong depolarization block whereby amplification provides negative feedback to clamp total spike output. In contrast, the local field potential and spike responses of insect ORNs both peak at saturating odor concentrations. As such, blocking PPK25-mediated amplification not only reduces LFP response but also total spike number. Therefore, signal amplification in insect ORNs may predominantly serve to modulate the gain of neuronal output (Ng, 2019).

What is the functional significance of PPK in the Or47b and Ir84a ORNs? Notably, these are the only ORN types known to promote courtship in D. melanogaster males, whose fertility and courtship drive increase and peak at about days of age. Male mating drive is highly influenced by external olfactory cues, including the availability of mates and food signaled by Or47b and Ir84a ORNs, respectively. Remarkably, the responses of both ORN types exhibit age-dependent plasticity, which is coordinated by the same reproductive hormone-juvenile hormone-through upregulation of PPK expression in older males. The expression level of PPK in turn determines the ORN response magnitude, with striking impacts on courtship. Therefore, flexibility over this biologically salient behavior is afforded by the dynamic regulation of PPK expression, which heightens males' sensitivity to food and mate odors at their age of peak fertility. This upregulation of PPK provides a molecular mechanism for how sex-specific refinements of olfactory circuits are achieved via hormonal signaling (Ng, 2019).

The critical role of the intracellular CBM in PPK function argues that Ca2+/CaM activates the channel by directly interacting with this motif. Such regulation contrasts sharply with previously reported mechanisms, in which Ca2+/CaM indirectly modulates ENaC activity through intermediate proteins. For example, in cultured Xenopus cells, Ca2+/CaM can inhibit ENaC currents by interacting with MARCKS (myristoylated alanine-rich C kinase substrate) to modulate channel open probability and also by activating CaMKII to regulate ENaC apical trafficking. Together, these findings highlight the complexity of interactions between Ca2+/CaM and neuronal DEG/ENaC (Ng, 2019).

The results described in this study further advance understanding of DEG/ENaC activation. The gating mechanisms for this family of sodium channels are known to be highly diverse: some open in response to mechanical stimuli; others to extracellular ligands; and still others are constitutively active. In light of these findings, it is possible that other members of the DEG/ENaC superfamily may also be directly activated by intracellular second messengers, allowing them to function as transduction channels to amplify sensory inputs. In support of this notion, similar N-terminal intracellular CBMs were bioinformatically identified in multiple members of the DEG/ENaC superfamily across species-including worm, fruit fly, mosquito, mouse, and human-suggesting that those channels have the potential to function as Ca2+-activated transduction channels (Ng, 2019).

DEVELOPMENTAL BIOLOGY

Embryonic

Maternally derived Calmodulin mRNA is homogeneously distributed throughout the early Drosophila embryo, but these maternal transcripts are lost by maximal germ band extension. Zygotic transcription of the gene in mid- to late-stage embryos is restricted to neural cell precursors and their progeny in both the central and peripheral nervous systems. Although all neuroblasts express CaM mRNA, certain neuroblasts within each hemisegment show distinctly higher levels than others. The pattern of differential expression is the same for all hemisegments, from cephalic segment two to abdominal segment eight. For each of these hemisegments, all four of the neuroblasts in the outer, lateral row express higher Calmodulin transcript levels. The third and fourth neuroblasts of the inner, medial row also have higher expression level, as does one neuroblast within the intermediate row. Axon tracts of the commissures and connectives are very lightly stained. Three pairs of neurons within the ventral nerve cord have markedly higher levels of CaM mRNA than other neurons. These cells may be some of the paired midline neurons within three of the abdominal segments. All sensory neurons of the Peripheral nervous system express CaM, whereas none of the support cells do. Transcript levels are lower in precursor cells than in differentiated neurons. Activation of Calmodulin transcription during embryonic development appears to mark a commitment to a neural fate. The two transcripts from the Calmodulin gene are differentially expressed during embryogenesis. The longer Calmodulin mRNA is a nervous tissue-specific transcript. This suggests that neural-specific regulation of polyadenylation site usage occurs (Kovalick, 1992).

Calmodulin is present in all cells at all times. In addition to this constitutive level, the amount of Calmodulin is highly regulated according to the tissue or stage of development. The combined level of the transcripts is developmentally regulated, and the relative amounts of the two transcript size classes (1.65 kb and 1.9 kb) are differentially regulated during development. The spatial distribution of Calmodulin transcripts has been examined by in situ hybridization in sections of adults and developmentally staged whole mount embryos. Calmodulin transcripts are evenly distributed early in embryogenesis. The region of the developing midgut is labeled. Stage 15 embryos show sharper anterior localization of label associated with the developing anterior sensory organs. A relatively high level of Calmodulin transcripts can also be seen in the brain. In late stages of embryogenesis, high levels accumulate in the developing nerve cord (Hanson-Painton, 1992).

The expression of the Drosophila calmodulin (CAM) gene is surprisingly complex. The nervous system, which shows intense transcription in embryogenesis, contains no detectable transcripts at the end of larval life, with the exception of expression in the ring gland in the third instar larval stage. New expression takes place at pupariation. This activation occurs at the time of a known burst of ecdysone secretion and is therefore a candidate for a relatively direct hormonal effect. From the mid-pupal stages, regional differences in hybridization intensity are detectable throughout the CNS. Most noticeable, neurons in the brain cortex show stronger hybridization than those in the optic lobes. In addition, marked cell-to-cell variation in hybridization is seen in the central brain, presumably reflecting functional differences between neurons. The gut shows no expression in early embryogenesis, but high levels of expression throughout the larval stages, but none during pupal reorganization. In particular, hybridization to the proventriculus and middle midgut is very intense. The pharyngeal epithelium and salivary glands also show strong expression, with the gastric caecae and anterior midgut showing somewhat lower levels and the Malpighian tubules containing only moderate transcription levels. In contrast, CAM expression in the thoracic muscles drops significantly on transition from pupal to adult life. In the testis, transcription is strongly up-regulated prior to meiosis. In the ovary, strong expression is seen in the nurse cells with even more intense expression in the columnar follicle cells encasing the oocyte. Levels of expression in the oocyte cytoplasm are lower than in the nurse and follicle cells. Growing cells show lower transcript levels than most differentiated tissues and in general, cells with intense exocytotic or endocytotic activity show the highest mRNA levels (Andruss, 1997).

Larval

Seventy-six genes have been identified that are strongly expressed in the Drosophila ring gland during development. For nine of these, further studies of expression pattern, mutant phenotype and molecular nature identify the genes as strong candidates to carry out an important role in endocrine functions controlling development. Two of the genes identified encode products that have already been implicated in the functioning of prothoracic glands in other insects. The Calmodulin gene is expressed exclusively and at high levels in the ring gland of third-instar larvae, suggesting an important, presumably endocrine function for calmodulin in that tissue, as has already been suggested for lepidopterans. Calmodulin and other Ca²⁺-binding proteins are integral to the transduction of a wide range of Ca²⁺-dependent signals; there is clear evidence for the Ca²⁺ dependence of ecdysteroid molting hormone (EC) production in the Manduca larval prothoracic gland (PTG), at least for the commitment peak early in the last larval instar. It is known that Ca²⁺ activates prothoracic gland adenylate cyclase both directly and as a complex when bound to calmodulin. Since cAMP phosphodiesterase activity is low at this stage, cAMP is expected to accumulate. Both large and small PTTHs (see Bombyx and Manduca prothoracicotropic hormone) stimulate increased cAMP levels in PTG; a rise in cAMP levels occurs with PTTH-stimulated EC production in early last-instar PTG (Harvie, 1998 and references).

The catalytic subunit of protein kinase A (PKA or cAMP-PK) is also expressed in the Drosophila ring gland. This protein probably functions downstream of cAMP in the Ca²⁺-cAMP-dependent signaling pathway. PKA is activated in M. sexta PTGs by PTTH immediately prior to EC production. This is consistent with the idea that activation of the Ca²⁺-cAMP-dependent signaling pathway by PTTH leads to PKA-dependent phosphorylation of key proteins, including ribosomal protein S6, and that this causes changes in selective translation leading to increased EC production (Harvie, 1998 and references).

Adult

The vast majority of ovarian Calmodulin mRNA species are of the shorter type (Kovalick, 1992).

Elevated levels of Calmodulin transcripts are seen quite distinctly in the adult neural tissues. Accumulation is quite high in the cell bodies surrounding the lamina, the medulla, and the lobula, as well as the cell bodies of the subesophageal ganglion, the mass of neurons providing communication from the head organs to the rest of the neurons extending into the thorax. In the compound eye, the striations in the grain distribution follow the length of the ommatidia. The observed pattern suggests that different cells of the ommatidia accrue different amounts of Calmodulin transcript (Hanson-Painton, 1992).

A transcriptional reporter of intracellular Ca(2+) in Drosophila

Intracellular Ca(2+) is a widely used neuronal activity indicator. This study describes a transcriptional reporter of intracellular Ca(2+) (TRIC) in Drosophila that uses a binary expression system to report Ca(2+)-dependent interactions between calmodulin and its target peptide. In vitro assays predicted in vivo properties of TRIC. TRIC signals in sensory systems were show to depend on neuronal activity. TRIC was able to quantitatively monitor neuronal responses that changed slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis (PI) cells to food and arousal. Furthermore, TRIC-induced expression of a neuronal silencer in nutrient-activated cells enhanced stress resistance, providing a proof of principle that TRIC can be used for circuit manipulation. Thus, TRIC facilitates the monitoring and manipulation of neuronal activity, especially those reflecting slow changes in physiological states that are poorly captured by existing methods. TRIC's modular design should enable optimization and adaptation to other organisms (Gao, 2015).

Using cultured cells and multiple in vivo assays, this study found that TRIC reports changes in Ca2+ levels under diverse conditions in visual, olfactory and neuromodulatory systems. The results provide quantitative assessments for choosing TRIC variants with appropriate sensitivity and stringency, and proof of principle that TRIC can be used to express a circuit manipulator. Thus, TRIC acts as a useful complement to functional Ca2+ imaging by integrating changes in activity over long periods of time and offering genetic access to neurons on the basis of their activity (Gao, 2015).

Vertebrate immediate-early-gene (IEGs) which evolved to be expressed in a high signal-to-baseline ratio in response to neuronal activation, are widely used to report neuronal activity. However, as they rely on endogenous signaling networks, their response properties and cell-type biases are difficult to modify. TRIC can be considered a rationally designed IEG, by exogenously introducing a protein-peptide interaction to detect Ca2+. The modular design of TRIC renders it more amenable to optimization. TRIC reports a rise in nuclear Ca2+ levels, which have previously been used to monitor pan-neuronal activity in C. elegans, and also accompanies neuronal activation in mammalian neurons likely shuttled by Ca2+-binding proteins. The current experiments indicate that nuclear Ca2+ correlates with activity in diverse neuronal classes in flies. It is likely that not all cell types have the same efficiency in converting cytoplasmic Ca2+ signal to nuclear Ca2+ signal. Thus, TRIC efficiency and optimization may differ for different neuronal types (Gao, 2015).

While this manuscript was in review, a Ca2+ integrator (CaMPARI) was reported in which the ultraviolet conversion of emission spectrum of a fluorescent protein was engineered to be contingent on Ca2+ concentration. CaMPARI can capture neuronal activity on a shorter time scale than TRIC or IEG. However, access of neurons to ultraviolet may limit the use of CaMPARI in deep tissues, at least in large animals, whereas TRIC and IEG report neuronal activity in the entire nervous system non-invasively. Notably, unlike CaMPARI or IEG, TRIC offers genetic access to active neurons, allowing activity-based circuit manipulation (Gao, 2015).

The results underscore the importance of optimizing TRIC for specific neuronal types. In this study, TRIC was optimized for multiple cell types, and many variants were described that can help users in other cells. It is recommended that users begin with CaM/MKII-mediated TRIC in their neurons of interest. If TRIC signal is detected, the users can attempt QA-mediated or FLP-mediated regulation of the timing of TRIC onset. The signal-to-baseline ratio can be further improved by titrating expression of TRIC using QA, choosing reporters with different stabilities, or switching to nlsLexADBDo or the MKIIK11A variant. Stoichiometry can also be leveraged to boost TRIC signal (Gao, 2015).

With the current version of TRIC, the signal accumulates and decays over many hours. To detect shorter periods of neuronal activity, an important future goal is to increase signal strength while avoiding saturation by basal Ca2+ concentrations. One solution to this problem would be to restrict TRIC to a narrower time window than that offered by the QA- or the FLP-mediated strategy. For example, TRIC could be split into DBD-X, Y-target peptide and CaM-AD, where X and Y are two interacting modules controlled by light. One could then synchronize TRIC with a specific manipulation, or even trigger TRIC repetitively with specific behavioral features using feedback from automated tracking. To preserve phasic information about neuronal activity, reporters with faster decays than CD8::GFP could be used or the TRIC components could be destabilized with tags for protein degradation. Given that the current TRIC was able to interact with endogenous CaM and its target peptides, another important direction is to 'isolate' TRIC by co-engineering the CaM and MKII components to lose binding to their endogenous partners, but maintain their mutual interaction. Future TRIC optimization could be achieved using high throughput screens in cultured cells, which can predict in vivo performance (Gao, 2015).

Previous studies used Ilp2 immunostaining, epitope-tagged Ilp2 or a secreted GFP as indirect indicators of PI activity. The major conclusions of these studies were validated using TRIC. After enhancing the dynamic range of TRIC, additional insight was gained into how PI activity is regulated. In particular, given that PI cells affect diverse processes, how do these cells determine their output according to all relevant inputs? For example, an animal may encounter conflicting metabolic needs, such as conserving energy versus defending territory in an impoverished environment. Nutrient and OA comparison could be viewed as a minimal model of such a dilemma, as OA contributes to arousal and is necessary for 'fight or flight' in insects. PI cells exhibited graded, yet more readily saturated, responses to such events. In contrast, the linear PI response to nutrients extended over a wider range. These distinctions, as well as the additive interaction between yeast and OA, point to the independent operation of these two categories of inputs. To further survey the input landscape, one could genetically manipulate candidate receptors autonomously or candidate upstream neurons non-autonomously while monitoring PI activity using TRIC (Gao, 2015).

The physiological states of flies can change over hours to days and can be accompanied by changes in the activities of neurons expressing modulatory neurotransmitters or neuropeptides. Although previous work has focused on the targets of modulatory neurotransmitters, inputs to these cells remain largely unknown. In addition, there are ~75 predicted neuropeptides in flies, only a small subset of which have been examined. TRIC can be applied to neurons expressing specific transmitters or neuropeptides and tested in different physiological states (for example, the NPF neurons). It is noted that the current TRIC variants might not fit the dynamic range of all neuronal types, and it might be necessary to test other AD/DBD ratios or other MKII mutants following the examples in this paper of optimization for PI cells (Gao, 2015).

Finally, TRIC can report a rise of intracellular Ca2+ that accompanies any cellular, developmental or physiological processes in flies and can be adapted for similar use in other model organisms. TRIC modules can be introduced as transgenes or by viral vectors, and specific stoichiometry can be achieved by specifying the number of AD and DBD sequences in multi-cistronic constructs. TRIC expression can be made contingent on recombinase or other binary systems in model organisms, such as mice, where many Cre lines are available for spatiotemporal control, which can help refine activity monitoring and circuit manipulation in specific cell types (Gao, 2015).

EFFECTS OF MUTATION

Targeted disruption of Ca(2+)-Calmodulin signaling in Drosophila growth cones leads to stalls in axon extension and errors in axon guidance. Ca(2+)-Calmodulin (CaM) function is selectively disrupted in a specific subset of growth cones in transgenic Drosophila embryos in which a specific enhancer element drives the expression of the kinesin motor domain fused to a CaM antagonist peptide (kinesin-antagonist or KA, which blocks CaM binding to target proteins) or CaM itself (kinesin-CaM or KC, which acts as a Ca(2+)-binding protein). In both KA and KC mutant embryos, specific growth cones exhibit dosage-dependent stalls in axon extension and errors in axon guidance, including both defects in fasciculation and abnormal crossings of the midline. These results demonstrate an in vivo function for Ca(2+)-CaM signaling in growth cone extension and guidance and suggest that Ca(2+)-CaM may in part regulate specific growth cone decisions, including when to defasciculate and whether or not to cross the midline (Van Berkum, 1995).

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues that prevent axons crossing the midline (Fritz, 2000).

A novel CaM inhibitor, called kinesin-antagonist (KA), has been expressed using the neurogenic enhancer element of the fushi tarazu gene (ftzng) in a subset of CNS neurons that normally do not cross the midline. KA expression decreases endogenous CaM activation of target proteins in the growth cone and this leads to specific axon guidance defects including stalls at selected choice points, failure to fasciculate properly and abnormal crossing of the midline. robo and slit mutations and KA interact synergistically to increase the number of axon bundles abnormally crossing the midline. KA also induces axon bundles to cross the midline in the absence of Comm protein. Sos-dependent crossovers are enhanced by KA or by slit mutation. KA and Sos also interact to increase the number of axon bundles crossing in a comm mutant. Thus, the data demonstrate that both CaM and Sos signaling pathways are required to prevent certain axons crossing the midline (Fritz, 2000).

Whether CaM and Sos-mediated signaling is working directly downstream of Robo or in closely associated, but parallel signaling pathways to prevent axons from crossing is difficult to ascertain from this genetic data alone. If these signaling pathways lie downstream of Robo, the data suggest that both CaM and Sos are activated upon Slit binding to Robo, and result in growth cone repulsion. Interestingly, increased levels of calcium have been implicated in growth cone retraction and growth cone collapse, two ways in which a growth cone may respond to a repulsive agent. In addition, retrograde actin flow, which leads to filopodial retraction, is stimulated by CaM activation of myosin light chain kinase. Two other CaM target proteins, cAMP adenylyl cyclase and phosphodiesterase, regulate cAMP cellular concentrations thus altering neuronal response to Netrin 1 and other guidance cues. Activation of a Sos signaling pathway can affect cytoskeletal dynamics by activating various GTPases known to regulate growth cone behavior and axon guidance. Moreover, the cytoplasmic tail of Robo, known to be essential for signaling function, has a tyrosine residue that could recruit Sos via Drk or dreadlocks (dock), another SH2-SH3 adapter protein that affects axon guidance. Alternatively, Robo may bind Enabled, a known substrate for Abelson tyrosine kinase (Abl), which has been implicated in commissure formation. If Sos binds to phosphotyrosine residues on Ena (also via an adapter protein) it could be indirectly recruited to Robo (Fritz, 2000 and references therein).

Another possibility is that a disruption in both the CaM and Sos signaling pathways indirectly causes abnormal crossovers. CaM has been identified as a player downstream of several guidance molecules. Indeed, the gaps in the longitudinal connectives observed with increasing copies of KA in a comm mutant or in KA robo mutants, which are not seen in robo mutants alone, suggest CaM may function downstream of other guidance cue receptors to allow extension through the connective. Once these signals are attenuated by expression of KA, axons may inadvertently cross the midline. However, if CaM only functions in cell adhesive mechanisms within the connectives, it is difficult to explain why axons cross the midline in comm mutants when no other axons cross and the presence of Slit is still being read by Robo (Fritz, 2000 and references therein).

Since CaM and Sos appear to interpret a midline repulsive cue, the existence of an additional midline repulsion system working in parallel to Robo represents an interesting possibility. In robo mutants, axons cross the midline but then move to the longitudinal connective, instead of collapsing at the midline as observed in slit mutants. It has been suggested that this occurs because the continued presence of Slit at the midline is detected by a second receptor system, and candidate genes include a second robo gene or karussel. As the data shows, heterozygous slit mutations interact very strongly with single copies of KA, Sos or KA Sos together, to force axons across the midline. The interaction between Sos and slit mutations, especially when compared to the lack of Sos and robo interaction, is particularly striking. It seems that if the activity of both repulsion systems is decreased due to the reduction of a common ligand (Slit), a disruption in CaM and/or Sos signaling dramatically increases midline crossing errors. Most of these results, including the synergistic effects of KA and Sos, robo and slit mutations, the robo-like phenotype of KA Sos mutants, and the enhancement of crossovers in comm mutants can be explained by a parallel decrease in both midline repulsive systems upon disruption of the CaM and Sos signaling pathways. Thus while the mechanisms by which CaM and Sos contribute to an axon guidance decision at the midline remain unclear, the data clearly indicate that CaM and Sos signaling pathways are critical to the transduction of repulsive information at the midline (Fritz, 2000 and references therein).

Three systems are involved in Drosophila phototransduction and there are two messengers for Drosophila light excitation. InsP3, generated by phospholipase C, could function as the first messenger, acting to trigger the amplification of the light signal by causing the release of Ca2+ from the endoplasmic reticulum via the ryanodine receptor. Thus InsP3 based amplification is the first system. Ca2+, in turn, acts as a second messenger by acting to engender its own release. This function of calcium serves as the second system activated in the photosynthetic cascade. Calmodulin is involved in regulating Ca2+ stores in Drosophila, inactivating the response, and thus acting as the third system involved in the photosynthetic cascade. Treatment of Drosophila photoreceptor cells with ryanodine and caffeine disrupt the current induced by light, whereas subsequent application of calcium-calmodulin (Ca-CaM) rescues the inactivated photoresponse. In calcium-deprived wild-type Drosophila and in calmodulin-deficient transgenic flies, the current induced by light is disrupted by a specific inhibitor of Ca-CaM. Furthermore, inhibition of Ca-CaM reveals light-induced release of Ca2+ from intracellular stores. Thus it appears that functional ryanodine-sensitive stores are essential for photoresponse. Calcium release from these stores appears to be a component of Drosophila phototransduction, and Ca-CaM regulates this process (Arnon, 1997b).

The regulatory protein Calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of Calmodulin in an intact animal, a Calmodulin null mutation was generated. Maternal Calmodulin supports Calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. Two pronounced behavioral abnormalities specific to the loss of Calmodulin are detected in these larvae: swinging the head and anterior body, which normally occurs in the presence of food, is three times more frequent in the null animals. Even more striking, most locomotion in Calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. The finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some Calmodulin mutations in Paramecium. Thus these results suggest evolutionary conservation of a role for Calmodulin in membrane excitability and linked behavioral responses (Heiman, 1996).

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. Mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region were generated and studied. One of these mutations (Cam7) produces a striking pupal lethal phenotype involving failure of head eversion. Cam7 mutants have smaller pupal cases than normal, with a distinctive "Michelin-man" phenotype of deeply indented rings corresponding to the junctions of larval body segments. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. It is suggested that this head eversion phenotype reflects specific disruption of an interaction of Cam with Myosin light chain kinase (MLCK). The wing vein phenotype indicates a role for Cam in suppression of vein formation. coracle, a Drosophila homolog of the mammalian Protein 4.1, a component of the plasma membrane cytoskeleton and a known Cam binding protein in vertebrates, could be involved in the wing vein phenotype. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations (Nelson, 1997).

Mutations have been generated in the single Drosophila Calmodulin gene and the effects of these mutations then examined on behavior, synaptic transmission at the larval neuromuscular junction, and structure of the larval motor nerve terminal. Flies hemizygous for Cam^3c1, a mutation in the first Ca2+-binding site, exhibit behavioral, neurophysiological, and neuroanatomical abnormalities. In particular, adults exhibit defects in locomotion, coordination, and flight. The effects on motor neuron function and transmitter release of many behavioral mutations, particularly those affecting ion channels, are enhanced by application of the K+ channel-blocking drug quinidine. This drug, when applied at a concentration of 0.1 mM, completely and specifically blocks the delayed rectifier K+current in the Drosophila larval muscle. Application of quinidine enhances the effects of ion channel mutations, such as Shaker and Hyperkinetic, on the duration of motor nerve terminal depolarization and transmitter release. The phenotypes of other excitability mutants, such as inebriated and pushover, are also enhanced by quinidine application. Application of quinidine has no significant effect on excitatory junctional current (EJC) amplitude in the wild-type control larvae or in larvae heterozygous for Cam mutations. In contrast, quinidine application to Cam^3c1/Cam^null at the three lowest external [Ca2+] levels tested causes an approximately threefold increase in EJC amplitude. The muscle responsiveness to neurotransmitter is shown to be normal, and that the increased amplitude EJCs observed in Cam^3c1/Cam^null larvae reflect increased transmitter release. One might imagine that impaired activation of a K+ channel in Cam^3c1/Cam^null might have no observable phenotypic consequences under otherwise normal conditions; however, in combination with quinidine, which could block a distinct, functionally redundant K+ channel, this effect could lead to increased nerve terminal depolarization and increased Ca2+ influx into the nerve terminal. The observation that the effects of Cam^3c1/Cam^null occur only at low external [Ca2+] is consistent with this view: the effects of most excitability mutations, including Shaker, Hyperkinetic, and inebriated, are also revealed only at low external [Ca2+]. This observation has been proposed to result from the activation of a Ca2+-activated K+ current at higher external [Ca2+]. Alternatively, Cam^3c1/Cam^null could be defective in Ca2+ buffering. In this view, a broadening of the action potential conferred by quinidine application combined with reduced Ca2+ buffering as a consequence of Cam3c1/Camnull could lead to increased transmitter release (Arredondo, 1998).

Synaptic bouton structure at motor nerve terminals is altered. Structural abnormalities are present in the nerve terminals of Cam^3c1/Cam^null larvae in muscle 13 of abdominal segments 3 - 5. In particular, rather than cascading into a string of distinct type I and II boutons as in the control larvae, the terminal arbor of the Cam^3c1/Cam^null larvae forms a thickened, or large, misshapen structure with few distinct boutons. The large structure results in a reduced number of boutons and a nearly complete lack of terminal branching in muscle 13. In comparison to control larvae, no abnormalities in the structure of nerve terminals on muscles 6, 7, or 12 have been observed. Despite this altered synapse morphology, muscle 13 synaptic transmission in Cam^3c1/Cam^null larvae resembles the muscle 6 properties. This observation raises the possibility that this defective bouton might still be functional. Although mutations in several neuronal signaling genes confer defects in the pattern of motor neuron innervation of the target muscle, the morphological defects of Cam^3c1/Cam^null at the nerve terminal differ from any that have been reported previously and do not appear to result from the same mechanisms as the "activity-dependent" increases in synaptic bouton number or axonal branching Rather, the phenotype observed appears to result from defects in the formation of distinct boutons at the proper locations along the muscle surface. It is unclear why this defect in bouton formation is observed only in muscle 13. Proper synapse formation on muscle 13 may be more sensitive to altered CaM function, or perhaps distinct mechanisms control bouton formation in muscle 13 vs. other muscles. Analysis of the genes required for proper bouton formation is less characterized than for axon pathfinding or growth cone guidance. The results presented here suggest a role for CaM in this process (Arredondo, 1998).

These effects are distinct from those produced by altering the activity of the CaM target enzymes CaM-activated kinase II (CaMKII) and CaM-activated adenylyl cyclase (CaMAC or Rutabaga). Mutations in rutabaga reduce facilitation and post-tetanic potentiation at the larval neuromuscular junction, whereas activation of an inhibitory domain of CaMKII confers a number of behavioral, electrophysiological, and anatomical defects, including defects in courtship conditioning, an abnormal spontaneous firing of motor axon action potentials, increases in axon branching and transmitter release, and a reduction in facilitation and augmentation. Cam3c1/Camnull larvae do not show these phenotypes; e.g., Cam^3c1/Cam^null larvae possess a normal number of axon branches and display normal EJC amplitude (in the absence of quinidine) and normal paired pulse facilitation. Similarly, whereas application of quinidine to Cam^3c1/Cam^null substantially increases evoked transmitter release, quinidine application has little or no effect on CaMKII-inhibited larvae. Thus, it is unlikely that the Cam^3c1 mutation is exerting its effects via either CaMAC or CaMKII, but rather via an alternative target. One alternative target, the Drosophila CaM-activated protein kinase Caki, is expressed in the central nervous system; mutants defective in caki exhibit behavioral defects related to those described here (Martin, 1996). The catalytic domain of Caki shares homology (41%) with type II CaM kinases, while the C-terminal part is divergent. Constitutively expressed Caki protein is enzymatically active. In situ hybridization shows that during embryogenesis, larval and pupal life, transcription of caki is restricted almost exclusively to the central nervous system. In the adult head, immunohistochemistry reveals Caki protein in the lamina, the neuropil of the medulla, lobula, lobula plate and in the central brain. Mutant caki flies show reduced walking speed in 'Buridan's paradigm'. Thus, the Cam^3c1 mutation might affect Ca2+ buffering or interfere with the activation or inhibition of a CaM target, distinct from CaMKII or CaMAC (Arredondo, 1998).

Working with mutants for two calcium binding sites, a study to assess the ability of Drosophila Calmodulin to form complexes with model target peptides melittin and mastoparan has shown that unlike the wild-type complex, the 1:1 protein:melittin complexes formed by mutants of the C-terminal sites are unable to bind a second molecule of melittin. In contrast, a site 2 mutant shows increased ability to bind two molecules of melittin. For the shorter peptide mastoparan, most mutants form aberrant complexes that are best interpreted in terms of a model in which mastoparan interacts with both terminal domains of Calmodulin. For two of the target enzymes of Calmodulin, the three mutants which form mastoparan complexes most similar to the wild-type complex are also the best enzyme activators (Mukherjeam, 1993).

The kinetics of calcium dissociation from two groups of site-specific mutants of Calmodulin from Drosophila have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator Quin 2. The BQ series of mutants consists of four proteins in which one of the four bidentate glutamate residues (Glu12 of each of the four calcium binding loops) has been replaced by glutamine. In the BK series of mutants, the corresponding glutamate has been replaced by lysine. Calcium-dissociation kinetics of proteins with a mutation in site I or II (N-terminal domain) are consistent with a model in which the mutation weakens binding at the non-mutated N-terminal partner site and has a small, but significant, effect on the kinetic properties of sites III and IV (C-terminal domain). The proteins with a mutation in site III or IV show a large effect, with decreased Ca2+ dissociation rate from the unmodified N-terminal Ca(2+)-binding sites I and II. A structural interpretation has been proposed, based on enhanced interactions between the domains when the affinity of individual sites have been dramatically reduced by mutation. This effect is greatest for the mutations in the C-terminal domain, which appear to destroy the co-operativity of Ca2+ binding at sites III and IV. The results show that site-specific mutation can have surprisingly far-ranging effects on kinetic properties of Calmodulin. The kinetic analysis also shows that studies of specifically engineered mutants may in principle help to unmask the values of intrinsic rate constants for the wild-type protein, not normally observable in the process of Ca2+ dissociation (Martin, 1992).

Ca2+ binding to the wild-type protein is best modeled as two pairs of sites with a higher affinity pair that shows strong cooperativity. For all but one of eight mutant proteins, only three Ca(2+)-binding events can be detected. In three of the amino-terminal mutants, the three residual sites are (i) a pair of relatively high affinity sites and (ii) a weakened low affinity site. For all four carboxyl-terminal mutations, the residual sites are three relatively low affinity sites. In general, mutations to sites 2 and 4 prove more deleterious than mutations to sites 1 and 3. The Ca(2+)-induced conformational changes in the vicinity of Tyr-138 are relatively undisturbed by mutations of site 1. However, the changes to Tyr-138 in the carboxyl-terminal site mutants indicate that upon disruption of the cooperative binding at the high affinity sites, conformational change in the carboxyl terminus occurs in two phases. It appears that binding of Ca2+ to either carboxyl-terminal site can elicit the first phase of the response but the second phase is almost abolished when site 4 is the mutated site. The final conformations of site 3 and 4 mutants are thus significantly different (Maune, 1992a).

In the absence of Ca2+, the helical content of these mutant Calmodulins is close to that of the wild-type protein. In the presence of excess Ca2+, Calmodulins with a mutation in the N-terminal sites show Ca(2+)-induced increases in helicity that are similar to those of the wild-type protein. In contrast, much less additional helix is induced by Ca2+ in Calmodulins with mutations in the C-terminal sites, with the two mutations to site IV showing a particularly poor response. Ca(2+)-induced changes to the environment of the single tyrosine of Drosophila Calmodulin (Tyr-138 in site IV of the C-terminal domain) have been monitored via CD at 280 nm. The signal from this residue is significantly altered in the Ca(2+)-free form of almost all these mutants, including those in the N-terminal domain. This indicates significant interaction between the N- and C-terminal domains of these mutants. There is a strong coupling between conformational change and cooperative Ca2+ binding at the two C-terminal sites (Maune, 1992b).

Calmodulin (CaM) is an essential component of calcium signaling in multicellular organisms. Null mutations of the Drosophila CaM gene (Cam) were used in combination with clonal analysis and immunolocalization to examine the effects of loss of Cam function in the ovarian germline and developing embryo. These studies have uncovered unexpected and striking movements of CaM protein within these tissues. In the ovary, evidence for transfer of CaM from an external source, across plasma membranes, into the germline cells was obtained. In late embryogenesis, maternally derived CaM protein relocalizes dramatically within the nervous system of both wildtype and Cam null embryos - a process that may also involve movement across cell membranes. These findings indicate dynamic, unsuspected elements to the in vivo functions of CaM in the whole organism (Andruss, 2004).

Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila

A mutation (Cam⁷) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. The biochemical properties of V91G D-CaM were investigated. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described the this paper establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM (Wang, 2004).

Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-β and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought (Kucherenko, 2008).

The screens described in this paper revealed some expected interactors, Dys, Cam and Khc. Calmodulin, a calcium binding protein required for muscle and neuronal functions has previously been shown to interact with mammalian the Dg-Dys complex. However, whether reduction of Cam activities suppresses or enhances the muscular dystrophy phenotype is not totally clear. Targeted inhibition of Cam signaling exacerbates the dystrophic phenotype in mdx mouse muscle while genetic disruption of Calcineurin improves skeletal muscle pathology and cardiac disease in ä-sarcoglycan null mice. Since reduction of Cam showed suppression of the phenotypes caused by reduction of the long forms of dystrophin in the Drosophila wing, it will be interesting to analyze whether reduction of Cam will suppress the Drosophila Dys muscle phenotype as well. Khc involvement in Dg-Dys complex is also expected since work in mammalian system has shown that Khc can bind Dystrobrevin, a component of Dg-Dys complex. It will be interesting to test in the future whether Drosophila Dystrobrevin can similarly bind Khc and what the functional significance of this interaction is in muscles and neurons. In oocyte development Khc is required as early as is Dys and Dg. It is, therefore, interesting to test the potential requirement of dystrobrevin in this process and to further dissect the Khc function in this complex during early polarity formation (Kucherenko, 2008).

Abelson tyrosine kinase and Calmodulin interact synergistically to transduce midline guidance cues in the Drosophila embryonic CNS

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision (Hsouna, 2008).

The developmental defects observed in the formation of the CNS axon scaffold clearly point to individual and co-operative roles for CaM- and Abl-dependent signaling pathways during axon guidance at the midline. Moreover, the range of defects suggest that both CaM and Abl have multiple roles during the transduction of midline attractive and repulsive cues, and probably converge to regulate key aspects of the cytoskeletal dynamics underlying axon outgrowth and steering (Hsouna, 2008).

When the signaling strength of either pathway is individually decreased, the predominant phenotype is ectopic midline crossing errors of pCC/MP2 axons. Ectopic midline crossing errors is also the primary defect in embryos experiencing a mild, but simultaneous, reduction of both CaM and Abl signaling. CaM and Abl appear to work together in the same neurons to transduce guidance cues because ectopic crossovers are replicated if a kinase inactive Abl transgene (ftz_ng-Abl^KN) is co-expressed with the CaM inhibitor in the same neurons. It also appears that the level of Abl activity is particularly important. When both copies of the endogenous abl gene are mutated, expression of even one copy of the CaM inhibitor is sufficient to induce major defects in the axon scaffold. The converse, one copy of abl⁴ in homozygous iCaM^KA (inhibitor of CaM-KA were KA stands for a novel competitive inhibitor called Kinesin-antagonis) embryos, does not significantly affect the frequency of ectopic crossovers. Together, these data are consistent with the earlier hypothesis that both signaling pathways function to transduce midline repulsive cues (Hsouna, 2008).

Robo, the receptor for the midline repellent Slit, is expressed in pCC/MP2 neurons from the onset of axonogenesis and its activity is required to prevent them from crossing the midline. Loss-of-function robo mutations enhance the frequency of ectopic crossovers induced by either iCaM^KA expression or abl mutations. Thus, the observation that a simultaneous decrease in CaM and Abl synergistically increases ectopic crossovers was anticipated. Mechanistically, Abl is known to bind to, and phosphorylate, Robo to potentially inhibit its activity. In addition, Enabled, a known substrate for Abl, binds to Robo and is required to signal midline repulsive activity. While little is known about how CaM contributes to Robo signaling, the meandering crossovers observed in robo mutants are replicated when iCaM^KA is combined with loss-of-function mutations in sos and Sos is now known to bind to Robo. A direct role for Sos in Robo signaling is also supported by the observation that ectopic midline crossovers occur in sos abl double mutants (Hsouna, 2008).

However, it is unlikely that CaM and Abl are operating solely downstream of Robo during midline guidance. When the level of CaM and Abl activity is substantially reduced in embryos using multiple copies of iCaM^KA and abl alleles, the frequency of crossovers increase but, in addition, commissures begin to fuse and gaps in the longitudinal connectives form. These latter defects are difficult to explain solely on the basis of a disruption in Robo signaling, since they are not generally evident in robo null embryos. Thus, the efficacy of other guidance mechanisms functioning at the midline must also be affected in the CaM and Abl mutants. One obvious candidate is Netrin-dependent midline attraction (Hsouna, 2008).

Netrin is a major midline attractant required for commissure formation in the Drosophila CNS, and Frazzled is the Netrin receptor guiding many commissure axons across the midline. In the absence of Fra, most posterior commissures do not form correctly. However, it is suspected that an alteration in Fra activity is not responsible for the fused commissures and longitudinal gaps observed in iCaM^KA and abl mutants. First, in abl and fra double mutants, a loss of Abl activity in fra mutants exacerbates commissure loss, and second, expression of iCaM^KA still induces crossovers in the absence of Fra. Most vertebrate literature also predicts that CaM-dependent enzyme activity increase during Netrin-dependent attraction, not decrease, as occurs with iCaM^KA expression (Hsouna, 2008).

There is, however, growing evidence that at least one other Netrin-dependent receptor is functioning during the midline guidance of pCC/MP2 neurons. While pioneering the longitudinal connective, pCC/MP2 neurons follow an axon trajectory delineated by Netrin localized along commissure axons by Fra. To project past these Netrin-rich commissures, midline attraction must be briefly inhibited by Robo activity. If this inhibitory signal fails, pCC/MP2 axons cross the midline using the newly emerging commissures and leave gaps in the longitudinal connective. This is, in fact, similar to the defects observed in iCaM^KA and abl mutants. Importantly, while they are responding to Netrin, most pCC/MP2 neurons do not appear to express Fra. Thus, in addition to inhibiting Robo-dependent midline repulsion, a combined loss of CaM and Abl activity may be preventing Robo from blocking this Netrin-dependent attraction at the segmental boundary. Testing this hypothesis awaits characterization of the Netrin-dependent receptor that is functioning at the segmental boundary. The absence of Fra expression in pCC/MP2 neurons may also explain 1) why iCaM^KA and abl induce crossovers of pCC/MP2 neurons even though fra and abl interact to reduce commissure formation, and 2) why iCaM^KA induces pCC/MP2 axons to cross the midline even in the absence of Fra (Hsouna, 2008).

Interestingly, this Netrin-dependent, but Fra-independent attraction near the segmental boundary is known to be sensitive to IP₃ levels, which are presumably leading to an increase in intracellular calcium. Thus, the ability of these neurons to remain on the correct side of the midline is quite sensitive to the level of calcium, and now CaM, signaling. Calcium and/or CaM may function downstream of specific receptors or more generally as second messengers governing basic cell processes, such as motility. Certainly, inhibiting even a small amount of CaM activity (using one copy of iCaM^KA) sensitizes these neurons to over-expression of Fra. Moreover, the frequency of crossovers (57%) observed in this experiment is approximately the same as observed when wild type Fra is over-expressed in a heterozygous robo mutant (55%). This implies that expression of a single copy of iCaM^KA is reducing Robo activity by half, a conclusion difficult to reconcile with previous data. Therefore, it is suspected that expression of iCaM^KA is altering the spatial and temporal regulation of calcium-dependent activity underlying growth cone movement and steering. In the case of pCC/MP2 neurons, this appears to preferentially result in ectopic midline crossovers, a defect which is further enhanced when the levels of receptor for midline attraction (Fra) or repulsion (Robo) are genetically manipulated (Hsouna, 2008).

An alteration in key calcium-dependent regulatory events would also be further exacerbated by a simultaneous loss in Abl activity, especially since Abl is a key regulator of the actin and myosin dynamics underlying growth cone advance. While not previously appreciated, there is some evidence linking CaM and Abl activity during axon guidance in the embryonic axon scaffold. For example, heterozygous abl mutations suppress the ectopic midline crossing errors induced by expression of an activated Myosin Light Chain Kinase (a CaM-dependent enzyme) even when Fra is co-expressed. In addition, actin dynamics are likely to be important since both iCaM^KA and abl mutations interact with Profilin loss-of-function mutations to alter axon path finding. This study specifically demonstrates a strong, synergistic interaction between CaM- and Abl-dependent signaling during in vivo development of the embryonic CNS. Clearly, it will be important to identify where these key signaling pathways converge to regulate actin and myosin dynamics and how these regulatory events contribute to axon guidance decisions at the midline (Hsouna, 2008).

New Dystrophin/Dystroglycan interactors control neuron behavior in Drosophila eye.

The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Genetic screens have been performed using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, this study has analyzed the DGC modifiers' function in the developing Drosophila brain and eye. Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, the function of previously screened components and their genetic interaction with the DGC in this tissue were determined. This study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. It was determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. These data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton (Marrone, 2011).

The roles that Dys and Dg play in disease have been apparent for some time since their disruption or misregulation has been closely linked to various MDs. Dg depletion results in congenital muscular dystrophy-like brain malformations associated with layering defects and aberrant neuron migration. These defects arise due to extracellular matrix protein affinity problems that influence neuronal communication and result in learning and memory defects. Similar to brain layer formation, the migration of R1-R6 growth cones into the lamina occurs in a similar manner where glia cells that migrate from progenitor regions into the lamina provide a termination cue to innervating axons. In Drosophila Dys and Dg are expressed in the CNS, PNS and visual system and both proteins are required for proper photoreceptor axon guidance and rhabdomere elongation. This work has identified novel components implicated in the process of eye-neuron development. Moreover, it was found that Nrk, Mbl, Cam and Capt genetically interact with Dys and/or Dg in visual system establishment (Marrone, 2011).

The proteins Mbl, Capt, Cam, Robo, Lis1 and Nrk have been shown previously to be associated with the nervous system, and this study has additionally found that mutations in chif, SP2353, CG34400 and vimar cause abnormal photoreceptor axon pathfinding and/or differentiation phenotypes. Lis1 has been shown to bind microtubules in the growth cone, and the human Lis1 homologue is important for neuronal migration and when mutated causes Lissencephaly, a severe neuronal migration defect characterized by a smooth cerebral surface, mental retardation and seizures. This study has found that Lis1^RNAi/GMR-Gal4 mutants have abnormally formed lamina plexuses, shortened rhabdomeres, and retinal vacuoles. Chif has been shown to regulate gene expression during egg shell development and is related to a DNA replication protein in yeast. The human ortholog for SP2353 (AGRN) is involved in congenital MD development. Drosophila SP2353 is a novel agrin-like protein that contains Laminin G domains, which makes it a potential new extracellular binding partner for Dg. CG34400 encodes for a protein homologues to human DFNB31 (Deafness, autosomal recessive 31) that causes congenital hearing impairment in DFNB31 deficient people and mouse whirlin, that causes deafness in the whirler mouse. Hearing loss has been as well demonstrated in association with various forms of muscular dystrophy. Vimar has been shown to regulate mitochondrial function via an increase in citrate synthase activity (Marrone, 2011).

Mbl is a Drosophila homologue of the human gene MBNL1. Mutations of this gene cause myotonic dystrophy and are associated with the RNA toxicity of CUG expansion diseases protein. This study shows that Mbl deficiency results in similar phenotypes to Dys and Dg loss of function, and to specifically interact with Dys in axon projections which is in accord with the Dys specific interaction seen in muscle. Dys has multiple isoforms, and the variability of DMD patients to have mental impairment has been linked in part to small Dys isoform mutations, which leads to speculation that Dys is a target for Mbl mediated splicing (Marrone, 2011).

Interestingly, Mbl isoforms have been demonstrated to regulate splicing of α-actinin, which belongs to the spectrin gene superfamily that also includes dystrophins. α-actinin and Capt, the Drosophila homologue of Cyclase-associated protein (CAP) are actin-binding proteins in the growth cone. Capt was first identified in yeast and is highly conserved throughout eukaryotic evolution. The main known function of Capt is to act in the process of actin recycling by working in conjunction with Actin Depolymerization Factor (ADF a.k.a. Cofilin) to help displace Cofilin from G-actin during depolymerization. It has already been reported that ADF/Cofilin has a role in retinal elongation. The actin cytoskeleton is a major internal structure that defines the morphology of neurons, and Capt has already been shown to be required to maintain PNS neuronal dendrite homeostasis in Drosophila via kinesin-mediated transport. Additionally, Capt has been found to lead to excessive actin filament polymerization in the eye disc and to cause premature differentiation of photoreceptors. The rate of axon projection is much slower than the rate of microtubule polymerization during axonal growth, implying that depolymerization/polymerization of actin is important during pathfinding. This study has also shown that Capt interacts with Dys and is necessary for proper projection of photoreceptor axons in the developing brain, and when absent, eyes develop with abnormal rhabdomeres. Furthermore, capt^RNAi mutants exhibit overgrowth of photoreceptor axons, and it is believed that a possible explanation for this is improper turnover of actin (Marrone, 2011).

Importantly, proteins that can be regulated by Ca²⁺ to organize actin filament bundles and to promote filament turnover include α-actinin and (ADF)/Cofilin, respectively. Cam functions as an intracellular Ca²⁺ sensor, and when Ca²⁺-Cam was selectively disrupted in a subset of neurons in Drosophila embryos, stalls in axon extension and errors in growth cone guidance resulted. Actin turnover is highly regulated by Ca²⁺ levels, and many proteins are Ca²⁺-mediated to regulate motility and axon guidance. The results and those from prior studies suggest that Cam is a major functional player of Ca²⁺ regulation in growth cones. Since it was shown here that mutations in Cam and capt have similar phenotypes in photoreceptor axon pathfinding and rhabdomere development, it is postulated that actin dynamics is the link between these two proteins and the phenotypes described here. Due to the importance of Cam for actin dynamics, its interaction with both Dg and Dys suggests that the DGC coordinates the actin cytoskeleton in the developing eye (Marrone, 2011).

The last gene identified in this work is Nrk. Recently various kinases, channels and other enzymes have been shown to associate with the DGC, although only a few of these interactions have been confirmed in vivo. Since Nrk is a component found to interact with Dys in photoreceptor axon pathfinding, it is most likely that it functions as a receptor to sense guidance cues rather than as a molecule affecting actin cytoskeletal rearrangement. The data here hint that Dg and Nrk could be two receptors integral to transferring signals important for neuronal layering (Marrone, 2011).

It is concluded that dynamic rearrangement of the actin cytoskeleton is crucial for the central and peripheral nervous system establishment, which depends on proper neuron migration and differentiation. This process requires not only the cell autonomous regulation of neuron motility, but also the interaction between the migrating cell and its underlying substrate. This interaction is often dependent on the signaling transduced via the ECM. The DGC and other factors are believed to be mediators of actin dynamics in growing axons and during neuronal cell morphogenesis, and this study found components that interact with Dys and/or Dg in both of these activities (see The DGC coordinates actin cytoskeleton remodeling). Additionally, disruption in gene expression of these components results in the same phenotypes seen with Dys and Dg mutants in the developing and adult eye. The data lead to the conclusion that the DGC is involved in signaling to cause cytoskeletal rearrangement and actin turnover in growth cones. Since many cases of muscular dystrophies are associated with mental retardation, it is believed that it is important to understand the role of the DGC in axon migration because understanding of this process could aid in finding an adequate therapy for this aspect of the disease's physiology. Since the human brain continues to develop well after gestation, and evidence shows that nerves maintain plasticity throughout an individual's lifespan, therapies could be devised that reverse these defects after birth (Marrone, 2011).

EVOLUTIONARY HOMOLOGS

Drosophila Calmodulin differs from the mammalian protein at only three residue positions: 99, 143 and 147 (Smith, 1987).

Drosophila's proteins related to calmodulin and other CaM related proteins

The EF-hand motif is present in dozens of characterized proteins belong to a large family. Many, but not all EF-hand proteins bind Ca++. Identified EF-hand proteins include Calmodulin, Troponin C, the myosin Essential light chain, the myosin Regulatory light chain, nonvertebrate Troponin, Ca-dependent protein kinase, Calpain and Calcineurin.

Calpains are calcium-dependent proteases believed to participate in calcium-regulated signal pathways in cells. Calpains have been found in vertebrates in ubiquitous as well as tissue-specific locations. A highly tissue-specific calpain gene has been characterized in Drosophila: CalpA, at 56C-D on the second chromosome. The encoded protein is found in a few neurons in the central nervous system, in scattered endocrine cells in the midgut, and in blood cells. In the blood cell line mbn-2, calpain is associated with a granular component in the cytoplasm. The expression of this protein is more restricted than that of the corresponding transcripts, which are widely distributed in the central nervous system, digestive tract, and other tissues. The sequence of CalpA is closely related to that of vertebrate calpains, but an additional segment is inserted in the calmodulin-like carboxy-terminal domain. This insert contains a hydrophobic region that may be involved in membrane attachment of the enzyme. Differential splicing also gives rise to a minor transcript that lacks the calmodulin-like domain (Theopold, 1995).

Using low-stringency hybridization and polymerase chain reaction (PCR)-based DNA amplification, four Drosophila melanogaster genes have been isolated: three of these encode troponin-C isoforms, and the fourth specifies a protein closely related to calmodulin. Two of the troponin-C genes, located (respectively) within the 47D and 73F subdivisions of chromosomes 2 and 3 encode very closely related isoforms. The isoform specified by the 47D gene accumulates almost exclusively in larval muscles, while the one encoded by the 73F gene is present in both larvae and adults. The third gene, located within the 41C subdivision of chromosome 2, encodes a more distantly related troponin-C isoform that accumulates only within adults. The fourth gene, which encodes the calmodulin-related protein, is located within the 97A subdivision of chromosome three. This protein has a different primary sequence from that of conventional calmodulin, which is specified by a gene located within the 49A subdivision of chromosome 2 (Fyrberg, 1994).

Pulses of ecdysone at the end of Drosophila larval development that signal the onset of metamorphosis dramatically reprogram gene expression. Ecdysone directly induces several early puffs in the salivary gland polytene chromosomes; these, in turn, activate many late puffs. Three early puffs (at 2B5, 74EF, and 75B) have been studied at the molecular level. Each contains a single ecdysone primary-response gene that encodes a family of widely expressed transcription factors. The 63F early puff differs in significant ways from the previously characterized early puff loci: The 63F puff contains a pair of ecdysone-inducible genes that are transcribed in the larval salivary glands: E63-1 and E63-2. E63-1 induction in late third instar larvae appears to be highly tissue-specific, restricted to the salivary gland. E63-1 encodes a novel Ca(2+)-binding protein related to calmodulin. The discovery of an ecdysone-inducible Ca(2+)-binding protein provides a foundation for integrating steroid hormone and calcium second messenger signaling pathways and generates an additional level for potential regulation of the ecdysone response (Andres, 1995).

A novel EF-hand protein of Dictyostelium discoideum, termed CBP2, is composed of 168 amino acids and contains four consensus sequences that are typical for (Ca2+)-binding EF-hand domains. The protein sequence exhibits only minor similarities to other calmodulin-type proteins from Dictyostelium. The genomic DNA harbors two short introns; their positions suggest that the gene is unrelated to the EF-hand proteins from the calmodulin group. Northern blot analysis shows that the mRNA level is significantly increased during development. Polyclonal antibodies raised against the recombinant protein recognize a protein of about 20 kDa. Like the mRNA, the protein is also more abundant in developing cells. Overlay experiments with 45Ca2+ indicate that the EF-hands in fact have (Ca2+)-binding activity. The recent description of CBP1, another calmodulin-type Dictyostelium protein that is upregulated during development, suggests that D. discoideum contains a family of EF-hand proteins that have specific functions during distinct steps of development (Andre, 1996).

Analysis of Calmodulin structure using engineered proteins

Three engineered mammalian calmodulins (CaMs) were constructed in which the two EF hand pairs were either substituted for one another or exchanged: CaMNN, the C-terminal EF hand pair (residues 82-148) was replaced by a duplication of the N-terminal pair (residues 9-75); CaMCC, the N-terminal pair was replaced by a duplication of the C-terminal pair; CaMCN, the two EF had pairs were exchanged. Skeletal muscle myosin light chain kinase (skMLCK) activity is activated to 75% of the maximum level by CaMCC and to 45% of the maximum level by CaMCN and is not significantly activated by CaMNN; Smooth muscle myosin light chain kinase activity (gMLCK) is fully activated by CaMCN and is not significantly activated by either CaMNN or CaMCC. Cerebellar nitric oxide synthase activity (nNOS) is fully activated by CaMNN and CaMCN and is not significantly activated by CaMCC. These results indicate that the EF hand pairs contain distinct but overlapping sets of determinants for binding and activation of enzymes, with the greater degree of overlap in determinants for binding. While the structural changes associated with swapping the EF hand pairs do not affect activation of nNOS or gMLCK activities, they significantly reduce activation of skMLCK activity, indicating that this process requires specific determinants in CaM outside the EF hand pairs (Persechini, 1996a).

Deletion of residues 2-8 from the N-terminal leader sequence in calmodulin abolishes calmodulin-dependent activation of skeletal muscle myosin light chain kinase activity and reduces calmodulin-dependent activation of smooth muscle myosin light chain kinase activity to approximately 50% of the maximum level measured at a saturating calmodulin concentration. Calmodulin-dependent activation of cerebellar nitric oxide synthase activity is not affected by this deletion. Overlapping tripeptide deletions from the leader sequence indicate that a three amino acidic cluster contains the determinants necessary for activation of myosin light chain kinase activity. Based on enzyme kinetic analyses, deletions in the leader sequence have little or no effect on the apparent affinities of calmodulin for the synthase or the two kinases. Since the N-terminal leader does not appear to play a significant structural role in the complexes between calmodulin and peptides representing the calmodulin-binding domains in the two kinases, these results indicate that it participates in secondary interactions with these enzymes that are important to activation, but not to recognition or binding of calmodulin (Persechini, 1996b).

CaM (4 cTnC) is a calmodulin-cardiac troponin C chimeric protein containing the first, second, and third calcium-binding EF-hands of calmodulin (CaM) and the fourth EF-hand of cardiac troponin C (cTnC). CaM (4 cTnC) shows 2-fold-enhanced carboxy-terminal Ca2+ affinity (relative to CaM) and also exhibits impaired activation of the CaM-regulated enzymes smooth muscle myosin light chain kinase (smMLCK), neuronal nitric oxide synthase (nNOS), and phosphodiesterase (PDE). Additional chimeras were constructed, replacing most of CaM helix 7, Ca2+-binding loop 4, and helix 8 with the corresponding helices and loops of cTnC. Point mutants in the fourth EF-hand of CaM were also constructed. Replacement of CaM's fourth loop with the corresponding loop of cTnC enhances Ca2+ affinity by over 3-fold through an increase in the Ca2+ on rate and also reduces the cooperativity of Ca2+ binding. In contrast, substitution of CaM helix 7 or 8 modestly decreases Ca2+ affinity by increasing the Ca2+ off rate, without impairment of cooperativity. All three of the helix and loop chimeras fully activate PDE, with minor shifts in Kact. CaM (helix 7 cTnC) shows a significantly impaired ability to activate smMLCK and nNOS, whereas the other two chimeras retain about 80% of the maximal smMLCK and nNOS activation observed with CaM (George, 1996).

Calmodulin subcellular location

Many important enzyme activities are regulated by Ca2+-dependent interactions with calmodulin (CaM). Some of the most important targets for CaM action are in the nucleus, and Ca2+-dependent CaM translocation into this organelle has been reported. Hormone-evoked cytosolic Ca2+ signals occur physiologically as oscillations, but, so far, oscillations in CaM concentration have not been described. Fluorescent-labeled CaM was loaded into pancreatic acinar cells and the fluorescence monitored in various regions by confocal microscopy. Sustained high concentrations of the hormone cholecystokinin or the neurotransmitter acetylcholine evoke a transient movement of cytosolic CaM from the basal nonnuclear area into the secretory granule region and, thereafter, a more substantial and prolonged translocation of CaM into the nucleoplasm. About 50% of the CaM that binds Ca2+ translocates into the nucleus. At a lower hormone concentration, evoking Ca2+ oscillations, regular spikes of increased CaM concentration are seen in the secretory granule region with mirror image spikes of decreased CaM concentration in the basal nonnuclear region. The nucleus is able to integrate the Ca2+ spike-evoked pulses of CaM translocation into a sustained elevation of the nucleoplasmic concentration of this protein (Craske, 1999).

Many targets of calcium signaling pathways are activated or inhibited by binding the Ca2+-liganded form of calmodulin (Ca²⁺-CaM). A test was performed of the hypothesis that local Ca²⁺-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca²⁺-CaM concentration.

Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca²⁺-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produce transient increases in free Ca²⁺-CaM concentration that are of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium leads to equalization of the nuclear and cytosolic free Ca²⁺-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM shows that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca²⁺-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca²⁺-CaM concentration gradient and of CaM translocation can be reversed by expressing a Ca²⁺-CaM-binding protein at high concentration in the cytosol. It is concluded that subcellular differences in the distribution of Ca²⁺-CaM-binding proteins can produce gradients of free Ca²⁺-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca²⁺-CaM concentrations, and thus the local activity of Ca²⁺-CaM targets. Free Ca²⁺-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca²⁺-CaM. From a regulatory perspective, this suggests that cells may control the amplitude of nuclear Ca²⁺-CaM signals by increasing or decreasing the concentration and composition of CaM-binding proteins in different cellular regions (Teruel, 2000).

Calmodulin is a core proteins of the yeast spindle pole body

The spindle pole body (SPB) is the microtubule organizing center of Saccharomyces cerevisiae. Its core includes the proteins Spc42, Spc110 (kendrin/pericentrin ortholog), calmodulin (Cmd1), Spc29, and Cnm67. Each was tagged with CFP and YFP and their proximity to one another was determined by fluorescence resonance energy transfer (FRET). FRET was measured by a new metric that accurately reflected the relative extent of energy transfer. The FRET values established the topology of the core proteins within the architecture of SPB. The N-termini of Spc42 and Spc29, and the C-termini of all the core proteins face the gap between the IL2 layer and the central plaque. Spc110 traverses the central plaque and Cnm67 spans the IL2 layer. Spc42 is a central component of the central plaque where its N-terminus is closely associated with the C-termini of Spc29, Cmd1, and Spc110. When the donor-acceptor pairs were ordered into five broad categories of increasing FRET, the ranking of the pairs specified a unique geometry for the positions of the core proteins, as shown by a mathematical proof. The geometry was integrated with prior cryoelectron tomography to create a model of the interwoven network of proteins within the central plaque. One prediction of the model, the dimerization of the calmodulin-binding domains of Spc110, was confirmed by in vitro analysis (Muller, 2005).

The spindle pole body is the microtubule organizing center of Saccharomyces cerevisiae (Jaspersen, 2004). Two SPBs establish the bipolar mitotic spindle, a defining event of mitosis that allows the stable transmission of equivalent genetic material to the mother and daughter cell at the time of cell division. This role of the SPB is carried out by the centrosome in higher eukaryotes (Muller, 2005).

The structure of the SPB is reviewed by Jaspersen (2004). Briefly the ultrastructure observed by electron tomography consists of a series of stacked layers embedded in the nuclear envelope. The inner plaque is the area where the microtubules dock to the SPB; this plaque harbors the gamma-tubulin complex and the N-terminus of Spc110. The central plaque and the IL2 layer are the two core layers. This core is composed of 5 proteins. Spc29 and Cmd1 reside in the central plaque. Spc42 is thought to begin within the central plaque, but terminate in the IL2 layer. The C-terminus of Spc110 is in the central plaque where it binds Cmd1. The C-terminus of Cnm67 lies in the IL2 layer where it binds Spc42 and links the SPB core to the outer plaque. The outer plaque is the cytoplasmic boundary of the SPB where the astral microtubules nucleate from a second region of gamma-tubulin. Based on primarily two-hybrid interactions the SPB core proteins are typically depicted as components lying along a linear path that proceeds from Spc110 to Spc29 to Spc42 to Cnm67 (Muller, 2005).

The ultrastructure of the SPB is clearly quite different from the centrosome. Centrioles are not present and the SPB remains inserted in the nuclear envelope during mitosis. Yet both have in common the gamma-tubulin complex, Spc110/kendrin/AKAP-450, calmodulin, centrin, and Sfi1p. (The latter two proteins are part of the SPB half-bridge, a domain involved in SPB duplication. Despite differences in gross anatomy, the SPB and centrosome likely share an underlying structure. To date the only component of either the SPB or centrosome whose structure is solved at atomic resolution is calmodulin. The paucity of structural information has limited the understanding of the molecular functions performed by individual SPB proteins. Without crystals or well behaved soluble proteins, the available research tools to probe the SPB structure or any large macromolecular complex are few (Muller, 2005).

This study used a hybrid approach that combined in vivo live-cell FRET measurements with previous cryo-EM analysis. CFP and YFP were used as FRET donor and acceptor and attached to the components of the SPB. Initially FRET values were classified as either positive or negative for energy transfer as judged by a comparison to carefully designed controls. This binary classification system allowed mapping of the ends of proteins within the architecture of the SPB. Next the positive values were subdivided into classes. The classification specified a unique geometry for the SPB components that was not only consistent with previous structural and genetic studies, but broadened the understanding of SPB organization (Muller, 2005).

The FRET results suggest that the Il2 layer and central plaque form an integrated meshwork of proteins with Spc42 closely associated with all components of the central plaque. The general features of the core proteins of the IL2 and central plaque, based on the current results and the general literature, are as follows. The N-terminus of Spc42 begins at the inner boundary of the central plaque, forms a coiled-coil domain that defines the spacing of the gap between layers, enters the IL2 layer, and finally loops back to end at the internal face of the IL2 layer. Remarkably, even though the N-terminal domain before the coiled coil is only ~60 amino acids long, the N-terminus is in close proximity to the C-termini of Spc29, Cmd1, and Spc110. Cnm67 begins at the outer plaque, penetrates the IL2 and ends in close proximity to the C-terminus of Spc42. The N- and C-termini of Spc29 both lie on the inner face of the central plaque. Cmd1 is situated near the C-terminal end of Spc110, consistent with in vitro binding experiments, genetic and two-hybrid results. Finally Spc110, which at its N-terminus binds the gamma-tubulin complex extends from the inner plaque through the central plaque and ends in close juxtaposition to the C-terminus of Spc42. All the termini of the central plaque and IL2 layer proteins lie along the internal edges of the IL2 and central plaque layers, facing the space between the two layers (Muller, 2005).

The SPB is organized around a hexagonal lattice of Spc42. The arrangement of Spc42 in the Il2 layer was suggested by analysis of cryoelectron micrographs of both SPB cores and two-dimensional crystals of Spc42 that arise in vivo upon Spc42 overexpression. Because the N-terminus of Spc42 is situated in the central plaque, the arrangement of Spc42 in the IL2 layer necessarily imposes the same organization on the location of Spc42 in the central plaque. Cryoelectron microscopy has not revealed this implied organization of the central plaque. However the visualization of the Spc42 arrangement in the IL2 relied upon the contrast between regions of high protein density and pockets of low or no density. If, as supported by the FRET results, the components of the central plaque are densely packed, a uniform and high protein density would mask the organization in electron micrographs (Muller, 2005).

The Spc42 lattice provided a template that enabled the FRET-based geometry of the core proteins to be taken and and a model to be generated for the organization of the central plaque. The model suggests that Spc42 and Spc29 form the heart of the central plaque. A strong association between Spc29 and Spc42 is well documented. Spc29 has a robust two-hybrid interaction with the N-terminus of Spc42. In an Spc110-226 mutant, Spc29 remains associated with Spc42 under the conditions in which Spc110-226, calmodulin, and the gamma-tubulin complex pull away from the SPB. Finally, Spc29 is seen with Spc42 at the satellite of the SPB. In this model Spc29 lies along the path of Spc42 and together they form a ring of protein around the center of the hexagonal unit in the central plaque (Muller, 2005).

At the center of the hexagonal unit is placed a trimer of Spc110 dimers as they unravel from their coiled coil motif. In the model Spc110 enters the central plaque through the ring of Spc29 and Spc42. Two-hybrid analysis suggested that Spc29 binds to Spc110 between the end of the coiled coil and the start of the Cmd1-binding domain, from positions 811 to 898. This region overlaps Region II of Spc110 (position 772-836), a domain that plays a role in locking Spc110 in place during mitosis. The FRET model is consistent with Spc29 and Spc42 acting as a clasp to surround and lock Spc110 in place. However the central plaque must not only lock Spc110 in place to withstand the push and pull of mitosis, but also must be organized in a way that facilitates the remodeling of the SPB during G1/S-phase when 50% of Spc110 turns over. Therefore any locking mechanism must be reversible and the interaction between Spc110 and Spc29 must be dynamic (Muller, 2005).

Calmodulin and the C-terminal domain of Spc110 are positioned to reinforce lateral stability of the central plaque. This is evident when the hexagonal unit is tessellated to form a mosaic lattice of the central plaque components. Calmodulin and the C-terminal domain of Spc110 from one hexagonal unit are juxtaposed with their counterparts in the adjoining hexagonal units. The dimerization of the C-terminal Spc110/Cmd1 domain was confirmed in vitro. Surprisingly even though calmodulin is a highly conserved component of the SPB, it is not required. An SPC110-407 mutant of S. cerevisiae that lacks the calmodulin-binding domain is still viable. One explanation is that the integrity of the SPB is maintained through structurally redundant lateral connections in IL2 layer and central plaque (Muller, 2005).

The tessellation of the repeat unit prompts the question of what determines the lateral limits of the SPB. How is the repeat symmetry broken and the boundary with the nuclear envelope established? One clue may come from a comparison of the dimensions of the SPB with the cluster of nuclear microtubules that originate at the SPB. The SPB is circular with an average diameter of ~165 nm for the central plaque from a diploid and therefore an area of ~2.1 x 10⁶Ų. A diploid would have ~35 microtubules emanating from the SPB (32 kinetochore microtubules and a three pole-to-pole microtubules. Microtubules have a cross-sectional diameter of 25 nm, so the minimal total area occupied by 35 microtubules (hexagonal packing with a packing density of 91% is 1.9 x 10⁶Ų. Even assuming some spread at the inner plaque, the SPB has almost the minimal area required to attach the nuclear microtubules. One mechanism that could minimize both the size of the SPB and the size of the bundle of microtubules would be feedback between microtubule attachment and Spc110 turnover. A removal of Spc110 molecules that are not nucleating microtubules would break the lattice symmetry, leaving Spc42 and Spc29 to interact with other proteins of the nuclear envelope. Spc110 is only added to the SPB after the insertion of Spc42 and Spc29 into the nuclear envelope, so the edge of the SPB does not require Spc110. The mechanism and role of Spc110 turnover is an area of continued research (Muller, 2005).

Calcineurin: a Calmodulin regulated phosphatase

Animals sense and adapt to variable environments by regulating appropriate sensory signal transduction pathways. Calcineurin (see Drosophila Calcinerin plays a key role in regulating the gain of sensory neuron responsiveness across multiple modalities. C. elegans animals bearing a loss-of-function mutation in TAX-6, a calcineurin A subunit, exhibit pleiotropic abnormalities, including many aberrant sensory behaviors. The tax-6 mutant defect in thermosensation is consistent with hyperactivation of the AFD thermosensory neurons. Conversely, constitutive activation of TAX-6 causes a behavioral phenotype consistent with inactivation of AFD neurons. In olfactory neurons, the impaired olfactory response of tax-6 mutants to an AWC-sensed odorant is caused by hyperadaptation, which is suppressible by a mutation causing defective olfactory adaptation. Taken together, these results suggest that stimulus-evoked calcium entry activates calcineurin, which in turn negatively regulates multiple aspects of sensory signaling (Kuhara, 2002).

Two types of cation channels, TAX-4/TAX-2 and OSM-9, are expressed in the AWC olfactory neurons. Genetic analyses suggest that odor sensing activates primary olfactory transduction through the TAX-4/TAX-2 channel, which allows calcium entry to activate AWC neurons, whereas odor-provoked calcium influx through the OSM-9 channel only affects olfactory adaptation. tax-6 animals are hyperadaptable to AWC-sensed isoamyl alcohol. Exposure to isoamyl alcohol for only 10 min is sufficient for tax-6 animals to adapt. This hyperadaptable phenotype and the partially defective olfactory response of tax-6 mutants to isoamyl alcohol are both completely suppressed by an osm-9 mutation. These results suggest that TAX-6 represses OSM-9-dependent olfactory adaptation in AWC. Taken together, two possible models are proposed for the role of TAX-6 in AWC signaling. TAX-6 could be activated by calcium entry through the primary signal transduction channel TAX-4/TAX-2 upon activation of the odorant (IAA) receptor, and the activated TAX-6 could inhibit the adaptation machinery. Alternatively, TAX-6 could be activated by the odorant (IAA)-evoked calcium influx through the OSM-9 channel, and the activated TAX-6 could negatively regulate opening of the OSM-9 channel that is required for isoamyl alcohol adaptation (Kuhara, 2002).

These models on the role of TAX-6 as a negative regulator for OSM-9-dependent olfactory adaptation in AWC might paradoxically imply that TAX-6 could be a positive regulator for TAX-4/TAX-2-dependent primary sensory signaling. If TAX-6 is a direct positive regulator of AWC primary transduction, at least partially defective olfactory responses to isoamyl alcohol could be expected in osm-9 tax-6 double mutants. It was found, however, that osm-9 tax-6 mutants show completely normal olfactory response to isoamyl alcohol. This result argues against a direct positive role of TAX-6 in AWC primary signaling. The results on osm-9 tax-6 mutants are also inconsistent with a direct negative regulatory role of TAX-6 in AWC primary signaling. If that were true, hyperattractive olfactory responses to isoamyl alcohol would be seen in osm-9 tax-6 mutants (Kuhara, 2002).

The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. Interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390. Selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bind 45Ca2+ normally. Mutants BE-2 and BQ-2 interact with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390); they stimulate the phosphatase activity of the CaN A subunit. Mutant BE-1 has a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gives very little stimulation of CaN A phosphatase activity. It is concluded that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects of B subunit Ca2+ binding in stimulation of CaN A phosphatase activity (Watanabe, 1996).

The PMC1 gene in S. cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work has shown that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. Calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter is slightly diminished and a pmc1::lacZ reporter is induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. It is proposed that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions (Cunningham, 1996).

S. cerevisiae mutants that exhibit phenotypes (calcium resistance and vanadate sensitivity) similar to those of calcineurin-deficient mutants were classified into four complementation groups (crv1,2,3 and 4). Crv1 is allelic to cnb1, a mutation in the regulatory subunit of calcineurin. The nucleotide sequences of mutant crv2 are identical to those of BCK1/SLK1/SKC1/SSP31 and the sequences of mutant crv3 match those of MPK1/SLT2; both genes are involved in the MAP kinase cascade. A calcineurin-deletion mutation (delta cnb1), which by itself has no detectable effect on growth and morphology, enhances some phenotypes (slow growth and morphological abnormality) of crv2 and crv3 mutants. The phenotypes of crv2 and crv3 mutants are partially suppressed by Ca2+ or by overproduction of the calcineurin subunits (Cmp2 and Cnb1). Like the calcineurin-deficient mutant, crv2 and crv3 mutants are defective in recovery from alpha-factor-induced growth arrest. The defect in recovery of the delta cnb1 mutant is suppressed by overexpression of MPK1. These results indicate that the calcineurin-mediated and the Mpk1- (Bck1-) mediated signaling pathways act in parallel to regulate functionally redundant cellular events important for growth (Nakamura, 1996).

The catalytic subunit of Ca2+/calmodulin(CaM)-dependent protein phosphatase (calcineurin A, protein phosphatase 2B) was isolated from Dictyostelium discoideum. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N-terminal and C-terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts. Recombinant Dictyostelium calcineurin A was purified from Escherichia coli cells and shows similar enzymatic properties as does the enzyme from other sources. A band of approximately 80 kDa migrates on gels and possesses an endogenous CaM-binding activity. Both the mRNA for calcineurin A and the protein are expressed during the growth phase. During early development the abundance of the protein is reduced and then increases to peak after 10 h of starvation, when tight aggregates have formed (Dammann, 1996).

Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. Soluble rat brain phosphatases have been identified that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity have been observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A. The two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each is relatively insensitive to okadaic acid, is active only in the presence of CaCl2 and calmodulin, and contains a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcineurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A, or with cyclosporin A to inhibit calcineurin, increases apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites. These results indicate that both phosphatase 2A and calcineurin dephosphorylate sodium channels in rat brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity (Chen, 1995).

The M current regulates neuronal excitability, with its amplitude resulting from high open probability modal M channel behavior. The M current is affected by changes in intracellular calcium levels. It is proposed that internal calcium acts by regulating M channel modal gating. Intracellular application of a preactivated form of the calcium-dependent phosphatase calcineurin (CaN420) inhibits the macroscopic M current, while its application to excised inside-out patches reduces high open probability M channel activity. Addition of ATP reverses the action of CaN420 on excised patches. The change in M channel gating induced by CaN420 is different from the effect of muscarine. A kinetic model supports the proposition that shifts in channel gating induced by calcium-dependent phosphorylation and dephosphorylation control M current amplitude (Marrion, 1996).

Embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) were prepared to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appears to be normal in CNA alpha -/- mice. CNA alpha -/- T cells respond normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts have a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remain sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. Thus CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA (B. W. Zhang, 1996).

The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocks the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression are inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation does not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) is not inhibited by in vivo treatment with FK506 and that there is no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. Different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes (Wang, C. R., 1995).

A new facet of calcium signaling involves the nuclear import of the NF-AT transcription factors from their dormant position in the cytoplasm. The protein phosphatase calcineurin appears to play an essential role in activating NF-AT nuclear import, as the calcineurin inhibitors cyclosporin A and FK506 block dephosphorylation and nuclear import of NF-AT. Calcium signaling induces an association between NF-AT4 and calcineurin; these molecules are transported, as a complex, to the nucleus, where calcineurin continues to dephosphorylate NF-AT4. It is proposed that a nuclear complex of NF-AT4 and calcineurin maintains calcium signaling by counteracting a vigorous nuclear NF-AT kinase (Shibasaki, 1996).

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. Two new isoforms of the transcription factor NFAT1 are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol ester; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response (Luo, 1996).

Slow- and fast-twitch myofibers of adult skeletal muscles express unique sets of muscle-specific genes, and these distinctive programs of gene expression are controlled by variations in motor neuron activity. It is well established that, as a consequence of more frequent neural stimulation, slow fibers maintain higher levels of intracellular free calcium than fast fibers, but the mechanisms by which calcium may function as a messenger linking nerve activity to changes in gene expression in skeletal muscle have been unknown. Here, fiber-type-specific gene expression in skeletal muscles is shown to be controlled by a signaling pathway that involves calcineurin, a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase. Activation of calcineurin in skeletal myocytes selectively up-regulates slow-fiber-specific gene promoters (Chin, 1998).

The myoglobin (Mb) and troponin I slow (TnIs) genes are expressed selectively in slow, oxidative skeletal muscle fibers, whereas the muscle creatine kinase (MCK) gene is expressed most abundantly in the fast, glycolytic myofiber subtype. To test whether these genes might respond differently to a calcineurin-stimulated signaling pathway, skeletal myogenic cells were transfected with reporter genes linked to well-characterized control regions from these genes, along with an expression vector encoding a constitutively active (calcium-insensitive) form of calcineurin that retains sensitivity to inhibition by cyclosporin A. Transcriptional activity of the slow-fiber-specific myoglobin and TnIs promoters is stimulated in cultured skeletal myotubes (C2C12) by active calcineurin, as measured by expression of luciferasein cotransfection assays. In contrast, activity of the fast-fiber-specific MCK promoter, or of other strong (CMV) or weak (minimal TATA element) promoters, is unaffected by activated calcineurin. The induction of the myoglobin promoter in the presence of the calcineurin expression plasmid is inhibited by cyclosporin A. This result indicates the specificity of the response, since the effect of cyclosporin A is to bind cyclophilin and form a protein complex that binds calcineurin and inhibits its protein phosphatase activity. The same relative potency of calcineurin-dependent transactivation (myoglobin and TnIs is much more potent than MCK, CMV, or TATA promoters) is observed in Sol8 myotubes, a different myogenic cell line. In contrast, forced expression of activated calcineurin had no effect on promoter activity in undifferentiated myoblasts or in 3T3 fibroblasts, demonstrating a requirement for muscle-specific factors in the calcineurin-stimulated pathway for transcriptional control of the myoglobin and TnIs promoters. Inhibition of calcineurin activity by administration of cyclosporin A to intact animals promotes slow-to-fast fiber transformation (Chin, 1998).

Transcriptional activation of slow-fiber-specific transcription appears to be mediated by a combinatorial mechanism involving proteins of the NFAT and MEF2 families. The finding that the myoglobin and TnIs promoters can be transcriptionally regulated by a calcineurin-dependent mechanism suggests the participation of NFAT transcription factors in the signaling cascade. Examination of the complete nucleotide sequences of these functionally defined transcriptional control regions (2.0 and 4.2 kb, respectively) reveals two 8-bp elements within each that match the consensus-binding sequence for NFAT transcription factors. The response to activated calcineurin of the native promoter sequences was compared to that of mutated promoters in which these putative NFAT recognition elements were ablated by site-directed mutagenesis. Disruption of putative NFAT recognition elements within both the myoglobin and TnIs promoters diminishes the response to activated calcineurin, indicating that the transactivation mechanism is likely to involve DNA binding of NFAT proteins. Transduction of the calcineurin-directed signal to the native myoglobin and TnIs promoters exhibits a saturable dose-response relationship with respect to the activated calcineurin expression plasmid; diminished reporter gene activation was evident across the entire dose range examined. Some degree of calcineurin-dependent transactivation persists after ablation of identifiable NFAT binding sites within these transcriptional control regions. Thus, either cryptic binding sites for NFAT proteins that cannot be recognized by inspection of the DNA sequence are present, or calcineurin-dependent signaling to these promoters can be driven without direct DNA binding of NFAT proteins. Nuclear localization of NFAT proteins in skeletal myocytes is under the control of calcineurin. These results identify a molecular mechanism by which different patterns of motor nerve activity promotes selective changes in gene expression to establish the specialized characteristics of slow and fast myofibers (Chin, 1998).

Using a genetic screen in yeast, a new family of proteins conserved in fungi and animals has been identified that inhibits calcineurin function when overexpressed. Overexpression of the yeast protein Rcn1p or the human homologs DSCR1 or ZAKI-4 inhibits two independent functions of calcineurin in yeast -- the activation of the transcription factor Tcn1p and the inhibition of the H+/Ca2+ exchanger Vcx1p. Purified recombinant Rcn1p and DSCR1 binds calcineurin in vitro and inhibits its protein phosphatase activity. Signaling via calmodulin, calcineurin, and Tcn1p induces Rcn1p expression, suggesting that Rcn1p operates as an endogenous feedback inhibitor of calcineurin. Surprisingly, rcn1 null mutants exhibit phenotypes similar to those of Rcn1p-overexpressing cells. This effect may be due to lower expression of calcineurin in rcn1 mutants during signaling conditions. Thus, Rcn1p levels may fine-tune calcineurin signaling in yeast. The structural and functional conservation between Rcn1p and DSCR1 suggests that the mammalian Rcn1p-related proteins, termed calcipressins, will modulate calcineurin signaling in humans and potentially contribute to disorders such as Down Syndrome (Kingsbury, 2000).

Whole-cell recording in the superficial layers of the developing superior colliculus (sSC) reveals a large drop in NMDA receptor (NMDAR) current decay time synchronized across all neurons and occurring consistently between post-natal (P) day 10 and P11. Blocking the Ca2+/calmodulin-dependent phosphatase calcineurin (CaN) in the postsynaptic neuron can abolish this drop. The regulation is induced prematurely by 1-2 hr of electrical stimulation in P10 collicular slices only if CaN and NMDAR currents can be activated in the neuron. These data suggest that a long-lasting, CaN-mediated control of NMDAR kinetics is rapidly initiated by heightened activity of the NMDAR itself and demonstrate a novel developmental and tonic function of CaN that can play an important role in modulating the plasticity of the developing CNS (Shi, 2000).

It is likely that this developmental regulation of the NMDAR serves functions that are qualitatively different from desensitization in more mature brain. For example, the CaN effect on NMDARCs described here is large. During the synaptogenic period from P6-P21, NMDARC decay time decreases by ~18 ms. Close to half of this decrease is caused by the sudden appearance of CaN activity at P11. By the end of the following week, the slower incorporation of the NR2A subunit into sSC synaptic NMDARs could be responsible for the additional shortening of the NMDARC decay time. Rat eyes open on P13-P14, and the sudden increase in visual activity resulting from pattern visual could readily damage collicular neurons if the mechanism of CaN-dependent NMDARC downregulation did not exist. In addition, the rapid downregulation of NMDARC decay time on P11 probably reduces much of the amplification of synaptic function caused by NMDAR activity. This loss of amplification may account for the sustained decreases in spontaneous spiking activity reported in the sSC after P10 by other investigators, and it may also serve to abruptly limit ongoing synaptic plasticity in the sSC. In short, this developmental function of CaN appears to be an exceptionally rapid and potent homeostatic mechanism that uses an increase in Ca2+ through the NMDAR channel to tonically decrease the potency of the NMDAR posttranslationally. This could maintain a nontoxic level of intracellular free Ca2+ in the face of a sudden increase in the activity arriving at collicular neurons. It is likely that in the visual system the increase in CaN activity results from the first powerful activation of the central visual pathway by light. It is also possible that similar sudden increases in activity occur in other regions of the nervous system. This activity-dependent, tonic, CaN-mediated control system may be broadly distributed within the vertebrate CNS (Shi, 2000).

A final significant property of the developmental change in CaN activity reported here is that it is not associated with changes in total CaN protein levels. The only event necessary to initiate phosphatase activity may therefore be the activation exerted by the NMDAR itself. Nevertheless, CaN is an elaborately regulated enzyme. Thus, changes in the activity or the synaptic localization of AKAP proteins, FKBP or cyclophorin family members, DARPP-32, CHP, or Cabin 1 may be mediating an additional level of control. Alternatively, or in addition, a prolonged depression of kinase activity could contribute to both the onset and stability of the CaN effect in sSC NMDARCs. The prolonged CaN activation at sSC synapses may also arise from a fundamentally different mechanism, such as protection of the phosphatases Fe-Zn active center from oxidation by superoxide dismutase. Regardless of the precise mechanism through which the prolongation of synaptic CaN activity is exerted, these developmental data suggest an interaction that may be retained in the mature brain. Prolonged activation of synaptic CaN could protect neurons from excitotoxicity in the face of seizure, ischemia, trauma, or disease-induced tonic increases in NMDAR activity. Thus, interventions that amplify or maintain this response may prove useful in the clinical treatment of a variety of neurological dysfunctions (Shi, 2000).

Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV). Skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occur despite the fact that CnA* muscles display threefold higher calcineurin activity and enhance dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events (Dunn, 2000).

Regarding the potential identity of contractile activity-dependent signal transduction events, there is mounting evidence that calcineurin must interact with parallel calcium-sensitive signaling pathways in order to fully activate downstream target genes. For instance, calcineurin synergizes with phorbol ester-dependent pathways to stimulate the IL-2 promoter in T lymphocytes and the expression of atrial natriuretic factor in cardiomyocytes. Similarly, calcineurin acts in conjunction with CaM-dependent kinase IV to fully activate the myoglobin promoter in cultured skeletal myocytes and the Nur77 promoter in T lymphocytes. Moreover, retroviral-mediated gene transfer of CnA* induces skeletal myogenesis in vitro only in the presence of extracellular Ca2+. Additionally, there is evidence that MAP kinase pathways are activated in response to increased contractile activity and play a role in regulation of the slow fiber phenotype. In this context, MEF2 is an enticing candidate as an integrator of calcineurin and other activation-linked signal transduction pathways, since this transcription factor is both dephosphorylated by calcineurin and phosphorylated by various CaM kinases, ERK5, p38, and PKC (Dunn, 2000 and references therein).

An alternative possibility is that calcineurin signaling may converge with other activity-linked pathways via the association of GATA with NFAT. Indeed, activation of calcineurin promotes the association of these two transcription factors via the dephosphorylation of NFATc1 and increased expression of GATA-2 under conditions of skeletal myocyte growth. Consistent with findings from hypertrophic myocytes, this protein is upregulated in the plantaris in response to muscle overload, but not lowered by CsA treatment, suggesting that this transcription factor may be important for growth but not necessarily a gene target of calcineurin. The fact that GATA is also known to associate with MEF2, and that fiber hypertrophy is observed only when NFATc1 and MEF2 are dephosphorylated and GATA-2 increases, leads to the idea that NFAT, MEF2, and GATA proteins act in synergy to transactivate target genes that lead to fiber growth in response to OV. Future studies should help identify the particular permutations of these transcription factors involved in the activation of slow fiber-specific genes versus those modulating adult fiber size (Dunn, 2000 and references therein).

The threshold for hippocampal-dependent synaptic plasticity and memory storage is thought to be determined by the balance between protein phosphorylation and dephosphorylation mediated by the kinase PKA and the phosphatase calcineurin. To establish whether endogenous calcineurin acts as an inhibitory constraint in this balance, the effect of genetically inhibiting calcineurin on plasticity and memory was examined. Using the doxycycline-dependent rtTA system to express a calcineurin inhibitor reversibly in the mouse brain, it has been found that the transient reduction of calcineurin activity facilitates LTP in vitro and in vivo. This facilitation is PKA dependent and persists over several days in vivo. It is accompanied by enhanced learning and strengthened short- and long-term memory in several hippocampal-dependent spatial and nonspatial tasks. The LTP and memory improvements are reversed fully by suppression of transgene expression. These results demonstrate that endogenous calcineurin constrains LTP and memory (Malleret, 2001).

The main finding of this study is that the regulated inhibition of the phosphatase CN leads to enhanced LTP both in vitro and in vivo, and to improved learning and memory storage. The parallel in time course of the increased persistence of LTP in awake animals and of the memory improvement strongly suggest a correlation between the duration of LTP and memory storage. Improved cognitive performance was observed both on spatial and nonspatial hippocampal-dependent tasks, consistent with the multipurpose role of the hippocampus in human declarative memory. Moreover, with different tasks, different temporal components of memory were improved. Thus, the complex object recognition task, involving brief training sessions, multiple objects, spatial transfer, and object change, elicit a weak form of memory that is strengthened at early and intermediate time points by the CN inhibitor, but does not persist longer in mutants than in controls. By contrast, a more robust form of memory elicited by a more intense training is maintained and persists for over a week longer in mutants expressing the CN inhibitor when compared to controls (Malleret, 2001).

Facilitated learning and memory was also observed in the Morris water maze and was evident not only with traditional measurements of spatial performance, such as escape latency, but also with more specific aspects of performance such as the precision of navigation. These measures suggest that mutant mice expressing the CN inhibitor retain spatial information more efficiently than controls. The persistent memory for the first platform position associated with the efficient learning of a second platform position suggests an overall enhanced capacity for memory storage with the CN inhibitor. Further, the rapid adaptation to spatial changes observed in mutants expressing the CN inhibitor on both the Morris water maze and the object exploration task suggest increased cognitive flexibility, a process that depends on the hippocampus (Malleret, 2001).

One of the molecular mechanisms allowing transmitted signals to persist or decay is thought to be the balance between phosphatase and kinase activity. Much evidence suggests that PKA and CN specifically regulate this balance and thereby serve as a gate for LTP. In the current study, evidence in support of this model is provided by demonstrating that shifting the endogenous balance away from calcineurin activity positively modulates synaptic plasticity in a PKA-dependent manner. Further, the data indicate that altering CN activity transiently in the adult brain is sufficient to positively or negatively control synaptic plasticity and memory storage. The effects observed suggest that CN is essential both for early events of plasticity and memory and for downstream pathways that contribute to persistent changes in plasticity and memory storage (Malleret, 2001).

Mechanistically, early and transient forms of plasticity and memory are known to rely on the covalent modification of pre-existing proteins while long-term forms require activation of transcription factors such as CREB, and protein synthesis. One possible mechanism for the facilitatory effect of the CN inhibitor may be a decrease in the activity of PP1, a protein phosphatase positively regulated by CN through dephosphorylation of inhibitor-1 (I-1). PP1 inhibition has been shown to promote the induction of LTP, whereas increased PP1 activity, produced by genetic suppression of I-1, has been shown to affect certain forms of LTP in some hippocampal regions. Since PP1 is effective in modulating CaMKII, a kinase critical for the transmission of postsynaptic signals required for the induction of LTP, it is possible that increased CaMKII activity mediated by lower PP1 activity facilitates the induction of LTP. Raising the signal for the induction of LTP, through genetic upregulation of NMDA-R function, has been shown to enhance LTP, learning, and memory. The findings suggest that LTP and memory enhancements can be similarly achieved by relieving a constraint downstream of the NMDA-R and that this constraint is exercised by CN (Malleret, 2001).

The effect of the CN inhibitor on long-lasting changes in plasticity and memory may be mediated by modulation of transcriptional control. Thus, the prolonged maintenance of LTP and of memory may arise from augmented CREB transcriptional activity via reduced CREB dephosphorylation by PP1. In this context, it is important to note that unlike the phenotypes observed in Drosophila mutants expressing active CREB, the CN inhibitor does not convert labile memory into long-lasting memory. It is, however, able to strengthen or prolong different phases of memory, suggesting that CN inhibition modulates rather than mediates memory processes (Malleret, 2001).

The PKA and CN pathways may also interact antagonistically at sites other than CREB. For example, CN can inhibit specific isoforms of adenylyl cyclase required for PKA activation. Similarly, PKA and CN can regulate, in opposite ways, phosphorylation sites on key proteins in synaptic transmission, such as the NMDA-R or AMPA receptor. The effect of inhibiting CN may also occur through processes additional to or independent of the cAMP pathway. For instance, CN inhibitor may modulate Ca2+-dependent kinases such as CaMKII or PKC through control of intracellular Ca2+ mobilization by regulation of inositol 1,4,5-triphosphate receptors. Finally, the PKA/CN gate most likely represents only one of several activator/suppressor mechanisms regulating plasticity and memory (Malleret, 2001).

Several genetic approaches have been used to study the molecular mechanisms of hippocampal functions such as memory. Standard genetic techniques, however, have suffered from the limitation that the genetic modification is permanent. Here, the usefulness of the rtTA system for such studies has been demonstrated by showing that inducible and reversible transgene expression allows temporary improvement of complex cognitive functions and of brain plasticity. The ability to achieve such reversible improvements in the adult animal demonstrates that no permanent changes in neuronal circuits are involved and that the effects result specifically from molecular and biochemical changes elicited by a reduction in CN activity. In this context, the rtTA system could be further exploited to assess the timing of the requirement of CN in such processes, for instance in various stages of memory storage such as memory consolidation or retrieval since CN has been suggested to be involved in both processes. Transgene expression was found in several brain structures and, therefore, the contribution of other structures, in addition to the hippocampus, to the enhancement of long-term memory, cannot be excluded. Overall, however, these results may provide a clear target for potential treatment of learning and memory disorders (Malleret, 2001).

Calmodulin and the myristoylated alanine-rich C kinase substrate (MARCKS)

The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin and actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly during development. To evaluate a possible developmental role for MARCKS, the gene was disrupted in mice by using the techniques of homologous recombination. Pups homozygous for the disrupted allele lacked detectable MARCKS mRNA and protein. All MARCKS-deficient pups died before or within a few hours of birth. Twenty-five percent had exencephaly and 19% had omphalocele (normal frequencies, < 1%), indicating high frequencies of midline defects, particularly in cranial neurulation. Nonexencephalic MARCKS-deficient pups had agenesis of the corpus callosum and other forebrain commissures, as well as failure of fusion of the cerebral hemispheres. All MARCKS-deficient pups also displayed characteristic lamination abnormalities of the cortex and retina. These studies suggest that MARCKS plays a vital role in the normal developmental processes of neurulation, hemisphere fusion, forebrain commissure formation, and formation of cortical and retinal laminations. It is concluded that MARCKS is necessary for normal mouse brain development and postnatal survival (Stumpo, 1995).

MARCKS and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C (PKC) substrates that also bind calmodulin regulated by PKC phosphorylation. The kinetics of PKC-mediated phosphorylation and the calmodulin binding properties of intact, recombinant MARCKS and MRP were investigated and compared with previous studies of synthetic peptides spanning the PKC phosphorylation site/calmodulin binding domains (PSCBD) of these proteins. Both MARCKS and MRP were high affinity substrates for the catalytic fragment of PKC, and their phosphorylation occurs with positive cooperativity. Affinities are similar to the values determined from studies of their respective PSCBD peptides. Two-dimensional mapping of MRP and its synthetic PSCBD peptide yield identical patterns of tryptic phosphopeptides; as in the case of MARCKS, all of the PKC phosphorylation sites in MRP lie within the 24-amino acid PSCBD. Sequence analysis of tryptic phosphopeptides reveals that the first and third (but not the second) serines in the MRP PSCBD are phosphorylated by PKC. Both MARCKS and MRP bind dansyl-calmodulin with high affinity, with a Kapp of 4.6 and 9.5 nM, respectively. Phosphorylation of MARCKS and MRP by PKC disrupt the protein-calmodulin complexes, with half-lives of 4.0 and 3.5 min, respectively. These studies suggest that intact, recombinant MARCKS and MRP are accurately modeled by their synthetic PSCBD peptides with respect to PKC phosphorylation kinetics and their phosphorylation-dependent calmodulin binding properties (Verghese, 1994).

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), have shown that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. The kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique is described in this study. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, kon = 10(11) M-1 s-1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, kon is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (koff) is ~10 s-1 (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca2+-calmodulin decreases markedly the lifetime of the peptide on vesicles. These results suggest that Ca2+-CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. It is thought that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4,5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175) (Arbuzova,1997).

Ionomycin stimulates membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator phorbol ester. However, while phorbol ester (as expected) powerfully stimulates the phosphorylation of the PKCs' 85-kDa MARCKS protein, ionomycin unexpectedly does not. Instead, ionomycin reduces the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevents phorbol-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin requires external Ca2+ because they are both abolished by removing Ca2+ from the culture medium. It is thought that Ca2+/calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes that have been stimulated by raising the external Ca2+ concentration. In the present experiments, calmodulin prevents MARCKS phosphorylation by phorbol-stimulated PKCs in glioma cell lysates, and this blockade is lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. Physiologically more significant is the fact that pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevents ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and phorbol-stimulated cells. The effect of ionomycin on MARCKS phosphorylation is not due to the stimulation of the Ca2+/calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, does not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent phorbol-stimulated PKCs from phosphorylating MARCKS depends on whether ionomycin was added before, with, or after phorbol treatment. Maximum blockade occurs when ionomycin is added before phorbol but is less effective when added with or after phorbol. These results indicate that Ca2+/calmodulin can profoundly affect PKCs' signaling at the substrate level (Chakravarthy, 1995).

Calmodulin interaction with miscellaneous proteins

Calmodulin is involved in receptor-mediated endocytosis in yeast. A temperature sensitive calmodulin mutant is completely blocked for alpha-factor internalization almost immediatly upon shift to the restrictive temperature. The uptake step of receptor-mediated endocytosis in yeast is dependent on the calcium binding protein calmodulin (Cmd1p). In order to understand the role that Cmd1p plays, a search was carried out for possible targets among the genes required for the internalization process. Co-immunoprecipitation, two-hybrid and overlay assays demonstrate that Cmd1p interacts with Myo5p, a type I unconventional myosin. Analysis of the endocytic phenotype and the Cmd1p-Myo5p interaction in thermosensitive cmd1 mutants indicates that the Cmd1p-Myo5p interaction is required for endocytosis in vivo. However, the Cmd1p-Myo5p interaction requirement is partially overcome by deleting the calmodulin binding sites (IQ motifs) from Myo5p, suggesting that these motifs inhibit Myo5p function, and implying that endocytosis involves alleviation of calmodulin inhibition of myosin function. Genetic and biochemical evidence obtained with a collection of cmd1 mutant alleles strongly suggests that Cmd1p plays an additional role in the internalization step of receptor-mediated endocytosis in yeast, involving additional calmodulin targets (Geli, 1998).

Functional connections have been identified between Calmodulin and the yeast actin cytoskeleton. cmd1A, one of four intragenic complementing groups of temperature-sensitive yeast calmodulin mutations, results in a characteristic functional defect in actin organization. Among the complementing mutations, a representative cmd1A mutation (cmd1-226: F92A) is synthetically lethal with a mutation in MYO2 that encodes a class V unconventional myosin with calmodulin-binding domains. Gel overlay assay shows that a mutant calmodulin with the F92A alteration has severely reduced binding affinity to a GST-Myo2p fusion protein. Random replacement and site-directed mutagenesis at position 92 of calmodulin indicate that hydrophobic and aromatic residues are allowed at this position, suggests the importance of a hydrophobic interaction between calmodulin and Myo2p. To analyze other components involved in actin organization through calmodulin, mutations were isolated and characterized that show synthetic lethal interaction with cmd1-226; these "cax" mutants fall into five complementation groups. Interestingly, all the mutations themselves affect actin organization. Unlike cax2, cax3, cax4, and cax5 mutations, cax1 shows allele-specific synthetic lethality with the cmd1A allele. CAX1 is identical to ANP1/GEM3/MCD2, which is involved in protein glycosylation. CAX4 is identical to the ORF YGR036c, and CAX5 is identical to MNN10/SLC2/BED1. Several possibilities can be envisioned for the Myo2p function in actin organization. Myo2p itself may be involved in localizing or moving the actin cytoskeleton toward the growing tip. Alternatively, cargoes in secretory vesicles driven by Myo2p may anchor or stabilize the actin cytoskeleton. Myo2p might be capable of cross-linking actin because of the predicted and demonstrated ability of class V myosins to dimerize. There is growing evidence suggesting that myosins regulate the actin network in yeast and other organisms. For example, loss of myosin I function in yeast results in defective actin organization. It has been suggested that 95F myosin (a class VI unconventional myosin) in Drosophila may be involved in the formation of actin furrows through the transport of cytoplasmic components (Sekiya-Kawasaki, 1998).

Activated forms of the GTPases, Rac (See Drosophila Rac) and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. IQGAP1 is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two approximately 190-kD subunits per molecule plus substoichiometric calmodulin. IQGAP1 contains four potential calmodulin-binding IQ domains and a region homologous to catalytic domains of GTPase-activating proteins, or GAPs. Purified IQGAP1 binds directly to F-actin and cross-links the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibits binding of IQGAP1 to F-actin, and is more effective in the absence of calcium than in its presence.. Colocalization of IQGAP1 with cortical microfilaments is cytochalasin-D sensitve. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin (Bashour, 1997).

The A kinase-anchoring protein AKAP79 coordinates the location of the cAMP-dependent protein kinase (protein kinase A), calcineurin, and protein kinase C (See Drosophila PKC) at the postsynaptic densities in neurons. Individual enzymes in the AKAP79 signaling complex are regulated by distinct second messenger signals; however, both PKC and calcineurin are inhibited when associated with the anchoring protein, suggesting that additional regulatory signals must be required to release active enzyme. This report focuses on the regulation of AKAP79-PKC interaction by calmodulin. AKAP79 binds calmodulin with high affinity in a Ca2+-dependent manner. Both proteins exhibit overlapping staining patterns in cultured hippocampal neurons. Calmodulin reverses the inhibition of PKCbetaII by the AKAP79(31-52) peptide and reduces inhibition by the full-length AKAP79 protein. The effect of calmodulin on inhibition of a constitutively active PKC fragment by the AKAP79(31-52) peptide is shown to be partially dependent on Ca2+. Ca2+/calmodulin reduces PKC coimmunoprecipitated with AKAP79 and results in a 2.6 increase in PKC activity in a preparation of postsynaptic densities. Collectively, these findings suggest that Ca2+/calmodulin competes with PKC for binding to AKAP79, releasing the inhibited kinase from its association with the anchoring protein (Faux, 1997).

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence has been investigated using three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). There is a lower affinity for caldesmon in all the mutants. When Ca2+ is replaced by Ba2+ the affinity of calmodulin for caldesmon is further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase is virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retain their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflectes their decreased affinity. It is concluded that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule (Huber, 1996).

Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. In intact cells, CGS9343B, a calmodulin antagonist, inhibits basal and Ca2+-stimulated phosphorylation of phosphatidylinositol. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways (Joyal, 1997).

G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC50 approximately 50 nM) > GRK6 >> GRK2 (IC50 approximately 2 &mgr;M) >> GRK1. Calmodulin inhibits GRK5 by caussing a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor, even in the absence of calmodulin. An amino-terminal domain of GRK5 (amino acids 20-39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues (characteristics typical of calmodulin binding sites), and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity (Pronin, 1997).

The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. SRY exhibits calcium-dependent binding to CaM. Binding occurs via the HMG box; an SRY peptide of residues 57-80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence, AACAAT, but not by a mutated sequence. Fluorescence spectra of the SRY/CaM complex indicate 1:1 stoichiometry and that binding is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM. It is proposed that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors (Harley, 1996).

In addition to the well-characterized GTP-dependent nuclear transport observed in permeabilized cells, a mode of nuclear transport occurs that is GTP-independent at elevated cytoplasmic calcium concentrations. Nuclear transport under these conditions is blocked by calmodulin inhibitors. Recombinant calmodulin restores ATP-dependent nuclear transport in the absence of cytosol. Calmodulin-dependent transport is inhibited by wheat germ agglutinin consistent with transport proceeding through nuclear pores. It is proposed that release of intracellular calcium stores upon cell activation inhibits GTP-dependent nuclear transport; the elevated cytosolic calcium then acts through calmodulin to stimulate the novel GTP-independent mode of import (Sweitzer, 1996).

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases (See Drosophila Nos). The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains, termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin. Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." Co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS, expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin (Michel, 1997).

The protection against apoptosis provided by growth factors in several cell lines is due to stimulation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, which results in activation of protein kinase B (PKB; also known as c-Akt) and phosphorylation and sequestration to protein 14-3-3 of the proapoptotic Bcl-2-family member BAD. A modest increase in intracellular Ca2+ concentration also promotes survival of some cultured neurons through a pathway that requires calmodulin but is independent of PI(3)K and the MAP kinases. Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) activates PKB directly, resulting in phosphorylation of BAD on serine residue 136 and the interaction of BAD with protein 14-3-3. Serum withdrawal induces a three- to fourfold increase in cell death of NG108 neuroblastoma cells, and this apoptosis is largely blocked by increasing the intracellular Ca2+ concentration with NMDA (N-methyl-D-aspartate) or KCl or by transfection with constitutively active CaM-KK. The effect of NMDA on cell survival is blocked by transfection with dominant-negative forms of CaM-KK or PKB. These results identify a Ca2+-triggered signaling cascade in which CaM-KK activates PKB, which in turn phosphorylates BAD and protects cells from apoptosis (Yano, 1998).

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin, with a Kd value of 44 nM, was detected. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 does not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. The region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. A pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses (Masuko, 1999).

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca2+-binding proteins that associate with components of the SNARE complex. Ca2+/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP_77-90), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP_77-90, but not mutant peptides, inhibits catecholamine release from chromaffin cells monitored by carbon fiber amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibits Ca2+-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppresses secretion. In the presence of TeNT, exocytosis is restored by transfection of TeNT-resistant (Q76V, F77W) VAMP, but additional targeted mutations in VAMP_77-90 abolishes its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca2+-dependent exocytosis, possibly to regulate SNARE complex assembly (Quetglas, 2002).

Myosin VI (Drosophila homolog: Jaguar) contains an inserted sequence that is unique among myosin superfamily members and has been suggested to be a determinant of the reverse directionality and unusual motility of the motor. It is thought that each head of a two-headed myosin VI molecule binds one calmodulin (CaM) by means of a single 'IQ motif'. Using truncations of the myosin VI protein and electrospray ionization(ESI)-MS, it has been demonstrated that in fact each myosin VI head binds two CaMs. One CaM binds to a conventional IQ motif either with or without calcium and likely plays a regulatory role when calcium binds to its N-terminal lobe. The second CaM binds to a unique insertion between the converter region and IQ motif. This unusual CaM-binding site normally binds CaM with four Ca2+ and can bind only if the C-terminal lobe of CaM is occupied by calcium. Regions of the MD outside of the insert peptide contribute to the Ca(2+)-CaM binding; truncations that eliminate elements of the MD alter CaM binding and allow calcium dissociation. It is suggested that the Ca(2+)-CaM bound to the unique insert represents a structural CaM, and not a calcium sensor or regulatory component of the motor. This structure is likely an integral part of the myosin VI 'converter' region and repositions the myosin VI 'lever arm' to allow reverse direction (minus-end) motility on actin (Bahloul, 2004).

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. CaM-binding protein, designated regulator of calmodulin signaling (RCS), has been identified. G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acts as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocks GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increases this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B (Rakhilin, 2004).

AlphaII-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of alphaII-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of alphaII-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, the crystal structure of a complex between bovine calmodulin and the calmodulin-binding domain of human alphaII-spectrin has been solved. The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes (Simonovic, 2006).

Interaction of Calmodulin with CaMKII

Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, mutant CaMs were designed that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the 'closed' Ca2+-free conformation. Their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation, were measured. CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. These results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways (Shifman, 2006).

Conservation of Ca(2+)/calmodulin regulation across Na and Ca(2+) channels

Voltage-gated Na and Ca(2+) channels comprise distinct ion channel superfamilies, yet the carboxy tails of these channels exhibit high homology, hinting at a long-shared and purposeful module. For different Ca(2+) channels, carboxyl-tail interactions with calmodulin do elaborate robust and similar forms of Ca(2+) regulation. However, Na channels have only shown subtler Ca(2+) modulation that differs among reports, challenging attempts at unified understanding. In this study, by rapid Ca(2+) photorelease onto Na channels, this view of Na channel regulation has been reset. For cardiac-muscle channels (NaV1.5), reported effects from which most mechanistic proposals derive, no Ca(2+) modulation was observed. Conversely, for skeletal-muscle channels (NaV1.4), fast Ca(2+) regulation was uncovered eerily similar to that of Ca(2+) channels. Channelopathic myotonia mutations halve NaV1.4 Ca(2+) regulation, and transplanting the NaV1.4 carboxy tail onto Ca(2+) channels recapitulates Ca(2+) regulation. Thus, this study argues for the persistence and physiological relevance of an ancient Ca(2+) regulatory module across Na and Ca(2+) channels (Ben-Johny, 2014).

Apocalmodulin itself promotes ion channel opening and Ca(2+) regulatio

The Ca(2+)-free form of calmodulin (apoCaM) often appears inert, modulating target molecules only upon conversion to its Ca(2+)-bound form. This schema has appeared to govern voltage-gated Ca(2+) channels, where apoCaM has been considered a dormant Ca(2+) sensor, associated with channels but awaiting the binding of Ca(2+) ions before inhibiting channel opening to provide vital feedback inhibition. Using single-molecule measurements of channels and chemical dimerization to elevate apoCaM, this study found that apoCaM binding on its own markedly upregulates opening, rivaling the strongest forms of modulation. Examining adult mammalian heart cells this study found that CaM-mediated feedback on cardiac L-type Ca2+ channels is the dominant control factor in controlling the cardiac and neuronal action potential duration, a vital excitability parameter whose prolongation drives heart failure and neuronal Ca2+ overload. Upon Ca(2+) binding to this CaM, inhibition may simply reverse the initial upregulation. As RNA-edited and -spliced channel variants show different affinities for apoCaM, the apoCaM-dependent control mechanisms may underlie the functional diversity of these variants and explain an elongation of neuronal action potentials by apoCaM. More broadly, voltage-gated Na channels adopt this same modulatory principle. ApoCaM thus imparts potent and pervasive ion-channel regulation (Adams, 2014).

Calmodulin and vacuole fusion and endocytosis

The basic reaction mechanisms for membrane fusion in the trafficking of intracellular membranes and in exocytosis are probably identical. But in contrast to regulated exocytosis, intracellular fusion reactions are referred to as 'constitutive', since no final Ca2+-dependent triggering step has been observed. Although transport from the endoplasmic reticulum to the Golgi apparatus in the cell depends on Ca2+, as does endosome fusion and assembly of the nuclear envelope, it is unclear whether Ca2+ triggers these events. Membrane fusion involves several subreactions: priming, tethering and docking. Proteins that are needed for fusion include p115, SNAPs, NSF, SNAREs and small GTPases, all of which operate in these early reactions, but the machinery that catalyses the final mixing of biological membranes is still unknown. Ca2+ is released from the vacuolar lumen following completion of the docking step. Calmodulin is identified as the putative Ca2+ sensor and as the first component required in the post-docking phase of vacuole fusion. Calmodulin binds tightly to vacuoles upon Ca2+ release. Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing. Hence, activation of SNAREs is not sufficient to drive bilayer mixing between physiological membranes. It is proposed that Ca2+ control of the latest phase of membrane fusion may be a conserved feature, relevant not only for exocytosis, but also for intracellular, 'constitutive' fusion reactions. However, the origin of the Ca2+ signal, its receptor and its mode of processing differ (Peters, 1998).

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin [5-hydroxytryptamine (5-HT)] 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation is attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation. This is because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src, but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase have no effect on 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis is required for 5-HT1A receptor-mediated Erk1/2 activation, a role for CAM in receptor endocytosis is postulated. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuates 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevents agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. It is concluded that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects CAM's role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2 (Della Rocca, 1999).

Calmodulin and ion channels

NMDA (N-methyl-D-aspartate) receptors are excitatory neurotransmitter receptors in the brain critical for synaptic plasticity and neuronal development. These receptors are Ca2+-permeable glutamate-gated ion channels whose physiological properties are regulated by intracellular Ca2+. Calmodulin interacts with the NR1 subunit of the NMDA receptor. Calmodulin binding to the NR1 subunit is Ca2+ dependent and occurs with homomeric NR1 complexes, heteromeric NR1/NR2 subunit complexes, and NMDA receptors from brain. Calmodulin binding to NR1 causes a 4-fold reduction in NMDA channel open probability. These results demonstrate that NMDA receptor function can be regulated by direct binding of calmodulin to the NR1 subunit, and suggest a possible mechanism for activity-dependent feedback inhibition and Ca2+-dependent inactivation of NMDA receptors (Ehlers, 1996).

Ca2+ influx through N-methyl-d-aspartate (NMDA) receptors activates signal transduction pathways critical for many forms of synaptic plasticity in the brain. NMDA receptor-mediated Ca2+ influx also downregulates the gating of NMDA channels through a process called Ca2+-dependent inactivation (CDI). Recent studies have demonstrated that the calcium binding protein calmodulin directly interacts with NMDA receptors, suggesting that calmodulin may play a role in CDI. The mutation of a specific calmodulin binding site in the C0 region of the NR1 subunit of the NMDA receptor blocks CDI. Intracellular infusion of a calmodulin inhibitory peptide markedly reduces CDI of both recombinant and neuronal NMDA receptors. This inactivating effect of calmodulin can be prevented by coexpressing a region of the cytoskeletal protein alpha-actinin2, known to interact with the C0 region of NR1. Taken together, these results demonstrate that the binding of Ca2+/calmodulin to NR1 mediates CDI of the NMDA receptor and suggest that inactivation occurs via Ca2+/calmodulin-dependent release of the receptor complex from the neuronal cytoskeleton (Zhang, 1998).

Plasma membrane calcium pump (PMCA) isoforms 4CII (generated by splicing at the C-terminus) and 4BICI (a pump version lacking the 10th transmembrane domain) were expressed in Sf9 cells using the baculovirus system. The purified PMCA4CII has a 20-fold lower affinity for calmodulin than the PMCA4CI, the PMCA4 isoform of the erythrocytes' membranes, but manifests a higher activity in the absence of calmodulin. The amount of phosphoenzyme intermediate formed by PMCA4CII in the presence of Ca2+ alone is almost 3 times higher than in PMCA4CI. The isoform lacking the 10th transmembrane domain (PMCA4BICI) has no Ca2+-dependent ATPase activity, but is still able to form the phosphoenzyme intermediate starting from phosphate. When expressed in COS cells, this isoform is retained in the endoplasmic reticulum; changes in membrane architecture apparently occur during its expression; the C-terminal portion of the isoform is located in the cytosol, indicating that the deletion of the 10th transmembrane domain results in the loss of at least another transmembrane domain (Preiano, 1996).

In primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca2+ and Na+ into the neuron. To maintain Na+ homeostasis, the excess Na+ entering through the ion channel should be removed by Na+,K(+)-ATPase. Incubation of primary cultured cerebellar neurons with glutamate results in activation of the Na+,K(+)-ATPase. The effect is rapid, peaking between 5 and 15 min (85% activation), and is maintained for at least 2 h. Glutamate-induced activation of Na+,K(+)-ATPase is dose dependent: it is appreciable (37%) at 0.1 microM and peaks (85%) at 100 microM. The increase in Na+,K(+)-ATPase activity by glutamate is prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase is reversed by phorbol ester, an activator of protein kinase C, indicating that activation of Na+,K(+)-ATPase is due to decreased phosphorylation by protein kinase C. Either W-7 or cyclosporin (both inhibitors of calcineurin) prevents the activation of Na+,K(+)-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na+,K(+)-ATPase that has been previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na+,K(+)-ATPase (Marcaida, 1996).

Neonatal rat sympathetic neurons developing in tissue culture contain 5 nicotinic acetylcholine receptor transcripts: alpha 3, alpha 5, alpha 7, beta 2, and beta 4. When examined in culture, neurons express four of these transcripts (alpha 3, alpha 5, beta 2, and beta 4) at levels similar to those in neurons developing in vivo: alpha 3 mRNA levels increase two- to threefold over the first week, whereas the levels for alpha 5, beta 2, and beta 4 remain essentially constant. In contrast, alpha 7 mRNA levels drop by 60-75% within the first 48 hr and remain low. During the first week, the ACh-evoked current densities on these cultured neurons increase twofold and correlate well with the increase in alpha 3 mRNA levels. Depolarizing the neurons with 40 mM KCl for 1-2 d upregulates the alpha 7 gene; this specific change in alpha 7 mRNA level correlates with an increase in alpha-bungarotoxin (alpha-BTX) binding on the surface of the neurons. Depolarization has little effect on the expression of the other four transcripts, or on either the magnitude or kinetics of the ACh-evoked currents. Furthermore, activators or inhibitors of protein kinase A (PKA), protein kinase C (PKC), or tyrosine kinase do not affect nAChR transcript levels in these cultured neurons. The effect of membrane depolarization on alpha 7 expression is a result of Ca2+ influx through L-type Ca2+ channels, and it has been shown that alpha 7 is upregulated through a Ca2+/calmodulin-dependent protein kinase (CaM kinase) pathway. The identification of CaM kinase as a link between activity and neurotransmitter receptor expression may indicate a novel mechanism that underlies some forms of synaptic plasticity (De Koninck, 1995).

Intracellular Ca2+ inhibits voltage-gated potassium channels of the ether a go-go (EAG) family. To identify the underlying molecular mechanism, the human version hEAG1 was expressed in Xenopus oocytes. The channels lose Ca2+ sensitivity when measured in cell-free membrane patches. However, Ca2+ sensitivity can be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca2+. Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrate direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section reveal a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca2+-activated potassium channels, Ca2+ dependent, with an apparent KD of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition (Schonherr, 2000).

Calmodulin and calcium channels

Calmodulin binding sites can be defined for skeletal, cardiac, and brain ryanodine receptor (RYR) Ca2+ channels. Cardiac and brain RYR peptides corresponding to the calmodulin binding sites present in the skeletal RYR have been synthesized, and their interactions with calmodulin have been monitored. The central portions of the skeletal, cardiac, and brain RYR protomers display two calmodulin binding sites, one with high affinity and one with low affinity. Depending on the RYR model having 4 or 12 transmembrane segments, a third calmodulin binding site (CaM3) has been identified a few residues upstream from the putative transmembrane segment M1 or M5. Its affinity for calmodulin varies between the RYR isoforms: the cardiac RYR CaM3 displays a high CaM affinity, while the skeletal and brain RYR CaM3 both have low affinity, the lowest affinity being displayed by the brain isoform. The RYRs calmodulin binding site CaM1 encompasses the sequence Arg-His-Arg-Val(Ile)-Ser-Leu (PM1 peptides), which is phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase. Phosphorylation of RYR PM1 peptides occurs on the Ser, corresponding to amino acid number 2919, 3020, and 3055 of the brain, cardiac, and skeletal RYR protomers, respectively. Phosphorylation of the RYR PM1 peptides is inhibited by calmodulin binding, and the formation of the PM1 peptide-calmodulin complex is inhibited by peptide phosphorylation. These data indicate that the effect of calmodulin binding to RYR CaM1 may be regulated by the phosphorylation state of the Ser residue localized within the sequence Arg-His-Arg-Val(Ile)-Ser-Leu (Guerrini, 1995).

Fertilization of oocytes incites numerous changes relying on Ca2+ signaling. In inseminated ascidian eggs, an increase in the egg surface membrane, monitored by a change in electrical capacitance, is recorded at the onset of meiosis resumption. This membrane addition to the cell surface is controlled by calcium release through a ryanodine receptor (RyR), sensitive to cyclic ADP-ribose. Using confocal microscopy analysis of ascidian oocytes immunostained with anti-RyR antibody, this calcium channel has been shown to be asymmetrically located in the vegetal cortical zone. Interestingly, the increase in cell capacitance occurring at fertilization is correlated with a fluorescent signal, imaged by the marker of vesicle trafficking FM 1-43, located close to the RyR region. Two putative partners of RyR, namely an FK-506 binding protein (FKBP) and a calmodulin, are identified in these oocyte extracts by detection of enzyme activity and PCR amplification. Both are necessary to sustain ryanodine receptor activity in these oocytes since the membrane insertion triggered by fertilization is inhibited by the FKBP ligand rapamycin and by a calmodulin antagonist peptide. These findings suggest that exocytosis in ascidian eggs is triggered at fertilization by a functional Ca2+ release unit operating as a complex of several proteins, including a calmodulin and an immunophilin, around the intracellular calcium channel itself (Albrieux, 2000).

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. In sea urchin eggs, Calmodulin acts as a functional mediator of cADPR-triggered CICR, through the RyRLC cADPR-induced Ca2+ release. This consists of two phases, an initial rapid release phase and a subsequent slower release. The second phase is selectively potentiated by calmodulin, which in turn, is activated by Ca2+ released during the initial phase. Caffeine enhances the action of calmodulin. Calmodulin does not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling (Tanaka, 1995).

The association of an endogenous, Ca(2+)-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) has been demonstrated. The activity of this thiol-protease is dependent on Ca2+ ion. These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type. This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356-1367 of the RyR. The cleavage site, the calmodulin binding site, is found at residues 1383-1400. The RyR cleavage by the Ca(2+)-dependent thiol-protease is prevented in the presence of ATP (1-5 mM) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca2+ efflux. These data suggest a possible involvement for this specific cleavage of the RyR/Ca2+ release channel in the control of calpain activity (Shoshan-Barmatz, 1994).

Cisternae vesicles from rabbit skeletal muscle were fused into planar bilayers and the effect of calmodulin on single Ca2+ release channel (ryanodine receptor) currents was investigated. In the presence of 10(-7) and 10(-9) M free [Ca2+], nanomolar concentrations of calmodulin activates the channel by increasing the open probability of single-channel events in a dose dependent manner. The activatory effect of calmodulin was reversed by 10 microM ruthenium red. At high, 10(-5) M, free Ca2+ ion, calmodulin inhibits channel activity. Calmodulin overlays have been carried out using concentrations of Ca2+ ion similar to those used for the planar lipid bilayer assays. In the presence of 10(-7) M Ca2+ ion, calmodulin binds to the ryanodine receptor, to a region defined by residues 2937-3225 and 3546-3655. These results suggest that calmodulin may activate the Ca(2+)-release channel (ryanodine-receptor) by interacting with binding sites localized in the central portion of the RYR protomer (Buratti, 1995).

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors (see Drosophila InsP3R) were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly binds to multiple sites on InsP3 receptors and Ca2+ increases the binding by 190% +/- 10%; the half-maximal effect occurs when the Ca2+ concentration is 184 +/- 14 nM. In the absence of Ca2+, calmodulin causes a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect is similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibita the Ca2+ release from cerebellar microsomes evoked by submaximal (but not by maximal) concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways (Patel, 1997).

Cyclic nucleotide-gated (CNG) channels form a family of ion channels that are gated open by cAMP and cGMP. In photoreceptors and olfactory neurons, these channels serve as final targets for cGMP- and cAMP-signaling pathways that are activated by light and odorants, respectively. A functionally significant feature of CNG channels is their Ca2+ permeability. At physiological extracellular Ca2+ concentrations, Ca2+ carries a substantial fraction of the total current passing through CNG channels. Ca2+ entry through CNG channels is crucially important for both excitation and adaptation of vertebrate photoreceptors and olfactory neurons as Ca2+ controls the activity of several signaling enzymes, including the CNG channels themselves. Ca2+/calmodulin (CaM) attenuates the activity of rod and olfactory CNG channels by increasing their apparent K1/2 for cGMP and cAMP. This modulation is believed to serve as one of several Ca2+-mediated feedback mechanisms that terminate the electrical response and set the sensitivity of photoreceptor cells and olfactory neurons. The mechanism of this modulation has been examined using electrophysiological and biochemical techniques. Heteromeric channels, consisting of alpha- and beta-subunits, display a high CaM sensitivity similar to the native channel. Using surface plasmon resonance spectroscopy, two unconventional CaM-binding sites (CaM1 and CaM2) were identified, one in each of the N- and the C-terminal regions of the beta-subunit. Ca2+ co-operatively stimulates binding of CaM to these sites exactly within the range of Ca2+ concentrations occurring during a light response. Deletion of the N-terminal CaM1 site results in channels that are no longer CaM-sensitive, whereas deletion of CaM2 has only minor effects (Weitz, 1998).

Ca2+-induced inhibition of alpha1C voltage-gated Ca2+ channels is a physiologically important regulatory mechanism that shortens the mean open time of these otherwise long-lasting high-voltage-activated channels. The mechanism of action of Ca2+ has been a matter of some controversy: previous studies have proposed the involvement of a putative Ca2+-binding EF hand in the C terminus of alpha1C and/or a sequence downstream from this EF-hand motif containing a putative calmodulin (CaM)-binding IQ motif. Using site directed mutagenesis it has been shown that disruption of the EF-hand motif does not remove Ca2+ inhibition. The IQ motif binds CaM and disruption of this binding activity prevents Ca2+ inhibition. It is proposed that Ca2+ entering through the voltage-gated pore binds to CaM and that the Ca/CaM complex is the mediator of Ca2+ inhibition (Qin, 1999).

Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium. Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels. A calcium-dependent interaction is reported between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels. In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current. These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain. These results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity (Lee, 1999).

L-type Ca2+ channels support Ca2+ entry into cells; this event triggers cardiac contraction, controls hormone secretion from endocrine cells and initiates transcriptional events that support learning and memory. These channels are examples of molecular signal-transduction units that self-regulate through their own activity. Among the many types of voltage-gated Ca2+ channels, L-type Ca2+ channels in particular display inactivation and facilitation, both of which are closely linked to the prior entry of Ca2+ ions. Both forms of autoregulation have a significant impact on the amount of Ca2+ that enters the cell during repetitive activity, with major consequences downstream. Despite extensive biophysical analysis, the molecular basis of autoregulation remains unclear, although a putative Ca2+-binding EF-hand motif and a nearby consensus calmodulin-binding isoleucine-glutamine ('IQ') motif in the carboxy terminus of the alpha1C channel subunit have been implicated. Calmodulin is a critical Ca2+ sensor for both inactivation and facilitation, and the nature of the modulatory effect has been shown to depend on residues within the IQ motif important for calmodulin binding. Replacement of the native isoleucine by alanine removes Ca2+-dependent inactivation and unmasks a strong facilitation; conversion of the same residue to glutamate eliminates both forms of autoregulation. These results indicate that the same calmodulin molecule may act as a Ca2+ sensor for both positive and negative modulation (Zuhlke, 1999).

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a 'dominant-negative' manner. This demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family (Peterson, 1999).

The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca2+ influx through postsynaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. CaMKII is shown to directly bind to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKIIalpha by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca2+ influx but also close to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP (Leonard, 1999).

Homologs of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca2+ entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP3 receptors (IP3Rs) for activation. Trp4 and other Trp proteins also interact with IP3Rs. The IP3R-binding domain also interacts with calmodulin (CaM) in a Ca2+-dependent manner with affinities ranging from 10 nM for Trp2 to 290 nM for Trp6. In addition, other binding sites for CaM and IP3Rs are present in the alpha but not the ß isoform of Trp4. In the presence of Ca2+, the Trp-IP3R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP3Rs inhibits the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 µM) but not a low (5 µM) concentration of the Trp-binding peptide of the IP3R. These data support the view that both CaM and IP3Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP3Rs differ for each Trp (Tang, 2001).

Mammalian homologs of Drosophila Trp form plasma membrane channels that mediate Ca2+ influx in response to activation of phospholipase C and internal Ca2+ store depletion. Human Trp3 is activated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and interacting domains, one on Trp and two on IP3R. Trp3 binds Ca2+-calmodulin (Ca2+/CaM) at a site that overlaps with the IP3R binding domain. Using patch-clamp recordings from inside-out patches, it has been shown that Trp3 has a high intrinsic activity that is suppressed by Ca2+/CaM under resting conditions. Trp3 is activated by the following: a Trp-binding peptide from IP3R that displaces CaM from Trp3, a myosin light chain kinase Ca2+/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca2+/CaM. It is concluded that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP3Rs (Zhang, 2001).

The mechanism involved in [Ca2+]i-dependent feedback inhibition of store-operated Ca2+ entry (SOCE) is not yet known. Expression of Ca2+-insensitive calmodulin (Mut-CaM) but not wild-type CaM increases SOCE and decreases its Ca2+-dependent inactivation. Expression of TrpC1 lacking C terminus aa 664-793 (TrpC1deltaC) also attenuates Ca2+-dependent inactivation of SOCE. CaM interacts with endogenous and expressed TrpC1 and with GST-TrpC1 C terminus but not with TrpC1deltaC. Two CaM binding domains, aa 715-749 and aa 758-793, were identified. Expression of TrpC1delta758-793 but not TrpC1delta715-749 mimics the effects of TrpCdelta1C and Mut-CaM on SOCE. These data demonstrate that CaM mediates Ca2+-dependent feedback inhibition of SOCE via binding to a domain in the C terminus of TrpC1. These findings reveal an integral role for TrpC1 in the regulation of SOCE. A key issue that remains to be resolved is whether TrpC1 itself forms the pore of SOCC (Singh, 2002).

Ryanodine receptor (RyR) activation by cyclic ADP-ribose (cADPR, a naturally occurring metabolite of NAD+) is followed by homologous desensitization. Though poorly understood, this 'switching off' process has provided a key experimental tool for determining the pathway through which cADPR mediates Ca2+ release. Moreover, desensitization is likely to play an important role in shaping the complexities of Ca2+ signaling involving cADPR, for example, localized release events and propagated waves. Using the sea urchin egg, a role has been unmasked for calmodulin, a component of the RyR complex and a key cofactor for cADPR activity, during RyR/cADPR desensitization. Recovery from desensitization in calmodulin-depleted purified endoplasmic reticulum (microsomes) is severely impaired compared to that in crude egg homogenates. An active, soluble factor, identified as calmodulin, is required to restore the capacity of microsomes to recover from desensitization. Calmodulin mediates recovery in a manner that tightly parallels its time course of association with the RyR. Conversely, direct measurement of calmodulin binding to microsomes reveals a loss of specific binding during cADPR, but not IP3, desensitization. These results support a mechanism in which cycles of calmodulin dissociation and reassociation to an endoplasmic reticulum protein, most likely the RyR itself, mediate RyR/cADPR desensitization and resensitization, respectively (Thomas, 2003).

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca²⁺ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. R- and N-type channels undergo Ca²⁺-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca²⁺ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels -- independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca²⁺ activity, respectively (Liang, 2003).

γCaMKII shuttles Ca(2+)/CaM to the nucleus to trigger CREB phosphorylation and gene expression

Activity-dependent CREB (see Drosophila CrebB) phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at voltage gated CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. This study reports a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin from the surface membrane to the nucleus. This study shows that the shuttle protein is γCaMKII (see Drosophila CaMKII), its phosphorylation at Thr287 by βCaMKII protects the Ca(2+)/CaM signal, and CaN (see Drosophila Calcineurin) triggers its nuclear translocation. Both betaCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII, and CaN in multiple neuropsychiatric disorders (Ma, 2014).

Neuroligins/LRRTMs prevent activity- and Ca2+/calmodulin-dependent synapse elimination in cultured neurons

Neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs) are postsynaptic cell adhesion molecules that bind to presynaptic neurexins. This paper shows that short hairpin ribonucleic acid-mediated knockdowns (KDs) of LRRTM1, LRRTM2, and/or NL-3, alone or together as double or triple KDs (TKDs) in cultured hippocampal neurons, did not decrease synapse numbers. In neurons cultured from NL-1 knockout mice, however, TKD of LRRTMs and NL-3 induced an approximately 40% loss of excitatory but not inhibitory synapses. Strikingly, synapse loss triggered by the LRRTM/NL deficiency was abrogated by chronic blockade of synaptic activity as well as by chronic inhibition of Ca(2+) influx or Ca(2+)/calmodulin (CaM) kinases. Furthermore, postsynaptic KD of CaM prevented synapse loss in a cell-autonomous manner, an effect that was reversed by CaM rescue. These results suggest that two neurexin ligands, LRRTMs and NLs, act redundantly to maintain excitatory synapses and that synapse elimination caused by the absence of NLs and LRRTMs is promoted by synaptic activity and mediated by a postsynaptic Ca(2+)/CaM-dependent signaling pathway (Ko, 2011).

Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel

Calmodulin (CaM) in complex with Ca²⁺ channels constitutes a prototype for Ca²⁺ sensors that are intimately colocalized with Ca²⁺ sources. The C-lobe of CaM senses local, large Ca²⁺ oscillations due to Ca²⁺ influx from the host channel, and the N-lobe senses global, albeit diminutive Ca²⁺ changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca²⁺ sensing has remained unknown. This paper advances a theory of how global selectivity arises, and this proposal was experimentally validated with methodologies enabling millisecond control of Ca²⁺ oscillations seen by the CaM/channel complex. It was found that global selectivity arises from rapid Ca²⁺ release from CaM combined with greater affinity of the channel for Ca²⁺-free versus Ca²⁺-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed in this study, promise generalization to numerous molecules residing near Ca²⁺ sources (Tadross, 2008).

The tight coupling of Ca²⁺ sensors to Ca²⁺ sources affords rapid and privileged signaling, but requires special sensing capabilities. In the CaM/Ca²⁺-channel complex, these capabilities are exemplified by the contrasting spatial Ca²⁺ selectivities of the two lobes of CaM. This study establishes that these selectivities are achieved not by sensing Ca²⁺ in different locations, but by decoding distinct temporal characteristics of nanodomain Ca²⁺. Local selectivity of the C-lobe arises from a slow CaM mechanism that exploits channel proximity and responds to intense yet intermittent Ca²⁺ signals. This local selectivity mechanism likely pertains to all forms of C-lobe regulation of Ca²⁺ channels, including the Ca²⁺-dependent facilitation (instead of Ca²⁺-dependent inactivation) of Ca_V2.1 channels. Conversely, global selectivity of the N-lobe disregards local signals and responds to weaker Ca²⁺ signals from distant sources. This vital capability arises from an “slow quick slow” (SQS) mechanism highly attuned to the fractional presence, but not intensity of signals. In the SQS mechanism it is postulated that in state 1, apoCaM is bound to the apoCaM site. In state 2, apoCaM is a transiently dissociated. In state 3, CaM binds two Ca2+ ions to become Ca2+/CaM, which can then bind the Ca2+/CaM effector site, yielding Ca2+-dependent inactivation (state 4). This mechanism also explains the switching of N-lobe selectivity between global (Ca_V2) and local (Ca_V1) extremes (Tadross, 2008).

Ca²⁺ decoding often entails multiple CaM/protein interactions whose function may not be apparent when taken out of context. Hence, successful mechanistic dissection may require experimental tools tailored to the intact signaling complex. This was achieved by voltage block of high-P_O channels, which affords millisecond control of nanodomain Ca²⁺ signals of intact channels. Voltage block may be adapted to other Ca²⁺-permeable channels, which may well use SQS-like mechanisms, given the prevalence of apoCaM interaction and rapid Ca²⁺ release from CaM. The technique could also extend to sensors that do not regulate ionic current, if the Ca²⁺-regulated molecule is engineered for optical readouts. For example, this approach could be applied to activity-dependent signaling that triggers nuclear transcription (Tadross, 2008).

Calmodulin and Selectin

Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation. This downregulation occurs through activation dependent proteolysis at a membrane-proximal site. Calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface. The effects of the calmodulin inhibitors on L-selectin expression and function can be prevented by cotreatment with a hydroxamic acid-based metalloprotease inhibitor. These results suggest a novel role for calmodulin in regulating the expression and function of an integral membrane protein through a protease-dependent mechanism. It is thought that calmodulin is already bound to the L-selectin cytoplasmic tail in the resting cell and that removal of calmodulin through cell activation results in L-selectin shedding. These findings may have broader implications for other cell surface proteins that also undergo regulated proteolysis (Kahn, 1998).

Calmodulin and neural cells

Deflection of the mechanically sensitive hair bundle atop a hair cell opens transduction channels, some of which subsequently reclose during a Ca2+-dependent adaptation process. Myosin I in the hair bundle is thought to mediate this adaptation; in the bullfrog's hair cell, the relevant isozyme may be the 119-kDa amphibian myosin I beta. Because this molecule resembles other forms of myosin I, it was hypothesized that calmodulin, a cytoplasmic receptor for Ca2+, regulates the ATPase activity of myosin. To investigate the possibility that calmodulin mediates Ca2+-dependent adaptation, calmodulin action was inhibited and the results were measured with two distinct assays. Calmodulin antagonists increase photolabeling of hair-bundle myosin I by nucleotides. In addition, when introduced into hair cells through recording electrodes, calmodulin antagonists abolish adaptation to sustained mechanical stimuli. This evidence indicates that calmodulin binds to and controls the activity of hair-bundle myosin I, the putative adaptation motor (Walker, 1996).

CA2+-regulated protein kinases play critical roles in long-term potentiation (LTP). To better understand the role of Ca2+/calmodulin (CaM) signaling pathways in synaptic transmission, Ca2+/CaM was injected into hippocampal CA1 neurons. Ca2+/CaM induces significant potentiation of excitatory synaptic responses, which is blocked by coinjection of a CaM-binding peptide and is not induced by injections of Ca2+ or CaM alone. Reciprocal experiments demonstrate that Ca2+/CaM-induced synaptic potentiation and tetanus-induced LTP occlude one another. Pseudosubstrate inhibitors or high-affinity substrates of CaMKII or PKC block Ca2/CaM-induced potentiation, indicating the requirement of CaMKII and PKC activities in synaptic potentiation. It is thought that postsynaptic levels of free Ca2+/CaM is a rate limiting factor and that functional cross-talk between Ca2+/CaM and PKC pathways occurs during the induction of LTP (Wang, J. H., 1995).

One form of Long-term depression (LTD) that has been observed in the hippocampus requires activation of postsynaptic NMDA (N-methyl-D-aspartate) receptors, a change in postsynaptic calcium concentration, and activation of postsynaptic serine/threonine protein phosphatase 1 (PP1) or 2A (PP2A). The mechanism by which PP1 or PP2A is regulated by synaptic activity is unclear because these protein phosphatases are not directly influenced by calcium concentration. LTD induction may require activation of a more complex protein phosphatase cascade consisting of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, its phosphoprotein substrate, inhibitor-1, and PP1. This hypothesis was tested using calcineurin inhibitors as well as different forms of inhibitor-1 loaded into postsynaptic cells. These results suggest a signaling pathway in which calcineurin dephosphorylates and inactivates inhibitor-1. This in turn increases PP1 activity and contributes to the generation of LTD (Mulkey, 1994).

Calmodulin and Munc13 form a Ca2+ sensor/effector complex that controls short-term synaptic plasticity

The efficacy of synaptic transmission between neurons can be altered transiently during neuronal network activity. This phenomenon of short-term plasticity is (1) a key determinant of network properties; (2) is involved in many physiological processes such as motor control, sound localization, or sensory adaptation, and (3) is critically dependent on cytosolic [Ca²⁺]. However, the underlying molecular mechanisms and the identity of the Ca²⁺ sensor/effector complexes involved are unclear. This study identifies a conserved calmodulin binding site in UNC-13/Munc13s, which are essential regulators of synaptic vesicle priming and synaptic efficacy. Ca2+ sensor/effector complexes consisting of calmodulin and Munc13s regulate synaptic vesicle priming and synaptic efficacy in response to a residual [Ca²⁺] signal and thus shape short-term plasticity characteristics during periods of sustained synaptic activity (Junge, 2004).

Neurons transfer information at chemical synapses. Interestingly, synaptic activity does not only transmit information but also regulates synaptic strength. Such activity-dependent modification of synaptic performance, or synaptic plasticity, is essential for information processing, learning, and memory (Junge, 2004).

Short-term synaptic plasticity (STP) occurs during and after repetitive synaptic activity on a timescale of milliseconds to minutes. It is a key determinant of network processes and is involved in brain functions as diverse as motor control, sensory adaptation, sound localization, and cortical gain control. STP can be expressed either as short-term enhancement (STE) or short-term depression (STD), depending on the initial release probability (P_r) of the synapses involved. High P_r is usually associated with STD, while a low P_r favors STE (Junge, 2004 and references therein).

Depletion of a readily releasable pool of fusion-competent synaptic vesicles (RRP) is a major cause for STD. The generation of this RRP is absolutely dependent on the priming action of UNC-13/Munc13s. The level of STD under steady-state conditions of RRP depletion and replenishment is controlled by a Ca²⁺-dependent vesicle supply process, of which the molecular mechanism and significance for STP are poorly understood. Calmodulin (CaM) may mediate this Ca²⁺-dependent process by acting on a subpool of the RRP with high P_r (Junge, 2004 and references therein).

A second well-known form of STP is STE. Three major forms of STE, facilitation, augmentation, and potentiation, can be distinguished based on their lifetime. During sustained activity, the efficacy of release is increased in STE, but it is unclear whether this is due to increased vesicular P_r or RRP size or both. STE is critically dependent on increased concentrations of residual Ca²⁺ ([Ca²⁺]_res), which accumulates during action potential activity due to incomplete elimination. According to the original residual Ca²⁺ hypothesis, [Ca²⁺]_res was thought to act on the secretory Ca²⁺ sensor. However, given the differences in Ca²⁺ requirements of fast neurotransmitter release and STE, additional, high-affinity Ca²⁺ sensors likely contribute to STE. The identification of such high-affinity Ca²⁺ sensors whose characteristics are compatible with the Ca²⁺ dynamics in presynaptic terminals and of molecules that transduce the residual Ca²⁺ signal to the secretory machinery during STE is essential for a mechanistic understanding of STE (Junge, 2004 and references therein).

The Munc13 proteins (Munc13-1, the splice isoforms bMunc13-2 and ubMunc13-2, and Munc13-3) are candidate mediators of STP. Genetic studies in mouse, fly, and nematode have established an essential role for this presynaptic protein family in synaptic vesicle priming and RRP generation. Munc13s regulate the SNARE protein Syntaxin and promote SNARE complex formation and fusion competence of synaptic vesicles (Junge, 2004 and references therein).

By determining synaptic vesicle priming, Munc13s modify synaptic strength. The domain structure of Munc13s with several binding sites for second messengers and regulatory proteins indicates that this function is tightly regulated. Indeed, Munc13s are targets of the diacylglycerol (DAG) second messenger pathway. The C₁ domain function of Munc13-1 is essential for DAG and phorbol ester (PE) binding and PE potentiation of synaptic amplitudes in hippocampal neurons. Moreover, rescue experiments in Munc13-1/2 double knockout (DKO) neurons show that STE is prevalent in neurons that express only ubMunc13-2, while moderate STD is prominent in neurons expressing only Munc13-1. Thus, Munc13 isoforms can differentially control STP, but the relation of this phenomenon to the long-established role of [Ca²⁺]_res in STP is unknown (Junge, 2004 and references therein).

This study reports that Munc13-1 and ubMunc13-2 bind CaM in a Ca²⁺-dependent manner via an evolutionarily conserved CaM recognition motif. Using synaptic depression, frequency facilitation, and augmentation protocols in autaptic hippocampal neurons (a special type of neuron that incorporates synaptical positive feedback through recurrent collaterals of its own axons) as a model of STP, it is shown that CaM binding to Munc13 proteins causes increased priming activity and RRP sizes. It is concluded that activation of the CaM/Munc13 complex by [Ca²⁺]_res represents a molecular correlate for the phenomenon of Ca²⁺-dependent vesicle pool refilling. This mechanism controls STP characteristics and is likely to be evolutionarily conserved (Junge, 2004).

Calmodulin, CaMKII, LTP and neural plasticity

Experience-dependent plasticity can be induced in the barrel cortex by removing all but one whisker, leading to potentiation of the neuronal response to the spared whisker. To determine whether this form of potentiation depends on synaptic plasticity, long-term potentiation (LTP) and sensory-evoked potentials were studied in the barrel cortex of alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII)T286A mutant mice. Three different forms of LTP induction were studied: theta-burst stimulation, spike pairing, and postsynaptic depolarization paired with low-frequency presynaptic stimulation. None of these protocols produced LTP in alphaCaMKIIT286A mutant mice, although all three were successful in wild-type mice. To study synaptic plasticity in vivo, measured sensory-evoked potentials were measured in the barrel cortex, and it was found that single-whisker experience selectively potentiates synaptic responses evoked by sensory stimulation of the spared whisker in wild types but not in alphaCaMKIIT286A mice. These results demonstrate that alphaCaMKII autophosphorylation is required for synaptic plasticity in the neocortex, whether induced by a variety of LTP induction paradigms or by manipulation of sensory experience, thereby strengthening the case that the two forms of plasticity are related (Hardingham, 2003).

Calmodulin, mitosis and meiosis

Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this 'S-M' checkpoint control is fundamental to the maintenance of genomic integrity. Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the 'cut' phenotype. In an attempt to identify conserved molecules involved in the S-M checkpoint, a regulatable murine cDNA library was screened in S. pombe and cDNAs have been identified that induce the cut phenotype in cells arrested in S phase by hydroxyurea. One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbs mitotic spindle functions, although spindle pole duplication is apparently normal. Both aspects of the phenotype induced by this cDNA product, termed Sha1 (for spindle and hydroxyurea checkpoint abnormal), are suppressed by simultaneous overexpression of calmodulin. Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp). These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes (Craig, 1998).

It has been suggested by many studies that Ca2+ signaling plays an important role in regulating key steps in cell division. In order to study the down stream components of calcium signaling, the gene of calmodulin (CaM) was fused with that of green fluorescent protein (GFP) and it was expressed in HeLa cells. The GFP-CaM protein has similar biochemical properties as the wild-type CaM, and its distribution is also similar to that of the endogenous CaM. Using this GFP-tagged CaM as a probe, a detailed examination of the spatial- and temporal-dependent redistribution of calmodulin was conducted in living mammalian cells during cell division. The major findings are: (1) high density of CaM is found to distribute in two sub-cellular locations during mitosis; one fraction is concentrated in the spindle poles, while the other is concentrated in the sub-membrane region around the cell. (2) The sub-membrane fraction of CaM becomes aggregated at the equatorial region where the cleavage furrow is about to form. The timing of this localized aggregation of CaM is closely associated with the onset of cytokinesis. (3) Using a TA-CaM probe, it was found that the sub-membrane fraction of CaM near the cleavage furrow is selectively activated during cell division. (4) When a CaM-specific inhibitory peptide is injected into early anaphase cells, cytokinesis is either blocked or severely delayed. These findings suggest that, in addition to Ca2+ ion, CaM may represent a second signal that can also play an active role in determining the positioning and timing of the cleavage furrow formation (Li, 1999).

Elevation of intracellular free calcium causes mouse egg activation by initiating a cascade of interacting signaling pathways that, in unison, act to remodel the cytoplasmic compartment and the nuclear compartment of the egg. Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is tightly associated with the meiotic spindle and 5 min after egg activation there is a transient, tight association of calmodulin (colocalized with CaM kinase II) on the meiotic spindle. These correlative observations led to testing whether activation of CaM kinase II mediates the chromosomal transit into an anaphase configuration. Calcium and calmodulin, at physiological levels, along with ATP are capable of driving the spindle (with its associated CaM kinase II) into an anaphase configuration in a permeabilized egg system. The transit into anaphase is dependent on the presence of both calcium and calmodulin and occurs normally when they are present at a ratio of 4 to 1. Peptide and pharmacologic inhibitors of CaM kinase II block the transit into anaphase, both in the permeabilized egg system and in living eggs (inhibitors of protein kinase C do not block the transit into anaphase). Using a biochemical approach it was confirmed that CaM kinase II increases in activity 5 min after egg activation and a second increase occurs 45 min after activation at the approximate time that the contractile ring of the second polar body is constricting. This corresponds to the approximate time when calmodulin and CaM kinase II colocalize at several points in the activated egg, including the region containing midzone microtubules. CaM kinase II appears localized on midzone microtubules as soon as they form and may have a role in specifying the position of the contractile ring of the second polar body (Johnson, 1999).

Eukaryotic chromosome segregation depends on the mitotic spindle apparatus, a bipolar array of microtubules nucleated from centrosomes. Centrosomal microtubule nucleation requires attachment of gamma-tubulin ring complexes to a salt-insoluble centrosomal core, but the factor(s) underlying this attachment remains unknown. In budding yeast, this attachment is provided by the coiled-coil protein Spc110p, which links the yeast gamma-tubulin complex to the core of the yeast centrosome. The large coiled-coil protein kendrin is a human ortholog of Spc110p. Kendrin was identified by its C-terminal calmodulin-binding site, which shares homology with the Spc110p calmodulin-binding site. Kendrin localizes specifically to centrosomes throughout the cell cycle. N-terminal regions of kendrin share significant sequence homology with pericentrin, a previously identified murine centrosome component known to interact with gamma-tubulin. In mitotic human breast carcinoma cells containing abundant centrosome-like structures, kendrin is found only at centrosomes associated with spindle microtubules (Flory, 2000).

Recently, a similar role has been suggested for the Drosophila melanogaster abnormal spindle protein (Asp). Asp, a centrosomal protein containing potential calmodulin-binding sites, appears to regulate the mitotic spindle apparatus by tethering gamma-TURCs together. Despite the similarities between Asp and kendrin, the functions of these two proteins are likely distinct. Kendrin and Asp share no homology with one another, whereas kendrin is clearly related to pericentrin, which interacts with gamma-tubulin. The predicted structure of kendrin, like that of Spc110p, contains long central coiled-coil domains flanked by noncoiled ends, whereas the secondary structure of Asp is predicted to be primarily gamma-helical with short stretches of coiled-coil near its C terminus. Additionally, Asp is predicted to contain an actin-binding domain, a feature found in neither kendrin nor Spc110p. The calmodulin-binding site of kendrin is similar to that of S. cerevisiae Spc110p and of the Spc110p homologs identified in A. nidulans and S. pombe, whereas the IQ-type calmodulin-binding site of Asp is more similar to those found in myosins. Finally, Asp localizes to both the centrosome and the spindle and was initially purified as a microtubule-associated protein, whereas kendrin is restricted to the centrosome, as is Spc110p. These differences indicate that the activities of kendrin may be more similar to those of Spc110p than to those of Asp. Further analysis of the functional relationships among kendrin, pericentrin, gamma-tubulin, and Asp will shed light on the mechanisms controlling the complex process of mitotic spindle formation and should aid in the understanding of centrosomal abnormalities that accompany cancerous growth (Flory, 2000 and references therein).

REFERENCES

Search PubMed for articles about Drosophila Calmodulin

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