InteractiveFly: GeneBrief

Arrestin 2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Drosophila visual transduction has served as a paradigm to characterize G protein-coupled neuronal signaling. As in mammals, light-activated rhodopsin is phosphorylated and interacts with a rhodopsin regulatory protein, arrestin, which facilitates deactivation of the receptor. However, unlike mammalian phototransduction, light activation in Drosophila is coupled to stimulation of phospholipase C rather than a cGMP-phosphodiesterase. The visual arrestin, undergoes light-dependent trafficking in mammalian and Drosophila photoreceptor cells, though the mechanisms underlying these movements are poorly understood. In Drosophila, the movement of the visual arrestin, Arr2, functions in long-term adaptation and is dependent on interaction with phosphoinositides (PIs). However, the basis for the requirement for PIs for light-dependent shuttling has been unclear. This study demonstrates that the dynamic trafficking of Arr2 into the phototransducing compartment, the rhabdomere, requires the eye-enriched myosin III, NINAC. Defects in ninaC result in a long-term adaptation phenotype similar to that which occurs in arr2 mutants. The interaction between Arr2 and NINAC is PI dependent and NINAC binds directly to PIs. These data demonstrate that the light-dependent translocation of Arr2 into the rhabdomeres requires PI-mediated interactions between Arr2 and the NINAC myosin III (Lee, 2004a).

The activity of signaling cascades can be profoundly affected by modulating the levels of regulatory proteins in specialized cellular compartments. The concentrations of such proteins can be altered through changes in protein synthesis or degradation. However, a faster mechanism for regulating the levels of a signaling protein involves protein trafficking in response to agonist stimulation. Dynamic movements of signaling proteins are of particular importance to neurons, such as photoreceptors, which are highly polarized and respond to their external stimulus, light, with great rapidity (Lee, 2004a).

During the last two decades, it has become clear that several key signaling proteins in mammalian and Drosophila photoreceptor cells undergo light-dependent translocations in and out of the phototransducing compartments, the outer segments, and rhabdomeres, respectively. These include the Drosophila TRPL cation channel (Bähner et al., 2002) and the Gα and Gβ subunits (Sokolov, 2002; Kosloff, 2003), which are concentrated in the phototransducing compartments only in dark-adapted animals. Upon exposure to light stimulation, these proteins shuttle into the cell bodies or inner segments over the course of several minutes. Visual arrestin, which participates in termination of the photoresponse, also undergoes dramatic light-dependent translocation, though the direction of movement is opposite to that of the TRPL and the Gα and Gβ subunits. In response to light stimulation, arrestin migrates into the outer segments/rhabdomeres of vertebrate and Drosophila photoreceptor cells (Broekhuyse, 1985; Philp, 1987; Whelan, 1988; Kiselev, 2000; Lee, 2004a and references therein).

The light-dependent translocations of signaling proteins in photoreceptor cells appear to function in long-term light adaptation (reviewed in Arshavsky 2003; Hardie 2003). Photoreceptor cells adapt to increasing intensities of background illumination by increasing the rate of termination of the photoresponse. The rate of termination of the light response is relatively slow in animals that are initially dark-adapted. Prior exposure to background illumination accelerates the rate of response termination. However, mutations that decrease the rate of translocation of the Drosophila visual arrestin from the cell body to the rhabdomere cause corresponding defects in this mode of adaptation (Lee, 2003; Lee, 2004a and references therein).

A critical question concerns the mechanisms underlying the light-dependent shuttling of signaling proteins, such as visual arrestin. Since mammalian rhodopsin undergoes light-dependent phosphorylation, which promotes binding to visual arrestin (reviewed in Arshavsky, 2002), it has been proposed that visual arrestin becomes concentrated in the outer segments due to binding to phosphorylated rhodopsin. However, it has been shown recently that translocation of visual arrestin into the outer segments occurs normally in rhodopsin kinase-deficient mice (Mendez, 2003; Zhang, 2003). It has been shown that the major Drosophila visual arrestin, Arr2, binds to phosphoinositides (PIs) and that this interaction is necessary for light-induced trafficking. However, the basis for this PI requirement and the identity of proteins that promote the light-dependent translocation of Arr2 or any other signaling protein have not been previously identified (Lee, 2004a).

It has now been shown that the NINAC myosin III, which consists of linked protein kinase and myosin head domains (Montell, 1988), is required for movement of Arr2 into the rhabdomeres. NINAC is expressed as two isoforms, p132 and p174, which are detected exclusively in the cell bodies and rhabdomeres, respectively (Porter, 1992). While p174 is required for response termination (Porter, 1992), no role for p132 in the photoresponse has been described. This study shows that p132 is the primary isoform necessary for light-dependent trafficking of Arr2 into the rhabdomeres. Moreover, flies that do not express p132 display a defect in long-term adaptation, consistent with its role in Arr2 shuttling. This study shows that the interaction between Arr2 and PIs is required for Arr2 to interact with NINAC, NINAC is also a PI binding protein, and that these interactions promoted the light-dependent movement of Arr2 into the rhabdomeres. These data indicate that the light-dependent shuttling of Arr2 into the rhabdomeres requires an association with the NINAC myosin III that is mediated through PIs (Lee, 2004a).

In contrast to the Lee (2004a) study, a study by Satoh (2005) found no requirement for NINAC in Arr1 or Arr2 translocation. The Satoh study concludes that the two Drosophila photoreceptor arrestins mediate distinct and essential cell pathways downstream of rhodopsin activation. The majority arrestin, Arr2, quenches rhodopsin signaling, while Arr1 promotes light-induced rhodopsin endocytosis. It is proposed that Arr1 mediates an endocytotic cell-survival activity, scavenging phosphorylated rhodopsin, thereby countering toxic Arr2/Rh1 accumulation; elimination of toxic Arr2/Rh1 in double mutants could thus rescue arr1 mutant photoreceptor degeneration (Satoh, 2005). Light-dependent subcellular translocation of Gqalpha in Drosophila photoreceptors has been shown to be facilitated by NINAC (Cronin, 2004).

Therefore, NINAC represents the first protein required for light-dependent movement of any signaling protein in vertebrates or invertebrates. In the ninaC null mutant (ninaC^P235), there was no detectable increase in rhabdomeral Arr2, even after a 1 hr exposure to light. Consistent with the requirement for NINAC for Arr2 translocation, long-term light adaptation is severely disrupted in the ninaC null mutant, ninaC^P235. This defect is more pronounced than that observed in mutant flies expressing a derivative of Arr2 (Arr2^3K/Q), which displays a large reduction in PI binding (Lee, 2003). The stronger light adaptation phenotype in ninaC^P235 than in arr2^3K/Q is consistent with the findings that the PI/Arr2 interaction and Arr2 translocation is reduced but not eliminated in arr2^3K/Q photoreceptor cells (Lee, 2004a).

The current results also address a long-standing question concerning the role for the cell body-enriched isoform of NINAC p132. NINAC p174 is enriched in the rhabdomeres and is required for termination of the phototransduction (Porter, 1992). However, a role for p132 in the photoresponse had not been described, which was not surprising given that p132 is detected exclusively in the cell bodies and phototransduction takes place in the rhabdomeres. Nevertheless, while both NINAC proteins participate in the light-dependent translocation of Arr2 into the rhabdomeres, it was found that p132 is the primary isoform required. This conclusion is further supported by the long-term adaptation defect in ninaC^Δ132 flies (Lee, 2004a).

Both NINAC isoforms consist of linked protein kinase and myosin domains, either of which might promote Arr2 shuttling. It is suggested that the myosin domain functions in the trafficking of Arr2, since a lysine to arginine mutation in the protein kinase domain, which eliminates enzymatic activity in other protein kinases, has no impact on either Arr2 translocation or long-term adaptation. The relatively rapid movement of Arr2 into the rhabdomeres, which occurs over the course of a few minutes, suggests that the myosin motor activity may promote the movement. Human photoreceptor cells express a NINAC homolog, Myo3A (Dosé, 2000), which moves toward the plus end of actin filaments (Komaba, 2003). As is typical of other microvillar structures, the plus end of the filaments is oriented near the distal tips of the microvilli. By analogy to Myo3A, Drosophila NINAC also is likely to be a plus-ended myosin, which could potentially shuttle cargo from the cell bodies into the rhabdomeres. However, it was not possible to demonstrate that the NINAC motor activity per se was required for movement of Arr2; point mutations in conserved residues required for motor activity of other myosins, such as those in the actin and ATP binding sites, cause instability of NINAC in vivo (Lee, 2004a).

Translocation of Arr2 has been shown to require direct interaction with PIs (Lee, 2003); however, the basis of this requirement has been unclear. In the current work, the association of Arr2 and NINAC was shown to be dependent on PIs and NINAC was shown to bind PIs. In support of this conclusion, it was found that the Arr2/NINAC interaction is disrupted by detergent and further augmented by the addition of exogenous PIs. Furthermore, NINAC binds to PIs in vitro. The interaction between NINAC and Arr2 is dependent on PIs and not on calmodulin. Calmodulin could not serve as a link between NINAC and Arr2; it was shown that Arr2 does not bind calmodulin. Although the NINAC proteins used for the biochemistry were characterized as partially purified, p132 and p174 were the only proteins clearly seen on a Coomassie-stained gel. Therefore, all other proteins were present at substoichiometric concentrations and would therefore not be effective linkers between NINAC and Arr2. In addition, p132 has only one calmodulin binding site. Consequently, the association of Arr2 to p132 immobilized on calmodulin-agarose could not have occurred through a second calmodulin bound to p132 (Lee, 2004a).

It is proposed that p132 binds to PI-containing vesicles, which bind simultaneously to Arr2, facilitating the trafficking of Arr2 into the rhabdomeres. Interestingly, there is an age-dependent accumulation of vesicles in the cell bodies of ninaC^Δ132 (Hicks, 1996), which may result from the absence of p132-dependent vesicular movement into the rhabdomeres of these mutant flies. Although the null allele, ninaC^P235, displays slow termination of the light response and retinal degeneration, which could indirectly affect Arr2 movement, ninaC^Δ132 flies do not undergo retinal degeneration or exhibit defects in activation or termination of the photoresponse (Porter, 1992). While it cannot be excluded that changes in calmodulin distribution associated with elimination of p132 (Porter, 1992) contribute to the defect in Arr2 translocation, several observations support the conclusion that p132 functions directly in light-dependent movement of Arr2. These include the findings that p132 associates with Arr2, the demonstration that this association is dependent on PIs, and the previous report that the Arr2-PI interaction is required for translocation (Lee, 2003; Lee, 2004a).

A remaining question concerns the mechanism underlying the reciprocal translocation of Arr2 from the rhabdomeres to the cell bodies in the dark. While p132 is required for the rapid light-dependent shuttling of Arr2 into the rhabdomeres, it was dispensable for the dark-associated movement of Arr2 out of the rhabdomeres. One possibility is that the transfer of Arr2 from the rhabdomeres to the cell bodies may occur via myosin VI, which is a minus end-directed motor. However, photoreceptor cell-specific expression of a myosin VI antisense transgene, which has been shown to suppress myosin VI activity in vivo, has no impact on Arr2 shuttling back to the cell bodies. Alternatively, the retrograde movement of Arr2 out of the rhabdomeres may occur by diffusion, since this movement occurs on the order of several hours, while the NINAC-dependent trafficking into the rhabdomeres is essentially complete in 10 min (Lee, 2004a).

The current study raises the question as to whether light-stimulated translocation of vertebrate visual arrestin into the outer segment of photoreceptor cells occurs through interaction with a myosin or kinesin and, if so, whether the association is PI dependent. Mammalian visual arrestin binds to inositol phosphates (Palczewski, 1991) and associates with vesicle-like structures in the inner segments (McGinnis, 2002). Kinesin-II is one candidate that could potentially participate in light-stimulated trafficking, because both visual arrestin and opsin accumulate in the inner segments in KIF3A knockout mice (Marszalek, 2000). However, it is not known if the kinesin-II participates in light-induced translocation of visual arrestin or whether the observed effect on arrestin localization is due to retinal degeneration. Unlike kinesin-II-deficient photoreceptor cells, absence of NINAC p132 in Drosophila does not lead to retinal degeneration. It is intriguing to speculate that the vertebrate Myo3A or Myo3B might function in a manner analogous to NINAC. Consistent with this possibility are the observations that the cilium connecting the inner and outer segments contains actin, in addition to tubulin. Moreover, Myo3B is expressed in the retina, and Myo3A is enriched in photoreceptor cells in addition to the cochlea. Recently, one form of nonsyndromic deafness has been attributed to mutations in human MYO3A. Whether these individuals also have a defect in long-term light adaptation is an open question, which remains to be addressed (Lee, 2004a and references).

Gαq splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in Drosophila

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein-coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq¹ isoform protein playing a major role in fly phototransduction. However, Gαq¹ null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. This study isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, it was found that mutations in the Gαq gene disrupt light responses, and the Gαq3 isoform protein was demonstrated to be responsible for the residual light response in Gαq¹ null mutants. Moreover, this study reports that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation of stable Rh1-arrestin 2 (Arr2) complexes. These findings clarify essential roles for several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis (Gu, 2020).

Heterotrimeric G proteins and G protein-coupled receptors play pivotal roles in mediating a variety of extracellular signals to intracellular signaling pathways, such as hormones, neurotransmitters, peptides, and sensory stimuli. In the Drosophila visual system, light stimulation activates the major rhodopsin (Rh1) to form metarhodopsin, which in turn activates heterotrimeric G proteins and norpA gene-encoded phospholipase C (PLCβ). Activated PLC catalyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). IP3 induces the release of Ca2+ from intracellular Ca2+ stores, whereas both DAG and IP3 may trigger extracellular Ca2+ influx by opening transient receptor potential (Trp) and transient receptor potential-like (TrpL) channels on the cell membrane. The Gαq gene encodes several Gαq splice variants, among which the Gαq-RD variant generates Gαq¹ isoform protein, and other splice variants generate Gαq3 isoform protein. Although both strong alleles of norpA and trpl;trp double mutants show completely abolished photoresponses, the Gαq¹ null mutant allele (Gαq⁹⁶¹) still displays a residual light response. These data indicate that other Gαq splice variants, or the Gq α subunits encoded by additional genes, contribute to the residual light responses in Gαq¹ null mutants (Gu, 2020).

Intracellular Ca2+ homeostasis controlled by Gq signaling is also essential for photoreceptor cell survival. Mutations in phototransduction cascade components, such as those in trp and norpA, prevent normal light-induced Ca2+ influx, resulting in stable Rh1/Arr2 complex formation and severe rapid light-dependent retinal degeneration. Disruption of stable Rh1/Arr2 complexes by genetic removal of Arr2 or suppression of Rh1 endocytosis can suppress the retinal degeneration either in norpA or trp mutant flies. Rh1/Arr2 complex formation is thought to contribute to impaired Ca2+ influx-activated CaM kinase II, which usually phosphorylates Arr2 to release Arr2 from Rh1. However, neither Gαq¹ nor Gαq⁹⁶¹ mutants undergo rapid retinal degeneration, exhibiting only slight retinal degeneration after keeping them in 12-h light/12-h dark cycles for 21 days. The disparate retinal degeneration phenotype between Gαq and norpA mutant is therefore unclear (Gu, 2020).

This study isolated a mutant fly with no detectable light responses and revealed that mutations in the Gαq gene cause the defective light responses. Gαq3 is responsible for the residual light response in Gαq¹ null mutants, and depletion of all Gαq splice variants results in rapid light-dependent retinal degeneration due to formation of stable Rh1/Arr2 complexes. In addition, this study revealed that Gαq3 plays essential roles in Rh1 synthesis. This study clarifies the essential role of different Gαq splice variants in fly phototransduction, retinal degeneration, and rhodopsin synthesis (Gu, 2020).

In Drosophila photoreceptors, G proteins are essential to activate the phototransduction cascade. The Gαq gene encodes several Gαq splice variants, and Gαq¹ has been shown to function as the predominant G protein in fly phototransduction. This study identified a mutation (5501T/A) in the Gαq gene, which specifically mutates Val to Asp at residue 303 in Gαq¹ but not Gαq3 isoforms. Although Val is replaced with Ile at residue 303 in vertebrate Gαq proteins, the hydrophobicity at this position is evolutionally conserved. Structural analyses have shown that the V303 region localizes to the interface between Gα proteins and its downstream effector PLC. The change of a hydrophobic residue to a polar one may affect the interaction between these two proteins. A recent study has shown that Gαq^V303D mutant protein is unable to activate PLC in vivo (Gu, 2020).

Although the 5501T/A Gαq gene mutation largely contributes to abolished light responses, this mutation is not fully responsible for the abolished light responses in no detectable light response (nlr) mutants because both nlr/Gαq¹ and nlr/Gαq⁹⁶¹ flies still exhibited a residual light response similar to Gαq¹ and Gαq⁹⁶¹ mutants. These data also excluded the possibility that Gαq^V303D mutant protein dominantly suppresses the function of Gαq protein. Gαq¹ expression in nlr mutants largely recovers the light response, further excluding the possibility that abolished light responses in nlr mutants are due to the dominant suppression of Gαq^V303D mutant protein (Gu, 2020).

The Gαq gene encodes several Gαq splice variants, and Gαq^221c mutants disrupt the expression of all Gαq splice variants (21). An ERG recording revealed that Gαq^221c null mutant clones showed no light responses. Previous whole-cell voltage-clamp recordings showed that the photoresponse of Gαq¹ homozygous cells is larger than that of Gαq¹ heterozygous cells. These results indicate that other Gαq splice variants might contribute to the residual light response in Gαq¹ null mutants. This study demonstrates that Gαq3 contributes to the residual light response in Gαq¹ null mutants (Gu, 2020).

The Gαq gene encodes several Gαq splice variants. Originally, two cDNAs resulting from different Gαq gene splicing were isolated. These two cDNAs encode Gαq1 and Gαq2 isoform proteins, respectively. Functional studies demonstrated that Gαq1 mediates the light response, whereas Gαq2 has no effect on phototransduction. Subsequently, two additional Gαq splice variants were isolated. To date, seven total Gαq splice variants have been annotated in Flybase, and these splice variants encode three different isoform proteins, including Gαq1, Gαq3, and Gαq4. This study has demonstrated that Gαq3 also mediates phototransduction. Overexpression of Gαq3 in nlr mutants induced detectable light responses but failed to fully restore the light response. Interestingly, the rescue flies exhibited comparable ERG trace amplitude and dynamics as those of Gαq¹ and Gαq⁹⁶¹ flies. These results indicate that different Gαq isoform proteins play different roles in phototransduction. Gαq mediates retinal degeneration (Gu, 2020).

Mutations in most genes encoding components of the phototransduction cascade result in rapid retinal degeneration, except for Gαq hypomorphic allele Gαq¹ and Gαq¹ isoform null mutant allele Gαq⁹⁶¹. Previous studies have shown that both Gαq¹ and Gαq⁹⁶¹ mutants undergo slow light-dependent retinal degeneration due to slow accumulation of stable Rh1/Arr2 complexes. In these Gαq mutants, the small residual photoresponse may reduce Ca2+ influx, which partially activates CaM kinase II and leads to the slow release of Arr2 from Rh1. This study shows that nlr mutants undergo rapid light-dependent retinal degeneration similar to that observed in norpA mutants. Disruption of stable Rh1/Arr2 complexes formation prevented retinal degeneration in the mutants. Under normal conditions, the interaction between Arr2 and Rh1 is transient, because light-triggered Ca2+ influx may activate CaM kinase II, which subsequently phosphorylates Arr2 to release Arr2 from Rh1. In nlr mutants, photoresponses were completely abolished so that the normal rise in Ca2+ after light stimulation was blocked, causing stable Rh1/Arr2 complex formation and retinal degeneration. These observations and explanations are consistent with mutations such as trp and norpA (Gu, 2020).

This study has shown the first evidence that Gαq3 regulates Rh1 synthesis. Rh1 is transported to the plasma membrane by vesicular transport mechanisms regulated by a large number of trafficking proteins. Previous studies have shown that Gαq homologue CG30054 regulates inositol 1,4,5,-tris-phosphate receptor (IP3R) to mediate calcium mobilization from intracellular stores and promote calcium-regulated secretory vesicle exocytosis. Given that Gαq3 shows high sequence identity to CG30054, they may regulate Rh1 synthesis through promoting calcium-regulated secretory vesicle exocytosis (Gu, 2020).

REGULATION

Ca²⁺ influx acting via CaM and NINAC accelerates the binding of arrestin to metarhodoposin

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, 2004a). 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).

Protein Interactions

Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell

Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to metarhodopsin, the active form of rhodopsin that stimulates signaling via G-proteins. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. In contrast, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin (Arr2) further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the RdgC phosphatase. It is proposed that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells (Byk, 1993)

Arrestin binding determines the rate of inactivation of the G protein-coupled receptor rhodopsin in vivo

G protein-coupled receptor inactivation is a crucial feature of cellular signaling systems; this process determines the catalytic lifetime of the activated receptor and is necessary for response termination. Although previous work has indicated a class of models in which several sequential steps are required for receptor inactivation, the rate-limiting event is still unclear. In this paper, a theory is developed that describes the kinetics of inactivation of the G protein-coupled receptor rhodopsin based on the rate of arrestin binding and the theory is tested using a combination of genetic and electrophysiological techniques in Drosophila photoreceptors. The theory quantitatively describes the inactivation kinetics of activated rhodopsin in vivo and can be independently tested with molecular and spectroscopic data. The results demonstrate that the rate of arrestin binding determines the kinetics of receptor inactivation in vivo and thus is the event that controls signal amplification at the first step of this G protein-coupled transduction cascade (Ranganathan, 1995).

Two arrestin genes, arrl and arr2, have been isolated in Drosophila (Smith, 1990; Hyde, 1990; LeVine, 1990; Yamada, 1990), both of which are expressed in all photoreceptors (R1-R8) in the eye (Dolph, 1993) and whose protein products share extensive amino acid identity (51%). These arrestins have at least partially redundant functions in phototransduction (Dolph, 1993), although the Arr2 protein is severalfold more abundant than art1 (LeVine, 1990; Matsumoto, 1991). In Drosophila, as in most invertebrates, the inactive arrestin-bound metarhodopsin species is a stable complex in the dark, but can absorb another photon to isomerize to phosphorylated rhodopsin, thereby releasing arrestin (Byk, 1993). Phosphorylated rhodopsin is subsequently dephosphorylated to regenerate functional receptor molecules (Ranganathan, 1995 and references therein).

The stoichiometric requirement of arrestin in vivo for the inactivation of metarhodopsin was demonstrated through the isolation and characterization of Drosophila mutants defective in arrestin function (Dolph, 1993). Hypomorphic alleles were isolated in both arrestin genes, and electrophysiological analysis of these mutant photoreceptors revealed that a significant loss of arrestin function leads to defective metarhodopsin inactivation, resulting in abnormally slow deactivation of the light response. Abnormal metarhodopsin inactivation in arrestin mutants was demonstrated by a reduced threshold for induction of a pathological state of the Drosophila photoreceptor known as the prolonged depolarizing afterpotential (PDA). A PDA is a sustained photoresponse triggered in wild-type cells by substantial photoconversion (>20%) of rhodopsin to metarhodopsin. During a PDA, photoreceptors are refractory to further light stimuli and are said to be inactivated. A PDA can be terminated by the photoconversion of metarhodopsin back to rhodopsin, indicating that unregulated metarhodopsin activity sustains the afterpotential. Thus, a PDA represents the reversible saturation of metarhodopsin-inactivation mechanisms in the cell. The analysis of arrestin mutant photoreceptors shows that the amount of rhodopsin isomerization required to induce a PDA matches the amount of arrestin in the cell. Thus, the generation of excess metarhodopsin over free arrestin represents the basis of the PDA, and arrestin is required stoichiometrically for metarhodopsin inactivation in vivo (Ranganathan, 1995).

What is the functional significance of the binding of arrestin for the rate of receptor inactivation? Indeed, the current view of metarhodopsin inactivation presented above suggests a multistep inactivation process, in which one of several events may set the inactivation rate. For example, phosphorylation of metarhodopsin, arrestin binding, the dissociation of Galpha, or conformational changes induced in metarhodopsin by any of these steps could each represent rate-determining events. The resolution of this issue has important functional consequences, since the lifetime of activated rhodopsin determines the gain in the first step of phototransduction and thus influences the overall sensitivity of the photoreceptor cell. In addition, the rate of metarhodopsin inactivation contributes to determining the temporal resolution of phototransduction (Ranganathan, 1995).

The rate-limiting process for receptor inactivation would therefore represent an important control mechanism driving photoreceptor adaptation and deactivation. This paper formalizes a model for metarhodopsin inactivation in which the stoichiometric binding of arrestin determines the receptor inactivation rate. This simple bimolecular reaction scheme leads to specific predictions about the dependence of the kinetics of receptor inactivation on free arrestin concentrations. These predictions are fully supported by electrophysiological measurements of metarhodopsin inactivation in dissociated Drosophila photoreceptors, where cytosolic arrestin levels are manipulated using both genetic and physiological techniques. In addition, a further test of the model through independent measurement of internal parameters using molecular and spectroscopic techniques demonstrates a good fit with the electrophysiological data. These results show that arrestin binding determines the kinetics of metarhodopsin inactivation in vivo (Ranganathan, 1995).

Calcium/calmodulin-dependent kinase II phosphorylates Arrestin 1

Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca2+ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, Arrestin 1 (Phosrestin 1), is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. The identity of the protein kinase responsible for the phosphorylation of phosrestin I was sought, to link the transmembrane signaling with the light-adaptive inactivation response. Type II Ca2+/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. Partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca2+/calmodulin-dependent kinase II; the kinase activity exhibits regulatory properties that are unique to Ca2+/calmodulin-dependent kinase II, such as Ca2+ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca2+/calmodulin-dependent kinase II autoinhibitory domain. Ca2+/calmodulin-dependent kinase KII activity is present in Drosophila eye preparations. These results are consistent with the hypothesis that Ca2+/calmodulin-dependent kinase II phosphorylates phosrestin I. It is concluded that Ca2+/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation (Kahn, 1997).

A role for the light-dependent phosphorylation of visual arrestin

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in the Drosophila visual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a Ca²⁺/calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascade in vivo. Nine alleles of the Drosophila photoreceptor cell-specific arr2 gene have been isolated. Flies carrying each of these alleles undergo light-dependent retinal degeneration and display electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca²⁺/calmodulin-dependent protein kinase site. The phosphorylation mutant has very low levels of phosphorylation and lacks the light-dependent phosphorylation observed with wild-type Arr2. Interestingly, the Arr2 phosphorylation mutant is still capable of binding to rhodopsin; however, it is unable to release from membranes once rhodopsin has converted back to its inactive form. This finding suggests that phosphorylation of arrestin is necessary for the release of arrestin from rhodopsin. It is proposed that the sequestering of arrestin to membranes is a possible mechanism for retinal disease associated with previously identified rhodopsin alleles in humans (Alloway, 1999).

Although it has been known for quite some time that Arr2 is phosphorylated in a light-dependent manner, it is unclear just what role this phosphorylation serves. The invertebrate phototransduction cascade results in an increase in intracellular calcium. It has been proposed that the calcium- and light-dependent phosphorylation of Arr2 acts as the signal to bind and inactivate metarhodopsin, and, therefore, Arr2 phosphorylation serves to modulate the inactivation of the signaling cascade. However, this study has found that arrestin is able to bind to activated rhodopsin (metarhodopsin) in the absence of phosphorylation, arguing against this feedback-regulation model. Invertebrate Arr2 is a very basic molecule with a pK_a of ~8.7. This characteristic may allow it to rapidly interact with an exposed acidic surface on activated rhodopsin in the absence of any covalent modification. Instead, posttranslational modification is required to remove arrestin from rhodopsin. Phosphorylation of Arr2 when it is bound to rhodopsin may trigger a conformational change or add negative charge that enables the release of Arr2 (Alloway, 1999).

The finding that the phosphorylation of Arr2 is required for proper function brings up an apparent discrepancy: previously, the C terminus of Arr2 was determined to be nonessential. In a previous study (Dolph, 1993), a truncated form of Arr2 that lacked the last 45 aa of Arr2, including the serine at position 366, was generated; this mutated form of Arr2 was phenotypically normal. An identical situation occurs in the bovine system, where deletion of the C terminus of arrestin yields a protein that still binds to rhodopsin but has lost its binding specificity, binding to both photoactivated and nonphotoactivated rhodopsin. As such, it is believed that the truncated form of Arr2 is not defective in release from rhodopsin but instead binds indiscriminately to both the active and inactive forms of rhodopsin. The truncation mutant is phenotypically normal; it still has a higher binding affinity for the active form of rhodopsin (Alloway, 1999).

In the missense alleles generated from the screen, Arr2 is found associated with membranes under all light conditions. This finding clearly explains the null phenotype of these alleles. Because rhodopsin is approximately five times more abundant than arrestin, all of the Arr2 becomes bound to membranes, and no soluble Arr2 is available to bind and inactivate metarhodopsin. However, it is unclear whether the titration of arrestin or the formation of arrestin/rhodopsin complexes is the primary cause of retinal degeneration in these alleles. Interestingly, many recently characterized human dominant rhodopsin alleles that are associated with retinitis pigmentosa and stationary night blindness also have defects in arrestin binding. These alleles show continuous activity of rhodopsin in vitro; however, both in vitro and in vivo, these aberrant rhodopsin proteins are constitutively bound to arrestin. One possible mechanism for retinal degeneration in these dominant alleles is the titration of arrestin caused by its increased affinity for the aberrant rhodopsin proteins. In this way, photoreceptor cells undergo degeneration because of insufficient levels of soluble arrestin to quench newly formed metarhodopsin (Alloway, 1999).

The formation of stable rhodopsin-arrestin complexes induces apoptosis and photoreceptor cell degeneration

Although many different mutations in humans and Drosophila cause retinal degeneration, in most cases, a molecular mechanism for the degeneration has not been found. This study demonstrates the existence of stable, persistent complexes between rhodopsin and its regulatory protein arrestin in several different retinal degeneration mutants. Elimination of these rhodopsin-arrestin complexes by removing either rhodopsin or arrestin rescues the degeneration phenotype. Furthermore, it is shown that the accumulation of these complexes triggers apoptotic cell death and that the observed retinal degeneration requires the endocytic machinery. This suggests that the endocytosis of rhodopsin-arrestin complexes is a molecular mechanism for the initiation of retinal degeneration. It is proposed that an identical mechanism may be responsible for the pathology found in a subset of human retinal degenerative disorders (Alloway, 2000).

This paper demonstrates the existence of a novel mechanism to explain the light-dependent retinal degeneration that is observed in a subset of Drosophila visual system mutants. Mutations in three distinct genetic loci, norpA, arr2, and rdgB, result in the light-dependent formation of stable rhodopsin-arrestin complexes. Elimination of either member of this complex rescues the retinal degeneration in each of the three genetic backgrounds. In addition, it is shown that the formation of these stable rhodopsin-arrestin complexes triggers apoptotic cell death. Furthermore, endocytosis is essential for inducing cell death in norpA mutants, suggesting that the internalization of the rhodopsin-arrestin complexes is an early step in the initiation of apoptosis of retinal photoreceptors. It is possible that the excessive endocytosis saturates a downstream cellular function (for example, saturation of the early endosome or depletion of an endocytic protein), which signals the cell to undergo programmed cell death. Alternatively, the endocytosis may block a signal that protects the cell from apoptosis (Alloway, 2000)

The inactivation of the Drosophila phototransduction cascade is an extremely rapid event. Drosophila photoreceptors can shut off the light-activated currents in less than a 100 ms following termination of the light stimulus. One way in which photoreceptors have evolved to do this is at the level of the G protein-coupled receptor rhodopsin. Immediately upon activation, rhodopsin is multiply phosphorylated on its C terminus, and this phosphorylation greatly increases its affinity for the abundant soluble protein arrestin. Invertebrate Arr2 is a very basic molecule with a pK_a of ~8.7 and, therefore, has a high affinity for phosphorylated rhodopsin. Thus, this interaction of activated rhodopsin and arrestin occurs very rapidly, and the receptor is quickly inactivated. It is tempting to speculate that the posttranslational modifications of arrestin and rhodopsin are essential to eliminate these complexes. In such a model, it is necessary to phosphorylate Arr2, thereby making it less basic, as well as to dephosphorylate rhodopsin, thereby making it less acidic. Thus, the system is designed such that the influx of calcium activates CamKII, the kinase that phosphorylates arrestin, and rdgC, the phosphatase that dephosphorylates rhodopsin. Both of these steps are crucial, as evidenced by the fact that rdgC mutants and arr2(S366A) mutants both undergo rapid light-dependent retinal degeneration. Evidence suggests that, in the absence of these posttranslational modifications, stable rhodopsin-arrestin complexes persist in the cell and are instrumental in the pathology of retinal degeneration (Alloway, 2000)

The retinal degeneration induced by rhodopsin-arrestin complexes can be partially rescued by a temperature-sensitive allele of dynamin. This observation suggests that the rhodopsin-arrestin complexes are being removed from the photoreceptor rhabdomere by the endocytic machinery. A similar receptor internalization pathway occurs for the vertebrate β-adrenergic receptor. β-arrestin is involved in the inactivation and internalization of the β-adrenergic receptor. A small domain near the carboxyl terminus of β-arrestin directly interacts with clathrin and targets the β-adrenergic receptor-β-arrestin complex for internalization. This clathrin binding site is curiously missing in the visual arrestins. However, in spite of the absence of the clathrin-interacting motif, these complexes seem to have the ability to interact with the endocytic machinery. One attractive model is that visual arrestin serves as an AP2-like adaptor, just like β-arrestin, but recruits clathrin by a different mechanism. The question of whether the endocytic proteins are recognizing motifs in rhodopsin or arrestin awaits further studies. Many models have been proposed to explain retinal degeneration in humans, including constitutive activity of the phototransduction cascade, improper trafficking of photoreceptor cell components, and defects in the recycling of rhodopsin. The results described in this study implicate the endocytic pathway in retinal degeneration and suggest that excessive endocytosis can be a trigger for apoptotic cell death (Alloway, 2000)

The internalization of rhodopsin-arrestin complexes via receptor-mediated endocytosis could partly explain the membrane association of arrestin in certain mutant backgrounds. The enhanced membrane affinity of arrestin could in part be due to the rhodopsin-arrestin complexes rapidly interacting with the endocytic machinery. It is possible that, once the complex is associated with clathrin cages, the arrestin would be locked in the membrane-associated state. In support of this model, it has recently been demonstrated that the unphosphorylated form of arrestin directly interacts with clathrin in vitro. Therefore, it is possible that the role of arrestin phosphorylation is to block interactions with clathrin, and any mutant background that fails to phosphorylate arrestin yields complexes due to clathrin interactions. However, it is still essential that a stable rhodopsin-arrestin complex be formed initially to allow for the assembly of the endocytic proteins (Alloway, 2000)

A large number of mutations in the human rhodopsin gene have been isolated that are responsible for retinal disease. Interestingly, there are several mutations in human rhodopsin that form stable rhodopsin-arrestin complexes. These include a mutation in a highly conserved lysine in the seventh transmembrane domain that has been shown to cause autosomal-dominant retinitis pigmentosa. This mutant form of rhodopsin is found both in vivo and in vitro to be constitutively phosphorylated and tightly bound to arrestin. It had been hypothesized that the complexes between rhodopsin and arrestin may be instrumental in the retinal degeneration process. Possibly, as in Drosophila, these stable human rhodopsin-arrestin complexes are removed from the rod outer segments by the endocytic machinery, and this excessive endocytosis is the direct cause of apoptotic retinal degeneration (Alloway, 2000)

One question yet to be addressed is why photoreceptor cells have a mechanism for eliminating rhodopsin-arrestin complexes if, under nonpathological conditions, these complexes do not persist in the cell. One possible explanation is that arrestin has a secondary function to eliminate defective rhodopsin molecules from the photoreceptor cell. A rhodopsin molecule that becomes photochemically damaged may render the receptor nonfunctional or constitutively active. The presence of a constitutively active rhodopsin molecule could potentially be very detrimental and, therefore, necessitates its removal from the photoreceptor cell. Presumably, any rhodopsin molecule that becomes constitutively active will bind arrestin and generate a stable rhodopsin-arrestin complex. The stabily bound arrestin would target the dysfunctional rhodopsin molecule for endocytosis and degradation. In this manner, arrestin could function as a surveillance protein, eliminating defective rhodopsin molecules from the photoreceptor cell. However, in certain mutant backgrounds, the regulation of rhodopsin-arrestin complex formation is defective, and a large number of complexes are formed. The increased endocytosis of these complexes initiates apoptosis and retinal degeneration (Alloway, 2000).

A molecular pathway for light-dependent photoreceptor apoptosis in Drosophila

Light-induced photoreceptor apoptosis occurs in many forms of inherited retinal degeneration resulting in blindness in both vertebrates and invertebrates. Though mutations in several photoreceptor signaling proteins have been implicated in triggering this process, the molecular events relating light activation of rhodopsin to photoreceptor death are yet unclear. This study uncovers a pathway by which activation of rhodopsin in Drosophila mediates apoptosis through a G protein-independent mechanism. This process involves the formation of membrane complexes of phosphorylated, activated rhodopsin and its inhibitory protein arrestin, and subsequent clathrin-dependent endocytosis of these complexes into a cytoplasmic compartment. Together, these data define the proapoptotic molecules in Drosophila photoreceptors and indicate a novel signaling pathway for light-activated rhodopsin molecules in control of photoreceptor viability (Kiselev, 2000).

A combination of genetic and biochemical methods was used to reveal a novel light-dependent but G protein-independent signaling pathway regulating photoreceptor cell viability in Drosophila. In addition to its established function in inactivation of metarhodopsin (M), Arr2 acts as a clathrin adaptor protein to mediate the internalization of Arr2-M complexes. The analysis of rdgC³⁰⁶ mutants (that are unable to dephosphorylate metarhodopsin and display light-dependent retinal degeneration) shows that the Arr2-dependent internalization process is necessary for photoreceptor cell apoptosis and that the triggering condition for apoptosis is the long-term accumulation of Arr2-M-p complexes in an internal compartment. These findings are in strong agreement with work that show that formation and internalization of stable Arr2-M complexes are associated with photoreceptor apoptosis in Drosophila. This study shows that mutations in several phototransduction genes show Arr2-dependent retinal degeneration, suggesting that the Arr2-dependent internalization pathway is likely to be a common mechanism for triggering apoptotic photoreceptor cell death (Kiselev, 2000).

How does this pathway function in wild-type photoreceptor cells and how is apoptosis suppressed under normal physiological conditions? Two phosphorylation events control this process: (1) the phosphorylation of M promotes apoptosis through an unknown mechanism downstream of the internalization process and the RdgC phosphatase counteracts this process by efficient dephosphorylation of M, and (2) the phosphorylation of Arr2 suppresses apoptosis by disrupting clathrin interaction and preventing internalization of the receptor complexes. The net flux of the proapoptotic Arr2-M-p complexes through the internalization pathway is therefore determined by the quantity of metarhodopsin created upon light exposure, the activity of the RdgC phosphatase, and the phosphorylation status of Arr2. Interestingly, both the activity of the RdgC phosphatase and the phosphorylation of Arr2 are upregulated by the light-dependent increase in intracellular calcium that occurs during visual signaling in Drosophila photoreceptors. Thus, phototransduction appears to promote the survival of photoreceptor cells by reducing the accumulation of internalized Arr2-M-p complexes (Kiselev, 2000).

These results help clarify a number of unexplained and mysterious observations in the study of the photoreceptor signaling in Drosophila. For example, mutations that eliminate the phosphorylation domain of either rhodopsin or arrestin have no effect on the sensitivity or kinetics of light transduction. Nevertheless, these phosphorylation events occur rapidly upon light activation and require the investment of substantial metabolic resources since rhodopsin and arrestin are among the most abundant of photoreceptor proteins. It is suggested that these phosphorylation events are dedicated regulatory processes that apply primarily to the control of cell viability. The large commitment of metabolic energy in these steps presumably highlights the concept that like all cells, photoreceptors need to suppress the latent apoptotic machinery for survival (Kiselev, 2000).

How can internalized Arr2-M-p complexes trigger apoptosis? Several studies support for the idea that internalized, arrestin-bound GPCRs can trigger additional signaling events. For example, β-arrestin1 recruits c-Src to form three-protein complexes with activated β2 adrenergic receptors, and mediates the internalization of the entire complex through direct interaction with clathrin. Upon internalization, the complex triggers stimulation of mitogenic signaling through activation of the Ras-MAP kinase pathway. Cell survival has been shown to be promoted by this pathway through both suppression of the apoptotic machinery and transcriptional activation of prosurvival factors, and β-arrestin- and receptor internalization-dependent activation of MAP kinases is reported to be the basis for the antiapoptotic effects of substance P. Thus GPCRs can trigger a second round of signaling through arrestin-mediated internalization and may generally promote the survival of cells through activation of mitogenic signaling. One possibility is that Arr2-mediated internalization of dephosphorylated metarhodopsin in Drosophila photoreceptors plays a similar role in triggering a prosurvival signaling pathway and that phosphorylated M-Arr2 complexes may trigger the apoptotic machinery by disruption of this process (Kiselev, 2000).

The conservation of arrestin-mediated internalization between Drosophilarhodopsin and mammalian β receptors highlights the similarity in functional mechanisms throughout the GPCR superfamily. The stimulus-dependent phosphorylation at the carboxyl terminus is no exception; this property is shared by most GPCRs and suggests that RdgC-like receptor phosphatases may also be found in other signaling systems. Two mammalian homologs of RdgC, PPEF-1 and PPEF-2, have been cloned that show tissue distributions consistent with a role in several sensory signaling processes. Indeed, PPEF-2 is found exclusively in the retinal rod photoreceptors and in the pineal gland, although the role of this protein in regulating function of vertebrate rhodopsin is not yet established. However, retinal degeneration in transgenic mice expressing a constitutively active form of rhodopsin (K296E) does not result from excessive stimulation of the visual signaling cascade; instead the mutant rhodopsin is found in a constitutively phosphorylated form, tightly bound to arrestin molecules. The corresponding mutation in human rhodopsin is associated with one form of retinitis pigmentosa, suggesting that stable phosphorylated GPCR-arrestin complexes may also be proapoptotic in humans (Kiselev, 2000).

An interesting issue of the apoptotic mechanism in rdgC³⁰⁶ photoreceptors is the long but apparently reliable time delay in the commitment to cell death. For example, DPP analysis of a population of rdgC³⁰⁶ flies exposed to light shows no individuals with retinal degeneration until the fourth day, but then nearly all animals show complete degeneration over the next 2 days. Such fidelity in the timing of apoptosis in many independent animals is reminiscent of many human age-dependent macular degenerations in which affected individuals show a similar age of onset of symptoms, and argues that the timing of degeneration must be a well-regulated feature of the commitment process and not a simple, randomly distributed event. Since formation and internalization of the Arr2-M-p complexes proceeds with a time course of hours, and commitment to apoptosis requires days, it is concluded that the mechanism controlling the decision for cell death must reside in the multivesicular bodies (MVB) that represent the stable pool of internalized membranes in Drosophilaphotoreceptors. The detailed characterization of commitment to apoptosis in rdgC³⁰⁶ photoreceptors should provide insight into understanding the factors controlling programmed timing of apoptosis (Kiselev, 2000).

Loss of the phospholipase C induces massive endocytosis of rhodopsin and arrestin in Drosophila photoreceptors

A subset of visual transduction mutants in Drosophila melanogaster induce the formation of stable complexes between rhodopsin and arrestin. One such mutant is in a visual system-specific phospholipase C (PLC). The rhodopsin/arrestin complexes generated in PLC mutants induce massive retinal degeneration. Both arrestin and rhodopsin undergo light-dependent endocytosis in a PLC mutant background. Interestingly, the internalized rhodopsin is rapidly degraded, but the arrestin is fully stable. The data are discussed with respect to mechanisms of arrestin-mediated endocytosis and human retinal disease (Orem, 2002).

Light adaptation through phosphoinositide-regulated translocation of Drosophila visual arrestin

Photoreceptor cells adapt to bright or continuous light, although the molecular mechanisms underlying this phenomenon are incompletely understood. This paper reports a mechanism of light adaptation in Drosophila, which is regulated by phosphoinositides (PIs). Light-dependent translocation of arrestin is defective in mutants that disrupt PI metabolism or trafficking. Arrestin binds to PIP(3) in vitro, and mutation of this site delays arrestin shuttling and results in defects in the termination of the light response, which is normally accelerated by prior exposure to light. Disruption of the arrestin/PI interaction also suppresses retinal degeneration caused by excessive endocytosis of rhodopsin/arrestin complexes. These findings indicate that light-dependent trafficking of arrestin is regulated by direct interaction with PIs and is required for light adaptation. Since phospholipase C activity is required for activation of Drosophila phototransduction, these data point to a dual role of PIs in phototransduction (Lee, 2003).

A curious but longstanding observation is that visual arrestin undergoes a dramatic light-dependent translocation from the inner segment to the outer segment of rods and cones. However, the function of this dynamic movement has not been described. Drosophila visual arrestin, Arr2, also undergoes light-dependent shuttling between the cell bodies and the phototransducing portion of the photoreceptor cells, the rhabdomeres (Kiselev, 2000). This observation has provided the potential for using a genetic approach to address two unresolved issues: (1) the mechanisms regulating this movement and (2) the function for this light-driven translocation. Due to the slow time course of the light-induced movements, which occur over a few to many minutes, an interesting possibility is that the trafficking of arrestin could contribute to long-term adaptation, since the concentration of arrestin has been proposed to limit response termination Ranganathan, 1995). One well-known illustration of long-term adaptation is experienced upon entering and leaving a darkened room, such as a movie theater. Interestingly, this delay occurs over a similar time course as the light-dependent translocation of visual arrestins (Lee, 2003).

Consistent with the proposal that the regulated movement of arrestin contributes to slow adaptation, it was found that a reduction in the rate of Arr2 translocation has a major impact on a light-dependent component of the photoresponse. In wild-type, it was found that the rate of termination of the photoresponse is significantly faster in flies that have had prior exposure to light; however, this adaptation feature is virtually eliminated in arr2 null mutant flies. Of particular significance, termination of the ERG response is much slower in flies that have defects in the light-dependent movement of Arr2. Based on these findings indicating that light-dependent movement of Arr2 contributes to long-term adaptation in Drosophila, it is proposed that dynamic movements of mammalian visual arrestin may contribute to long-term adaptation in humans, in addition to other established mechanisms, such as chromophore regeneration (Lee, 2003).

Transducin also undergoes a light-driven translocation between the rod outer and inner segments, and this movement out of the outer segment is correlated with a reduction in the amplitude of the photoresponse. Similar light-dependent movement of the Gqα occurs in fly photoreceptor cells, and this translocation is dependent on the presence of the Gβγ. However, the effect of this translocation on adaptation has not been addressed. Recently, light-regulated translocation of the TRPL channel has been suggested as a novel mechanism for reducing the sensitivity to increasing intensities of light. Therefore, Drosophila appears to have at least two long-term light-adaptation mechanisms: light-dependent movement of Arr2 into the rhabdomeres for increasing the speed of termination of the photoresponse and light-dependent movement of TRPL into the cell body for enabling the photoreceptor cells to adjust the amplitude of their response to background light (Lee, 2003).

A second central question concerning the light-driven changes in the spatial distribution of Arr2 concerns the underlying mechanism. Several observations support the conclusion that the movements of Arr2, to and from the rhabdomeres, are regulated by PIs. The rate of Arr2 translocation is much slower as a result of alterations in the levels of expression of gene products, such as a PI-transfer protein (RDGB), CDS, and PTEN, that affect the metabolism or distribution of PIs. Furthermore, Arr2 binds to PIs in vitro, and mutations in this binding site causes translocation defects. In addition, endocytosis of β arrestin is mediated in vitro through interaction with PIs, though such a mode of regulation has not been demonstrated in vivo. In this report, evidence is provided that the movement of visual arrestin is regulated by PIs in photoreceptor cells in both directions, in and out of the rhabdomeres (Lee, 2003).

An issue raised by the current experiments is the identity of the PI or IP that interacts with and regulates the movement of Arr2 in vivo. It seems more likely that Arr2 binds to PIs than IPs, since the shuttling defects, which are observed in mutants affecting the PI-transfer protein or cds, are similar to those resulting from mutation of the PI/IP binding site in Arr2. PIP₃ is a prime candidate for regulating Arr2, since the IC₅₀ is lowest for PIP₃. Furthermore, overexpression of the phosphatase that hydrolyzes PIP₃ (PTEN) results in impairment of Arr2 translocation to the rhabdomeres but not shuttling to the cell bodies. These results support the conclusion that PIP₃ facilitates shuttling of Arr2 to the rhabdomeres but also indicate that PIP₃ does not affect movement of Arr2 from the rhabdomeres to the cell bodies. Given that translocation of Arr2 is disrupted by mutation of the PI binding site in Arr2, it is proposed that another PI, which remains to be identified, is required for this latter movement. The defect in Arr2 shuttling to the cell bodies in the PTEN null might result from an increased level of PIP₃, which competes with another PI required for movement to the cell bodies (Lee, 2003)

The demonstration that translocation of Arr2 is regulated by PIs addresses a lingering question concerning potential roles of PIs in photoreceptor cells. The Drosophila visual transduction cascade is among the most intensively studied GPCR cascades. During the last 30 years, many proteins and mutations have been identified that perturb PI signaling; however, the targets and mechanisms directly regulated by PIs have not been previously described (Lee, 2003).

The regulation of Arr2 shuttling by PIs occurs on the order of a few to many minutes. This is in contrast to the millisecond time scale, which operates in the activation of phototransduction. Although the specific activation mechanism involved in Drosophila phototransduction remains elusive, it is established that it depends on a PLCβ (NORPA). Thus, PLC-mediated hydrolysis of PIP₂ leads to rapid activation of the light-sensitive channels through the millisecond generation of PIP₂ metabolites or reduction in PIP₂ levels. Since adaptation occurs over a much slower timescale, regulation of this latter phenomenon exclusively by direct effects of second messengers on protein activities might be too rapid. Rather, regulation of adaptation by the translocation of signaling proteins provides a mechanism whereby changes in second messengers, such as PIP₃, result in delayed effects on the magnitude and the kinetics of signaling. Therefore, PIs appear to have the capacity to serve a dual role in activation and adaptation by modulating the activities and localization of signaling proteins (Lee, 2003).

A major unresolved issue in mammalian vision is the function of PIs in rods and cones, since cGMP rather than lipid second messengers mediate activation of mammalian phototransduction. This question arises in part from the observation that several enzymes regulating PIs, including p110 PI3-kinase and DAG kinase, are activated in mammalian photoreceptor cells in a light-dependent manner. Furthermore, rods and cones express homologs of many eye-enriched proteins that function in Drosophila phototransduction. These include a PLCβ4 and M-rdgB2. However, the functions of these genes in rods and cones have not been identified, despite the generation of mouse knockouts (Lee, 2003).

It is proposed that PIs are excellent candidates for regulating the intracellular translocation of mammalian photoreceptor proteins in general and visual arrestin in particular. Consistent with this proposal are the observations that mammalian visual arrestin undergoes a light-dependent translocation, which appears to occur through an active mechanism rather than via passive diffusion. Moreover, mammalian visual arrestin binds to IPs in vitro, although an interaction with PIs was not tested. If mammalian visual arrestin binds PIs, then disruption of PI metabolism may interfere with adaptation in rods and cones, similar to the defects in arr2^3K/Q, rdgB, cds, and PTEN mutant flies. Thus, it would be interesting to reevaluate the PLCβ4 and m-rdgB2 knockout mice for effects on arrestin translocation and light adaptation (Lee, 2003).

Previous reports have shown that stable Arr2/rhodopsin complex formation leads to retinal degeneration in norpA or rdgC flies (Alloway, 2000; Kiselev, 2000). Removal of the arr2 gene in a norpA or rdgC background partially suppresses the photoreceptor cell death. This partial suppression could be due to elimination of Arr2/rhodopsin complexes, reduction in endocytosis of rhodopsin, or disruption of some other Arr2 function. In this work, Arr2/rhodopsin binding and PI-regulated trafficking of Arr2 were uncoupled by expressing Arr2^3K/Q, which is defective in movement but not rhodopsin binding. Since the retinal degeneration in norpA is largely rescued in arr2^3K/Q flies, these results suggest that apoptosis in norpA results from endocytosis of Arr2/rhodopsin complexes rather than a defect in Arr2/rhodopsin binding. This conclusion is further supported by the finding that there was even greater suppression of the norpA degeneration in an arr2^3K/Q than in an arr2⁵ null background (Lee, 2003).

It is possible that endocytosis of stable arrestin/rhodopsin complexes may contribute to certain types of retinal degenerations in humans, as appears to be the case in Drosophila. If so, the findings in the current report that retinal degeneration is suppressed by interfering with the PI/Arr2 interaction raise the intriguing possibility that application of drugs that suppress PI production in the rods and cones or PI binding to visual arrestin may be an effective approach to suppress certain types of human retinal dystrophies (Lee, 2003).

Light-dependent translocation of visual arrestin regulated by the NINAC myosin III

To address whether NINAC is required for light-dependent trafficking of Arr2, the major visual arrestin, Arr2 spatial distribution was examined in wild-type and null ninaC mutant flies (ninaC^P235) at several time points after light stimulation. In dark-adapted wild-type and ninaC^P235 flies, Arr2 is dispersed throughout the photoreceptor cells, with a greater overall proportion of the Arr2 pool in the cell bodies than in the rhabdomeres. After a 5 min exposure of wild-type flies to blue light, which stably converts rhodopsin to light-activated metarhodopsin, Arr2 is concentrated in the rhabdomeres. However, in ninaC^P235 flies, Arr2 is not concentrated in the rhabdomeres, even after stimulating the flies for 1 hr with light. Elimination of NINAC does not have a global effect on rhabdomeral localization, since the spatial distributions of other rhabdomere-enriched proteins are similar in wild-type and ninaC^P235 photoreceptor cells. An exception is calmodulin; NINAC is the major retinal calmodulin binding target, and elimination of NINAC results in instability of calmodulin in both the cell body and rhabdomeral compartments (Porter, 1993a; Porter, 1995). In contrast to calmodulin, the overall concentration of Arr2 is unaffected in ninaC^P235 photoreceptor cells (Lee, 2004a).

To address the requirements for the individual NINAC isoforms for light-dependent shuttling of Arr2 into the rhabdomeres, immunostaining was performed on photoreceptor cells obtained from flies that expressed only p174 (ninaC^Δ132) or p132 (ninaC^Δ174) (Porter, 1992). After a 5 min pulse of blue light, the relative concentration of Arr2 in the ninaC^Δ132 rhabdomeres increased, although to a smaller extent than in wild-type. Following exposure of ninaC^Δ132 to blue light for 1 hr, Arr2 was enriched in the rhabdomeres. Thus, the kinetics of Arr2 trafficking in ninaC^Δ132 are retarded rather than eliminated as in the ninaC^P235 null mutant. Light-induced movement of Arr2 into the rhabdomeres is not impaired detectably in ninaC^Δ174; however, p174 appears to contribute to some extent to Arr2 translocation as elimination of both proteins in ninaC^P235 causes a more severe phenotype than disruption of p132 alone. These data indicated that p132 is the NINAC isoform most critical for light-dependent trafficking of Arr2 into the rhabdomeres. In contrast to these results, elimination of p132 has no effect on the dark-associated retrograde movement of Arr2 from the rhabdomeres to the cell bodies (Lee, 2004a).

The Arr2 movement could be due to a requirement for either the protein kinase or myosin domain. However, it could not be tested whether mutations in the myosin domain disrupt Arr2 translocation since deletion of this domain or point mutations in conserved residues, which are critical for the activity of other myosins, cause instability of the NINAC proteins (Porter, 1993b). Therefore, to determine whether the protein kinase domain was required, the corresponding lysine in NINAC (residue 45), which is required for ATP binding in other kinases, was changed to an arginine. It was found that both NINAC isoforms are expressed at the same levels in the transgenic flies, ninaC^K45R, as in wild-type. In addition, on the basis of electroretinograms (ERGs), which measure the summed light responses of all retinal cells, there is no apparent defect in activation or termination of the photoresponse. Of importance here, light-dependent translocation of Arr2 and long-term adaptation are indistinguishable between ninaC^K45R and wild-type flies. These data indicate that the protein kinase domain is dispensable for trafficking of Arr2 (Lee, 2004a).

The translocation of Arr2 is required for long-term light adaptation (Lee, 2003). Therefore, ninaC mutants that show defects in Arr2 trafficking should display reductions in light adaptation. To test this proposal, electroretinogram recordings, were performed. In wild-type flies, termination of the light response is relatively slow in flies that have been dark adapted for 4 hr, much faster after a 10 min pre-exposure to light, and further accelerated after 1 hr of prior light exposure. In contrast, it was found that ninaC^P235 flies display a defect in long-term adaptation; acceleration of the rate of termination of the photoresponse by pre-exposure to light is much less pronounced than in wild-type. The data further support the conclusion that NINAC plays a crucial role in Arr2 translocation and in long-term light adaptation (Lee, 2004a).

To determine whether there is a correlation between long-term light adaptation and light-dependent translocation of Arr2 in ninaC^Δ132 and ninaC^Δ174 flies, ERGs were performed using the long-term adaptation paradigm described above. It was found that the rates of termination of the photoresponse were similar between wild-type and ninaC^Δ174, while the rate of the termination of the photoresponse in ninaC^Δ132, after a 10 min pre-exposure to light, was intermediate between wild-type and the null ninaC^P235 flies. These results indicate that the rates of long-term adaptation are consistent with the light-dependent changes in Arr2 distribution in these flies (Lee, 2004a).

The preceding results raise the question as to how NINAC regulates the trafficking of Arr2. One possibility is that Arr2 can bind directly to NINAC. However, Arr2 and NINAC did not appear to associate directly in vitro or to co-immunoprecipitate from fly heads. Therefore, whether there might be an indirect interaction between NINAC and Arr2, which might be disrupted by the conditions used for the immunoprecipitations, was considered. To address this question, advantage was taken of the observation that the two NINAC isoforms are the major retinal calmodulin binding proteins, while Arr2 does not bind calmodulin. Therefore, whether Arr2 from fly heads might associate with calmodulin-agarose beads in a NINAC-dependent manner was tested. To perform this assay, pull-down assays were employed using extracts prepared from wild-type, ninaC^Δ132, ninaC^Δ174, and ninaC^P235 fly heads. Arr2 was detected in the bound fraction from wild-type, ninaC^Δ132, and ninaC^Δ174 but not from extracts prepared from null ninaC^P235 fly heads. Gα did not bind to calmodulin-agarose in either the presence or absence of NINAC, while a known retinal calmodulin binding protein, RDGC, bound to calmodulin-agarose in a NINAC-independent manner. These data indicated that Arr2 interacts with both isoforms of NINAC, since the binding to calmodulin-agarose is dependent on the presence of NINAC (Lee, 2004a).

Candidate molecules that could potentially contribute to Arr2/NINAC interactions and which are disrupted by amphiphilic detergents, such as TritonX, include PIs. PIs are especially attractive candidate intermediates linking Arr2 and NINAC, since Arr2 binds PIs and the Arr2/PI interaction is crucial for its light-dependent movement and for long-term light adaptation (Lee, 2003). If the Arr2/NINAC interaction requires PIs, then it should be disrupted using a derivative of Arr2 with three lysine to glutamine substitutions (arr2^3K/Q), which severely disrupt PI binding (Lee, 2003). It was found that using arr2^3K/Q extracts, Arr2 binding to the beads decreases dramatically (Lee, 2004a).

To provide a second assay to characterize the Arr2/NINAC interaction, pull-down assays were employed using NINAC proteins partially purified from fly heads and GST fused to the C-terminal portion of Arr2 (Arr2C-wt; amino acids 204-401), which contains the main Arr2/PI binding site (Lee, 2003). It was found that NINAC associates with GST-Arr2C-wt, but not to GST alone. Moreover, there was relatively little association between NINAC and GST-Arr2C^3K/Q. Consistent with the reciprocal pull-down assays using NINAC bound to beads, the interaction between GST-Arr2C-wt and NINAC was disrupted by addition of TritonX-100 to the buffer. The combination of the above data suggested that PIs mediates the association between Arr2 and NINAC (Lee, 2004a).

To directly test whether PIs promote the Arr2/NINAC interaction, whether addition of PIs enhance binding of partially purified NINAC to GST-Arr2-wt was considered. Phosphatidylinositol-4,5-bisphosphate (PIP₂) was used for these assays since PIP₂ is among the most abundant PIs in vivo and is among the PIs that bind most efficiently to Arr2 (Lee, 2003). While 0.1% TritonX-100 disrupts the Arr2/NINAC interaction, it was found that addition of 30 μM or more PIP₂ increases the association between both NINAC isoforms and GST-Arr2C-wt. Phosphatidylinositol (PIns, an uncharged phosphoinositide) or inositol hexaphosphate (IP₆, a highly charged inositol phosphate without a lipid moiety) do not augment the Arr2/NINAC interaction (Lee, 2004a).

The data indicating that PIs promote the physical association of Arr2 and NINAC raise the possibility that NINAC also binds PIs. Therefore, binding assays were performed using PIP₂ and PIP₃ beads. It was found that both partially purified isoforms of NINAC bind to PIP₂ and PIP₃ beads but not to control beads. Since NINAC associated with Arr2, the PI/NINAC interaction could potentially have been mediated through Arr2. However, it was found that NINAC partially purified from either null arr2⁵ or arr2^3K/Q flies bind effectively to either the PIP₂ or PIP₃ beads (Lee, 2004a).

Rhodopsin kinase activity modulates the amplitude of the visual response in Drosophila: The GPRK1 might have a phosphorylation-independent role affecting Rh1/Arr2 binding

A feature shared between Drosophila rhodopsin and nearly all other G protein-coupled receptors is agonist-dependent protein phosphorylation. Despite extensive analyses of Drosophila phototransduction, a determination of the identity and function of the rhodopsin kinase (RK) has been elusive. This study provides evidence that G protein-coupled receptor kinase 1 (GPRK1), which is most similar to the ß-adrenergic receptor kinases, G protein-coupled receptor kinase 2 (GRK2) and GRK3, is the fly RK. GPRK1 is enriched in photoreceptor cells, associates with the major Drosophila rhodopsin, Rh1, and phosphorylates the receptor. As is the case with mammalian GRK2 and GRK3, Drosophila GPRK1 includes a C-terminal pleckstrin homology domain, which binds to phosphoinositides and the Gßgamma subunit. To address the role of GPRK1, transgenic flies were generated that expressed higher and lower levels of RK activity. Those flies with depressed levels of RK activity display a light response with a much larger amplitude than WT. Conversely, the amplitude of the light response is greatly suppressed in transgenic flies expressing abnormally high levels of RK activity. These data point to an evolutionarily conserved role for GPRK1 in modulating the amplitude of the visual response (Lee, 2004b).

Phosphorylation of Rh1 has been suggested to stabilize arrestin/Rh1 complexes (Kiselev, 2000). Therefore, an examination was made to determine whether the association and/or dissociation of the major arrestin (Arr2) with Rh1 was affected in ogprk1 and ogprk1^K220R flies that exhibit high and low Rh1 phosphorylation, respectively. To characterize the Arr2/Rh1 interaction, an arrestin pelleting assay was used. As expected, in wild type most of the Arr2 binds to Rh1 after exposure to blue light, which stabilizes the active form of Rh1 and promotes the Rh1/Arr2 interaction. After an identical blue light treatment, slightly less Arr2 binds to Rh1 in ogprk1^K220R than in WT flies. Surprisingly, the proportion of bound Rh1/Arr2 is even lower in ogprk1 than in either wild type or ogprk1^K220R. Because the effects on the level of Rh1 phosphorylation are opposite in ogprk1 and ogprk1^K220R flies, these data suggested that the GPRK1 might have a phosphorylation-independent role affecting Rh1/Arr2 binding. In contrast to these results, no differences between fly strains in the dissociation of the Rh1/Arr2 complexes promoted by exposure to orange light (Lee, 2004b).

Suppression of constant-light-induced blindness but not retinal degeneration by inhibition of the rhodopsin degradation pathway

Continuous exposure to light, even at relatively low intensities, leads to retinal damage and blindness in wild-type animals. However, the molecular mechanisms underlying constant-light-induced blindness are poorly understood. It has been presumed that the visual impairment resulting from long-term, continuous exposure to ambient light is a secondary consequence of the effects of light on retinal morphology, but this has not been addressed. To characterize the mechanism underlying light-induced blindness, a molecular genetic approach was applied using Drosophila. The temporal loss of the photoresponse was found to be paralleled by a gradual decline in the concentration of rhodopsin. The decline in rhodopsin and the visual response are suppressed by a C-terminal truncation of rhodopsin, by mutations in arrestin, and by elimination of a lysosomal protein, Tetraspanin 42Ej (Sunglasses). Conversely, the visual impairment is greatly enhanced by mutation of the rhodopsin phosphatase, rdgC. Surprisingly, the mutations that suppressed light-induced blindness do not reduce the severity of the retinal degeneration resulting from constant light. Moreover, mutations known to suppress retinal degeneration did not ameliorate the light-induced blindness. These data demonstrate that the constant light-induced blindness and retinal degeneration result from defects in distinct molecular pathways. The results support a model in which visual impairment caused by continuous illumination occurs through an arrestin-dependent pathway that promotes degradation of rhodopsin (Lee, 2004c).

It is a longstanding observation that exposure of wild-type animals to constant light leads to retinal degeneration. In the current work, attempts were made to determine if constant light causes phototoxicity in Drosophila and, if so, whether the mechanism could be characterized further by using a genetic approach. As a sensitive and quantitative assay for phototoxicity, focus was placed on the effects of continuous light on the photoresponse by performing ERGs. It was found that flies maintained under continuous ambient light for many days gradually lose their visual response and eventually go blind (Lee, 2004c).

An important unanticipated finding was that the mechanism of light-induced blindness is distinct from that underlying light-induced retinal degeneration. Mutations such as arrestin2 and sun that suppress the light-induced blindness do not suppress the retinal degeneration resulting from exposure to the identical light conditions. In fact, a C-terminal deletion of rhodopsin (Rh1^Δ356), which significantly reduces the severity of visual impairment by continuous illumination, actually accelerates the morphological damage resulting from constant ambient light. The lack of suppression of the low-light-induced retinal degeneration in wild-type flies is striking because the rh1^Δ356 and the arr2^3K/Q alleles analyzed in this study greatly suppress certain genetically induced retinal degenerations (Lee, 2004c).

The results in the current study demonstrate that the loss of the photoresponse due to continuous light is not simply a secondary consequence of the retinal degeneration, which occurs in parallel with the visual impairment. Rather, light-induced apoptosis and blindness result from perturbations in different processes. This point is further illustrated by the findings that mutations in or overexpression of proteins known to suppress apoptosis in flies and other organisms do not ameliorate the light-induced blindness. Furthermore, although disruption of arrestin2 suppresses the visual defects caused by continuous illumination, mutations in arrestin actually cause retinal degeneration in the presence of cyclic light in both flies and the mouse (Lee, 2004c and references therein).

The combination of results presented in this study indicates that the low-light-induced blindness is due to a decline in rhodopsin levels. An indication that this is the case is that Rh1 is the only protein that declines in parallel with the visual impairment. Furthermore, mutations that either decrease or increase the severity of the Rh1 degradation cause a comparable suppression or enhancement of the visual impairment. However, genetic suppression of the light-induced decline in Rh1 levels does not reduce the retinal degeneration resulting from constant light. Thus, the decrease in the concentration of rhodopsin did not appear to underlie the retinal degeneration (Lee, 2004c).

A key question concerns the mechanism through which continuous low light causes a large reduction in Rh1 levels. The Rh1 degradation resulting from acute exposure to very bright light occurs through an arrestin-independent pathway, which remains to be defined. In contrast to these results, mutations in arrestin2 suppress the low-light-induced loss of Rh1. The differences in the mechanisms underlying bright- versus low-light-induced blindness are somewhat reminiscent of a recent mouse study demonstrating that retinal degeneration caused by extended exposure to low light is caused by a different mechanism than that for retinal degeneration caused by brief exposure to very bright light. However, the similarities between this recent report and the current study are limited because the light-induced loss of the ERG is not due to retinal degeneration. Although elimination of the trimeric G protein suppresses the retinal degeneration in the mouse, a hypomorphic allele of the Gα_q does not reduce the severity of low-light-induced blindness in the fly, although the amplitude of the ERG is reduced as a result of a decreased concentration of the G protein (Lee, 2004c).

Arrestin was originally characterized as a regulatory protein that functions in the inactivation of rhodopsin and other G protein-coupled receptors (GPCRs). Arrestin has been shown to bind clathrin and, under some conditions, to participate in endocytosis of GPCRs. The interaction between rhodopsin and arrestin is usually transient and typically does not lead to endocytosis of rhodopsin. However, in mutant backgrounds that remove the rhodopsin phosphatase (RDGC) or phospholipase C (NORPA), which is the effector for the G protein, the Drosophila rhodopsin Rh1 is stably bound to arrestin, leading to endocytosis. Once internalized through endocytosis, GPCRs are either recycled to the plasma membrane or degraded. In the case of the norpA and rdgC mutant flies, it is not known if the internalized Rh1 is ultimately recycled or degraded. Moreover, stable rhodopsin/arrestin complexes had not previously been observed in wild-type photoreceptor cells (Lee, 2004c).

The results in this work support a molecular model in which constant light leads to blindness through a multi-step process initiated by the formation of stable rhodopsin/arrestin complexes and culminating with the loss of the light receptor, Rh1. A continuous low or moderate level of illumination, in the absence of any mutation, promotes the formation of stable rhodopsin/arrestin complexes. The concentration of Rh1 gradually declines, through a process involving the photoreceptor cell enriched lysosomal protein Sunglasses (Lee, 2004c).

The preceding pathway underlying low-light-induced blindness is supported by genetic evidence. Deletion of the C terminus of Rh1 prevents the formation of stable rhodopsin/arrestin complexes, which result from certain genetic perturbations that dramatically disrupt phototransduction. In wild-type flies exposed to constant illumination, the truncated Rh1 (Rh1^Δ356) also interferes with the formation of Rh1/Arr2 complexes and greatly suppresses light-induced blindness. In addition, arrestin2 mutations suppress the light-induced decline in Rh1 and the impairment in the photoresponse. Elimination of the photoreceptor cell enriched lysosomal protein Sun also reduces the severity of the light-induced blindness, but to a lesser extent than in rh1^Δ356 or arr2^3K/Q mutant backgrounds (Lee, 2004c).

These data suggest that the formation of rhodopsin/arrestin complexes is the key step determining the extent of Rh1 degradation and visual impairment in response to constant light. Additional evidence in support of this model is that the harmful effect of continuous light on the photoresponse is accelerated significantly in a genetic background, rdgC, which increases Rh1/arrestin complexes and Rh1 degradation (Lee, 2004c).

It is proposed that mammalian visual impairment, which results from exposure to continuous but low-intensity light, may also occur through an arrestin-dependent mechanism and reductions in rhodopsin levels. According to this model, stable rhodopsin/arrestin complexes and endocytosis/degradation of rhodopsin do not normally occur to any significant extent in wild-type animals. Rather, as a result of continuous light, the rhodopsin concentration gradually decreases through an arrestin-dependent pathway. It will be interesting to determine whether mutations that affect arrestin trafficking in mammals also suppress visual impairment resulting from constant light (Lee, 2004c).

The model presented here differs from the 'equivalent-light hypothesis', which proposes that phototoxicity and retinal degeneration resulting from continuous light are due to constitutive activation of signaling by rhodopsin or other phototransduction molecules. Although there is compelling evidence that the equivalent-light hypothesis applies to certain forms of morphological degeneration in the retina, the data indicate that the light-induced blindness occurs through an increase in stable rhodopsin/arrestin complexes and degradation of rhodopsin. This conclusion is also supported by the observation that the hypomorphic allele of the Gα_q does not suppress the visual impairment in flies. Although it remains to be determined whether rhodopsin/arrestin complexes occur in wild-type mammals in response to continuous low light, it has been shown that intense levels of light cause a decline in rhodopsin levels in vertebrates. It will be interesting to address whether high- and low-light-induced degradation of mammalian rhodopsin occur through arrrestin-independent and arrestin-dependent mechanisms, respectively, as is the case in Drosophila (Lee, 2004c).

A relevant question is why a mechanism exists for formation of stable rhodopsin/arrestin complexes and degradation of rhodopsin if this phenomenon has negative consequences for the visual response. It has been suggested that endocytosis and degradation of stable rhodopsin/arrestin complexes may normally occur at very low levels and provide a quality control mechanism for eliminating photodamaged rhodopsins, which might otherwise accumulate in photoreceptor cells and have deleterious effects. Thus, the constant-low-light-induced blindness in wild-type animals would appear to be a pathological consequence resulting from excessive activity of a quality control mechanism, which is normally protective (Lee, 2004c).

A dominant rhodopsin mutation triggers two mechanisms of retinal degeneration and photoreceptor desensitization

A variety of rod opsin mutations result in autosomal dominant retinitis pigmentosa and congenital night blindness in humans. One subset of these mutations encodes constitutively active forms of the rod opsin protein. Some of these dominant rod opsin mutant proteins, which desensitize transgenic Xenopus rods, provide an animal model for congenital night blindness. In a genetic screen to identify retinal degeneration mutants in Drosophila, a dominant mutation was identified in the ninaE gene (NinaE^pp100) that encodes RH1, the rhodopsin that is expressed in photoreceptors R1-R6. Deep pseudopupil analysis and histology showed that the degeneration is attributable to a light-independent apoptosis. Whole-cell recordings revealed that the NinaE^pp100 mutant photoreceptor cells are strongly desensitized, which partially masks their constitutive activity. This desensitization primarily results from both the persistent binding of arrestin (ARR2) to the NINAE^pp100 mutant opsin and the constitutive activity of the phototransduction cascade. Whereas mutations in several Drosophila genes other than ninaE induce photoreceptor cell apoptosis by stabilizing a rhodopsin-arrestin complex, NinaE^pp100 represents the first rhodopsin mutation that stabilizes this protein complex. Additionally, the NinaE^pp100 mutation leads to elevated levels of G_qalpha in the cytosol, which mediates a novel retinal degeneration pathway. Eliminating both G_qalpha and arrestin completely rescues the NinaE^pp100-dependent photoreceptor cell death, which indicates that the degeneration is entirely dependent on both G_qalpha and arrestin. Such a combination of multiple pathological pathways resulting from a single mutation may underlie several dominant retinal diseases in humans (Iakhine, 2004).

Arrestin1 mediates light-dependent rhodopsin endocytosis and cell survival while Arrestin2 quenches rhodopsin signaling

Arrestins are pivotal, multifunctional organizers of cell responses to GPCR stimulation, including cell survival and cell death. In Drosophila norpA and rdgC mutants, endocytosis of abnormally stable complexes of rhodopsin (Rh1) and fly photoreceptor Arrestin2 (Arr2) trigger cell death, implicating Rh1/Arr2-bearing endosomes in pro-cell death signaling, potentially via arrestin-mediated GPCR activation of effector kinase pathways. In order to further investigate arrestin function in photoreceptor physiology and survival, Arr2’s partner photoreceptor arrestin, Arr1, was studied in developing and adult Drosophila compound eyes. Arr1, but not Arr2, was shown to be essential for normal, light-induced rhodopsin endocytosis. Also distinct from Arr2, Arr1 is essential for light-independent photoreceptor survival. Photoreceptor cell death caused by loss of Arr1 is strongly suppressed by coordinate loss of Arr2. It was found that Rh1 C-terminal phosphorylation is essential for light-induced endocytosis and also for translocation of Arr1, but not Arr2, from dark-adapted photoreceptor cytoplasm to photosensory membrane rhabdomeres. In contrast to a previous report, no requirement was found for photoreceptor myosin kinase NINAC in Arr1 or Arr2 translocation. This study concludes that the two Drosophila photoreceptor arrestins mediate distinct and essential cell pathways downstream of rhodopsin activation. It is proposed that Arr1 mediates an endocytotic cell-survival activity, scavenging phosphorylated rhodopsin and thereby countering toxic Arr2/Rh1 accumulation; elimination of toxic Arr2/Rh1 in double mutants could thus rescue arr1 mutant photoreceptor degeneration (Satoh, 2005).

Arrestins mediate and regulate cell responses to G protein-coupled receptor (GPCR) signaling. Recruited to activated GPCRs marked by conformational change and phosphorylation, arrestins uncouple G proteins from active receptors and promote receptor endocytosis, desensitizing cells. Endocytosed receptors, in complex with arrestins, initiate a second, non-G protein branch of GPCR signaling, scaffolding and activating kinases to 'signaling endosomes' that particularly engage cytoplasmic pathways. Arrestin-scaffolded kinases notably include c-Src family and mitogen-activated protein kinases (MAPKs), and their activation impacts numerous cell activities including chemotaxis, cell motility, and cell survival. In Drosophila photoreceptors, stable complexes of visual arrestin Arr2 with phosphorylated, active Rh1 metarhodpsin (M-p) cause massive, fatal endocytosis. While mechanisms of this cell death remain to be detailed, disruption of MAPK signaling is a likely suspect. In order to better understand how arrestins cooperate in cell physiology and survival, Arr2's partner visual arrestin, Arr1 was studied in developing and adult Drosophila photoreceptors (Satoh, 2005).

Upon stimulation, like GPCRs generally, both Drosophila Rh1 and vertebrate rhodopsins activate rhodopsin kinase (RK), which phosphorylates the receptor's C terminus. Vertebrate rhodopsin phosphorylation decreases signaling and recruits visual arrestin, whose binding further quenches activity. Elimination of C-terminal phosphorylation sites in a mouse rhodopsin truncation mutant prolongs photoresponses. Mutations of human RK cause Oguchi disease, a congenital stationary night blindness (Satoh, 2005).

The significance of Drosophila Rh1 phosphorylation for photoresponse deactivation is less clear. A phosphoregulated cycle of arrestin binding to, and release from, activated Rh1 recycles the receptor for another round of photodetection. However, flies lacking Rh1 C-terminal phosphorylation sites show normal photoresponse deactivation and Arr2 binds activated Rh1, metarhodopsin (M), without requiring phosphorylation. Indeed, Arr2 binds to M prior to phosphorylation, and hyperphosphorylation of Rh1 by rhodopsin kinase decreases Arr2 binding. Rh1 C-terminal phosphodeficient mutants rescue photoreceptor degeneration in norpA mutants lacking effector phospholipase C and rdgC mutants lacking rhodopsin phosphatase, but a role for Rh1 C-terminal phosphorylation in normal cell physiology remains to be determined (Satoh, 2005).

Like Drosophila photoreceptors, vertebrate rods express two visual arrestins: arrestin, with a long C terminus resembling fly Arr2, and p44, a splice variant whose truncated C terminus resembles the shorter Arr1. Like Arr1, p44 is a minority arrestin, present at levels approximately 10% those of full-length arrestin. Like Arr2, p44 binds unphosphorylated, as well as phosphorylated, active rhodopsin. Upon illumination, p44 redistributes to 'lipid raft' membrane microdomains, while arrestin does not. It has been proposed that the two rod visual arrestins mediate receptor shutoff in differing light regimes, p44 operating in dim illumination normal for rod cell function and arrestin acting in bright illumination. Like RK, mutations of human arrestin cause Oguchi disease, likely resulting from constant, low-level activation of the phototransduction cascade. Constitutive phototransduction cascade activity, 'equivalent light' that entrains the pathophysiology of intense, damaging illumination, may underlie several forms of retinal degeneration (Satoh, 2005).

Vertebrate rhodopsin is unusual among GPCRs in that receptor activation and arrestin recruitment does not promote endocytosis. Mammalian nonvisual cells commonly express two arrestins, β-arrestin1 and β-arrestin2, that target activated receptors for endocytosis at coated pits. Similar to mammalian nonvisual GPCRs, invertebrate rhodopsins are normally endocytosed upon activation; illuminated compound eye photoreceptors accumulate long-lived, rhodopsin-bearing endosomes. Arrestin has been localized to rhodopsin-bearing endosomes of Limulus photoreceptors, and Arr2 has been demonstrated to mediate the massive Rh1 endocytosis that kills norpA and rdgC mutant photoreceptors, but a functional requirement for arrestin in normal light-induced endocytosis has not been demonstrated (Satoh, 2005).

Arrestins translocate from resting cell cytoplasm to activated GPCRs at the plasma membrane; visual arrestins translocate to illuminated photosensory membrane organelles, vertebrate outer segments, and invertebrate rhabdomeres. Normal arrestin translocation in rods of mice lacking phosphorylated rhodopsin rules out simple diffusion to a light-dependent phospho-rhodopsin arrestin binding 'sink'. Translocation fails in photoreceptors deficient for the microtubule motor kinesin-II subunit, KIF3A, consistent with active arrestin transport to outer segments via the connecting cilium. However, recent experiments show arrestin translocation is energy independent, supporting translocation via diffusion to light-activated Rh* rhodopsin. Light-induced Arr2 translocation to Drosophila rhabdomeres promotes adaptation and has been reported to require the photoreceptor Myosin-III, NINAC (Satoh, 2005).

In the present work, Arr1 has been characterized in Drosophila photoreceptors. Arr1 is 7-fold less abundant than Arr2, and Arr1 loss by itself had no reported phenotype; its only reported phenotype is to prolong the photoresponse 10-fold in arr1;arr2 double mutants. This study finds that Arr1 is necessary for normal light-dependent endocytosis and that Arr1 loss causes light-independent photoreceptor cell death. Rh1 C-terminal phosphorylation is essential for Arr1 translocation to stimulated rhabdomeres and for light-induced Rh1 endocytosis. Unexpectedly, it was also found that both Arr1 and Arr2 translocate robustly to rhabdomeres of NINAC null ninaC^P235 flies. Elimination of Arr2 rescues photoreceptor death caused by loss of Arr1. These results demonstrate a vital role for Arr1 in rhodopsin endocytosis and cell survival (Satoh, 2005).

This study has found that the majority arrestin, Arr2, quenches rhodopsin signaling, while Arr1 promotes light-induced rhodopsin endocytosis. Both functions are cell essential. Arr2 loss leads to light-dependent cell death, and results of this study show that Arr1 loss blocks light-dependent rhodopsin endocytosis and causes light-independent cell death. Photoreceptor cell death following endocytosis of abnormally stable Arr2/Rh1 complexes suggests that they signal pro-cell death activity. Results in this study show that Arr2 removal rescues photoreceptor death caused by Arr1 loss. It is proposed that Arr1 normally captures phospho-rhodopsin and targets it to endocytic removal, inhibiting Arr2/Rh1 accumulation and its toxic endocytosis, thereby effecting a vital prosurvival activity (Satoh, 2005).

Results here showing Rh1 C-terminal phosphorylation promotes Arr1 binding and light-dependent endocytosis reveal a new role for Drosophila Rh1 phosphorylation. It is speculated that Rh1 phosphorylation promotes strong Arr1 binding, perhaps mediated by simultaneous engagement of activation and phosphorylation recognition domains as described for vertebrate visual arrestin. Durable Arr1/Rh1 complexes could capture phosphorylated Rh1 for endocytic removal, perhaps downregulating signaling that exceeds rdgC phosphatase capacity and/or promoting Rh1 turnover. It is notable that in normal flies, RLV immunofluorescence shows low levels of Arr2 along with consistently stronger Arr1 staining, suggesting that Arr1 may facilitate Arr2/Rh1 complex endocytosis as well as inhibit its accumulation (Satoh, 2005).

Drosophila photosensory membrane turnover is poorly understood. Unlike vertebrate outer segments, which are constantly renewed at their base as older, distal tip membrane is shed and phagocytosed by apposed retinal pigment epithelium (RPE), Drosophila photoreceptors do not show the circadian shedding of rhabdomere microvilli observed in some compound eyes, and fly eyes lack a phagocytic RPE equivalent. Drosophila Arr1 may mediate rhodopsin turnover, directing phospho-Rh1 to endocytosis and removal under basal, dark conditions as well as in response to light (Satoh, 2005).

A requirement for Arr1 in endocytosis is unexpected, given that its unusually short C-terminal lacks motifs that bind AP-2 and clathrin to promote clathrin-mediated endocytosis (CME). It remains to be determined if Arr1 targets Rh1 to already-nucleated clathrin-coated pits, or if Arr1 contains novel endocytosis-promoting domains. At 364 amino acids in length, Arr1 lacks a Ser366, whose phosphorylation in Arr2 decreases Rh1 binding, but sequence alignment suggests that Arr1 Ser361 may provide a comparable phospho-regulatory site. A third Drosophila arrestin, Kurtz, contains canonical C-terminal clathrin and AP-2 binding domains, but Kurtz is not expressed in the fly eye. GFP-clathrin light chain and α-adaptin localize at the rhabdomere base, suggesting that Rh1 is internalized by CME. Dynamin mediates scisson of invaginated-coated pits from the plasma membrane and dynamin loss inhibits CME. Restrictive temperatures decrease light-induced Rh1 endocytosis in temperature-sensitive dynamin shi^ts mutant flies, suggesting that CME participates in Rh1 endocytosis (Satoh, 2005).

Results of this study show that Arr2 is dispensable for light-induced endocytosis. Arr2 can promote endocytosis when norpA or rdgC mutants stabilize Arr2/Rh1 complexes, but in normal flies, these complexes are transient, destabilized by Arr2 phosphorylation that rapidly follows photostimulation. Arr2 phosphorylation following photostimulation also inhibits clathrin binding, further diminishing endocytic participation. Provocatively, although vertebrate arrestin-rhodopsin complexes are not normally endocytosed, a Retinitis Pigmentosa mutant rhodopsin forms stable arrestin complexes that are endocytosed and disrupt normal cell function (Satoh, 2005).

It is speculated that endocytic Arr2/M-p signaling kills arr1 mutant photoreceptors. Several observations show constitutive, light-independent Rh1 endocytosis: fluorescence microscopy of 7-day-old dark-reared adult wild-type photoreceptors reveals a low level of small Rh1-immunopositive large vesicles (RLVs). In the electron microscope, dark-reared photoreceptors show occasional multivesicular bodies (MVBs) and coated pits at the rhabdomere base. GFP-clathrin light chain and α-adaptin localize to the rhabdomere base of both dark- and light-exposed photoreceptors. Spontaneous rhodopsin activation has been observed in Drosophila, and, once formed, Drosophila M is stable for at least 40 min. It is speculated that Arr1 targets spontaneously activated M-p to basal endocytosis. Either alone or in competition with Arr2, Arr1 constantly scavenges M-p, and, by 3 days without this surveillance, Arr2/M-p complexes, stable in the absence of light-induced Arr2 phosphorylation, reach levels that cause massive and toxic endocytosis. Confocal immunofluorescence detects Arr2 in endosomes of degenerating arr1 mutant photoreceptors. Elimination of endocytosis in arr1;arr2 double mutants may thus rescue arr1 mutant degeneration. Constant surveillance over a rhabdomere's approximately 100 million receptors and timely capture and removal of inappropriately signaling receptors may be essential for normal photoreceptor physiology (Satoh, 2005).

Photoreceptor cell death in norpA mutants does not depend on apoptosis pathway proteins Rpr, Hid, Grim, and Dronc caspase, and expression of apoptosis inhibitor p35 does not rescue norpA or rdgC degeneration. Resemblance between Arr2-mediated Drosophila photoreceptor cell death and nonapoptotic, autophagic cell death of mouse striatal cells in response to neurokinin-1 receptor activation has been noted. Observations made in this study that the morphology of photoreceptor cell death in Arr1 mutants does not simply resemble apoptosis, that expression of antiapoptotic p35 gives only modest photoreceptor rescue, and a failure to detect activated caspase 3 or caspase 7 in dying photoreceptors, suggest that nonapoptotic, autophagic pathways may participate in arr1 mutant retinal degeneration. Arrestin-mediated GPCR signaling pathways intersect pathways of autophagy-regulated cell survival, potentially including AKT/PKB prosurvival signaling (Satoh, 2005).

The observation that Arr2 translocates normally in photoreceptors lacking NINAC differs from that of Lee and Montell (2004b), who report loss of Arr2 translocation in ninaC^P235 null mutant photoreceptors. This study finds, in contrast, robust light-induced translocation of Arr1 and Arr2 in ninaC^P235 photoreceptors. Some contribution of NINAC to Arr2 translocation may be evidenced by the increased frequency of Arr1 staining in ninaC^P235 rhabdomeres; if Arr2 competes with Arr1 and Arr2 delivery is decreased in mutants, Arr1 may be advantaged (Satoh, 2005).

Differences in genetic strain, illumination levels, and age have been ruled out as the reason for Arr2 translocation in ninaC^P235 observed here but not in Lee and Montell. The different results may be attributable in part to different immunolocalization methods; whole-mount confocal immunofluorescence shows excellent subcellular detail. These observations are otherwise in good agreement with previous reports of arrestin translocation. Results presented in this study are consistent with diffusion-based arrestin translocation, paralleling recent observations that mouse arrestin translocation does not require energy (Satoh, 2005).

Drosophila Arr1 and Arr2 share hallmark arrestin capacities: both bind and quench activated receptor signaling, and both can promote endocytosis. However, in normal cells, each has emphasized one of these two activities: Arr2 specializes in Rh1 signal quenching while Arr1 mediates endocytosis of activated Rh1. The mechanistic bases for this specialization remain to be determined, but the normal balance of both operations is cell essential. Arrestin pathways are broadly conserved across eukaryotes, and Drosophila photoreceptors offer a useful window into endocytosis and signaling, an 'inseparable partnership' that impacts virtually all cell physiology (Satoh, 2005).

An essential role for endocytosis of rhodopsin through interaction of visual arrestin with the AP-2 adaptor

A class of retinal degeneration mutants have been identified in Drosophila in which the normally transient interaction between arrestin2 (Arr2) and rhodopsin is stabilized and the complexes are rapidly internalized into the cell body by receptor-mediated endocytosis. The accumulation of protein complexes in the cytoplasm eventually results in photoreceptor cell death. The endocytic adapter protein AP-2 is essential for rhodopsin endocytosis through an Arr2-AP-2ß interaction, and mutations in Arr2 that disrupt its interaction with the ß subunit of AP-2 prevent endocytosis-induced retinal degeneration. If the interaction between Arr2 and AP-2 is blocked, this also results in retinal degeneration in an otherwise wild-type background. This indicates that the Arr2-AP-2 interaction is necessary for the pathology observed in a number of Drosophila visual system mutants, and suggests that regular rhodopsin turnover in wild-type photoreceptor cells by Arr2-mediated endocytosis is essential for photoreceptor cell maintenance (Orem, 2006).

The results demonstrate that Drosophila Arr2 plays a role as a mediator of rhodopsin endocytosis by interacting with the AP-2 adaptor complex. The AP-2 adaptor complex is required for the endocytosis of rhodopsin during certain pathological conditions and the disruption of this complex rescues norpA-mediated retinal degeneration. In addition, flies with a single point mutation in the AP-2 binding domain of Arr2, norpA-induced endocytosis of stable rhodopsin-arrestin complexes and the subsequent retinal pathology is blocked. Internalization of rhodopsin by an Arr2-AP-2 interaction is essential for photoreceptor cell viability (Orem, 2006).

Certain Arr2 variants bind tightly to rhodopsin. This results in the recruitment of the endocytic machinery and cell death via excessive rhodopsin endocytosis. In this study, an Arr2 variant (arr2^R393A) was found that binds more tightly to rhodopsin than wild-type Arr2. However, this mutant does not trigger extensive retinal degeneration. This is further evidence for the essential role of the Arr2-AP-2 interaction in receptor internalization. In this Arr2 background complexes are formed between Arr2 and Rh1, but since Arr2 cannot interact with AP-2 they are not internalized and no photoreceptor cell death is induced (Orem, 2006).

These data point to an essential role for the endocytosis of rhodopsin through Arr2 in the maintenance of photoreceptor cells. Previous work has implicated an essential role for Drosophila arrestins in endocytosis; however, these studies used mutations that either blocked all endocytosis in the photoreceptor cell or utilized loss-of-function mutants that could have other effects on the photoreceptor. Photoreceptor cell degeneration can be induced by inhibiting dynamin function with a dominant negative mutation or by blocking AP-2 function, as described in this study. However, it is possible that a global inhibition of endocytosis may halt the internalization of compounds essential for cell viability. Therefore, cell death may be unrelated to defects in the phototransduction cascade or the internalization of rhodopsin. In addition, retinal degeneration in Arr1 mutants may be due to the defect in endocytosis but pleiotropic affects associated with the loss of Arr1 may also contribute to the aberrant retinal morphology. By using Arr2 variants that are unable to internalize rhodopsin, the endocytosis of one protein (rhodopsin) was selectively blocked while leaving general endocytosis intact. Therefore, the retinal degeneration observed in this study is due solely to defects in rhodopsin internalization through its interaction with Arr2 (Orem, 2006).

One interesting question concerns the purpose of the essential role for rhodopsin-Arr2 endocytosis. One possibility is that this may be a mechanism to remove damaged rhodopsin from the cell. If rhodopsin is photochemically damaged in such a way that it becomes constitutively active, it would be deleterious to the cell, and would need to be removed. Presumably constitutively active rhodopsin would form a stable complex with Arr2 and be targeted for endocytosis through the interaction of arrestin with the AP-2 adaptor complex. This would provide a surveillance mechanism for the cell, whereby defective rhodopsin molecules are quickly and efficiently removed. Second, it may be an adaptive mechanism. In high light conditions the amount of activated rhodopsin may exceed the ability of arrestin to quickly decouple the metarhodopsin from the phototransduction pathway. This would lead to a loss of visual temporal resolution and be detrimental to cell viability. However, under high light conditions at any given time a higher percentage of the cellular arrestin will be bound to rhodopsin and increase the likelihood that the Arr2 bound to rhodopsin will interact with AP-2 and drive internalization of rhodopsin. This would serve to lower the concentration of rhodopsin available to activate the phototransduction cascade and thereby reduce sensitivity under conditions of intense illumination (Orem, 2006).

Light-induced translocation of Drosophila visual Arrestin2 depends on Rac2

Photoreceptor cells are remarkable in their ability to adjust their sensitivity to light over a wide range of intensities. Rapid termination of the photoresponse is achieved in part by shuttling proteins in and out of the light-transducing compartment of the photoreceptor cells. One protein that undergoes light-dependent translocation is the rhodopsin regulatory protein arrestin. However, the mechanisms coupling rhodopsin to arrestin movement are poorly understood. This study shows that light-dependent shuttling of the major arrestin in Drosophila photoreceptor cells, Arrestin2 (Arr2), occurs independently of known elements of the phototransduction cascade. Disruptions of the trimeric G protein, phospholipase Cβ, the TRP channel, or the Na⁺/Ca²⁺ exchanger did not influence Arr2 localization. Rather, it was found that loss of the small GTPase Rac2 severely impaired Arr2 movement and prolonged the termination of the photoresponse. These findings demonstrate that light-induced translocation of Arr2 occurs through a noncanonical rhodopsin/Rac2 pathway, which is distinct from the classical phototransduction cascade (Elsaesser, 2010).

Activity-dependent shuttling of signaling proteins between the cell surface and intracellular compartments is a widespread phenomenon which contributes to the magnitude and duration of signaling in neurons and many other cell types. One of the earliest demonstrations of activity-dependent translocation of signaling proteins from one cell compartment to another was the light-induced translocation of visual arrestin from the inner to the outer segments of rod photoreceptor cells over the course of a few minutes. Light-dependent shuttling of signaling proteins is an evolutionarily conserved phenomenon, as photostimulation also triggers the movement of the Drosophila visual arrestins from the cell bodies to the fly counterpart to rod outer segments, the rhabdomeres. The trimeric G proteins that function in mammalian and Drosophila phototransduction undergo light-dependent translocation as well, as does the Drosophila transient receptor potential-like (TRPL) channel. However, in contrast to the arrestins, these latter proteins shuttle out of the outer segments and rhabdomeres in response to light. The movements of these signaling proteins have important physiological consequences, as they contribute to light adaptation and termination of the photoresponse and thus are crucial for the ability of photoreceptor cells to adjust their sensitivity to the surrounding light conditions (Elsaesser, 2010).

The mechanisms and signaling pathways controlling the translocation of the Drosophila arrestins, G protein, and TRPL proteins have been explored but are incompletely understood. The light-dependent movement of the major visual arrestin, referred to as Arrestin2 (Arr2), requires interaction with PIP₃. In addition, the NINAC myosin III has been reported to contribute to the spatial reorganization of G_q, TRPL, and Arr2, although Arr2 depends on NINAC only under blue but not white light. Because light triggers the translocations, they would be expected to require activity of the phototransduction cascade. In flies, light-activated rhodopsin engages a heterotrimeric G protein, G_q, leading to stimulation of a phospholipase C (PLC) and opening of the TRP and TRPL cation channels. Visual arrestin binds to rhodopsin and attenuates signaling by dislodging the heterotrimeric G protein associated with the light-activated rhodopsin. Indeed, movement of TRPL requires G_q and PLC, although the light-dependent shuttling of G_q has been reported to occur independently of PLC, TRP, or TRPL (Elsaesser, 2010).

The current work found that the dynamic spatial redistribution of Arr2 from the cell bodies to the rhabdomeres required rhodopsin, but did not depend on any of the other known components of the phototransduction cascade. These include G_q, PLC, TRP, TRPL, the Na⁺/Ca²⁺ exchanger (CalX), and protein kinase C. Rather, it was found that the small GTPase Rac2 interacts with rhodospsin and is essential for the translocation of Arr2 into the rhabdomeres. As is the case with photoreceptor cells expressing Arr2 derivatives that do not translocate efficiently, mutations in rac2 cause a defect in termination of the photoresponse. These data indicate that the light-dependent movement of Arr2 depends on a parallel phototransduction cascade that is initiated by coupling of rhodopsin to Rac2 (Elsaesser, 2010).

Rhodopsin is the archetypal G-protein-coupled receptor, which defines a family of highly related visual pigments. Before the current work, all light-activated pathways known to be physiologically required in photoreceptor cells functioned through heterotrimeric G proteins. Alternative candidate effectors for rhodopsin were small GTPases, because mammalian rhodopsin interacts with a Rho family member and activates this small GTPase in a light-dependent fashion. However, no role has been ascribed for transducing light activity through a rhodopsin/small GTPase pathway (Elsaesser, 2010).

This study found that the light-induced movement of Arr2 from the cell bodies to the rhabdomeres was strictly dependent on Rac2. Moreover, termination of the photoresponse was severely impaired in rac2^δ flies. Thus, the findings indicate a signaling mechanism underlying one form of light adaptation which entails transduction of the light signal through a small GTPase, rather than engaging Rh1 with the G_q/PLC/TRP cascade (Elsaesser, 2010).

In addition to its role as a light receptor, rhodopsin plays a structural role during photoreceptor cell morphogenesis. This light-independent function of rhodopsin is mediated by Rac1. In contrast, the requirement for Rac2 for Arr2 movement did not appear to be due to a morphological defect because the ultrastructures of wild-type and rac2^δ null mutant photoreceptor cells were indistinguishable. Thus, although the roles of Rac1 and Rac2 are often considered interchangeable, Rac2 is specifically required for light-dependent translocation of Arr2 translocation in adult photoreceptor cells, whereas Rac1 has a structural role during development. Other nonredundant functions of Rac2 have been described in the Drosophila cellular immune response (Elsaesser, 2010).

It appears that two phototransduction pathways contribute to light-dependent movements of signaling proteins in Drosophila photoreceptor cells. The classical pathway is required for shuttling of TRPL because it is dependent on most elements of the phototransduction cascade including the PLC. However, the movement of Arr2 depends on a second noncanonical, Rac2-dependent pathway. Light-dependent shuttling of the Gα_q may also function through this second pathway because it occurs normally in mutants missing PLC, protein kinase C, or TRP (Elsaesser, 2010).

The current work raises questions concerning the nature of the proteins that function in concert with Rh1/Rac2 signaling in photoreceptor cells. Finally, because the mammalian Rac1, which is the GTPase most related to Drosophila Rac2, is activated in a light-dependent manner, it is proposed that rhodopsin/Rac signaling may be an evolutionarily conserved mechanism controlling light-induced arrestin translocation and light adaptation in mammalian rods and cones (Elsaesser, 2010).

Regulation of arrestin translocation by Ca2+ and myosin III in Drosophila photoreceptors

Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown. Using real-time fluorescent imaging of Arr2-green fluorescent protein translocation in dissociated ommatidia, this study shows that translocation is profoundly slowed in Ca(2+)-free solutions. Conversely, in a blind PLC mutant with ~100-fold slower translocation, rapid translocation was rescued by the Ca(2+) ionophore, ionomycin. In mutants lacking NINAC (calmodulin [CaM] binding myosin III) in the cell body, translocation remained rapid even in Ca(2+)-free solutions. Immunolabelling revealed that Arr2 in the cell body colocalizes with NINAC in the dark. In intact eyes, the impaired translocation found in trp mutants was rescued in ninaC;trp double mutants. Nevertheless, translocation following prolonged dark adaptation was significantly slower in ninaC mutants, than in wild type: a difference that was reflected in the slow decay of the electroretinogram. The results suggest that cytosolic NINAC is a Ca(2+)-dependent binding target for Arr2, which protects Arr2 from immobilization by a second potential sink that sequesters and releases arrestin on a much slower timescale. It is proposed that rapid Ca(2+)/CaM-dependent release of Arr2 from NINAC upon Ca(2+) influx accounts for the acceleration of translocation by phototransduction (Hardie, 2012).

Photoreceptors are highly polarized cells with membrane-rich photosensory compartments separated from the rest of the cell body. It has recently become widely recognized that several phototransduction proteins translocate between these compartments in response to light, representing a form of long-term light and dark adaptation. One of the best-studied examples is arrestin, which terminates the light response by binding to photo-isomerized rhodopsin (metarhodopsin). In dark-adapted photoreceptors most arrestin localizes to the cell body in both vertebrate and insect photoreceptors, but on illumination translocates to the photosensory compartmen. In fly photoreceptors the photosensory compartment is represented by the rhabdomere, a light-guiding, rod-like stack of ~30,000 densely packed apical microvilli loaded with rhodopsin and proteins of a phototransduction cascade mediated by heterotrimeric Gq protein, phospholipase C (PLC), and Ca²⁺-permeable 'transient receptor potential' (TRP) channels (Hardie, 2012).

Although the possible role of active transport by molecular motors remains debated, recent evidence in both vertebrate rods and Drosophila microvillar photoreceptors supports an essentially passive diffusional model of arrestin translocation, down gradients established by light-regulated 'sinks'. Recent studoes provided evidence that metarhodopsin (M) is the major light-activated sink in fly rhabdomeres by showing that the dominant arrestin isoform (Arr2) translocated in a 1:1 stoichiometric relationship to the number of rhodopsin photo-isomerizations (Satoh, 2010). This study also showed that Arr2 translocation was very rapid (τ ~10 s), but profoundly slowed in mutants of various phototransduction proteins including Gq, phospholipase C (PLC) (norpA), and the major Ca²⁺-permeable TRP channel (Satoh, 2010). The evidence suggested that Ca²⁺ influx via the light-sensitive channels was required to accelerate Arr2 translocation, possibly by releasing Arr2 from a Ca²⁺-dependent cytosolic sink (Satoh, 2010). However, direct evidence for the role of Ca²⁺ was lacking, while the identity of the putative cytosolic sink and the mechanism(s) mediating the acceleration remained unresolved (Hardie, 2012).

This study shows directly that Ca²⁺ is both necessary and sufficient to accelerate Arr2 translocation and provides evidence that the Ca²⁺-regulated cytosolic sink is the cytosolic isoform of NINAC, a calmodulin (CaM) binding myosin III. The evidence also suggests the existence of another potential Ca²⁺ dependent cytosolic sink, which sequesters and releases arrestin on a much slower timescale, and that NINAC protects Arr2 from sequestration and immobilization by this site. The data support a mechanism for the Ca²⁺-dependent translocation of Arr2 that is remarkably similar to a previously proposed disinhibitory mechanism of Ca²⁺-dependent inactivation of M (Liu, 2008) required for rapid termination of the light response (Hardie, 2012).

Previously studies have proposed that Ca²⁺ influx via the light-sensitive TRP channels is required for rapid Arr2 translocation, because the slow translocation in trp mutants could be rescued by genetic elimination of Na⁺/Ca²⁺ exchanger activity. The present study confirmed the role of Ca²⁺ directly by imaging Arr2-GFP translocation in dissociated ommatidia, and showing that extracellular Ca²⁺ is required for rapid Arr2 translocation. Ca²⁺ was not only required, but also sufficient to enable rapid translocation without any products of PLC activity, since the Ca²⁺ ionophore, ionomycin, fully rescued translocation in blind norpA mutants lacking PLC. Significantly, it was found that the requirement of Ca²⁺ for rapid translocation was obviated in null mutants of NINAC (CaM binding MyoIII), with the cytosolic p132 isoform of NINAC alone being sufficient to slow down translocation in Ca²⁺-free conditions. This suggests that cytosolic NINAC p132 acts as a Ca²⁺/CaM-dependent 'brake' on translocation by binding Arr2, releasing it in response to Ca²⁺ influx associated with the photoresponse. This conclusion was further supported by finding that the ninaC mutation rescued rapid translocation in trp mutants (in ninaC;trp double mutants). However, the failure to rescue translocation in norpA;ninaC mutants, except under special conditions, and the demonstration of significant slowing of translocation following prolonged dark adaptation in ninaC mutants also indicated the existence of a second Ca²⁺-dependent cytosolic sink (Hardie, 2012).

Despite an earlier study reporting that Arr2 translocation was impaired in ninaC mutants, as shown here and previously (Satoh, 2005; Satoh, 2010), Arr2 translocation, whether of endogenous Arr2 or GFP-tagged Arr2, appears essentially intact in ninaC-null mutants. In fact, far from being impaired, the results indicate that translocation can be rescued by ninaC mutations under conditions where translocation is slowed down by reduced Ca²⁺ influx. Although translocation was significantly slower in ninaC mutants following prolonged dark adaptation, it was never prevented and full translocation was always achieved within ~2-3 min of appropriate illumination (Hardie, 2012).

A novel phenotype of ninaC^P235-null mutants, and also ninaC^Δ174 mutants lacking only the rhabdomeric p174 isoform, was the complete absence of an early rapid increase in fluorescence routinely observed during the first ~500 ms of measurements of Arr2-GFP fluorescence from the DPP of wild-type photoreceptors or dissociated ommatidia. With a time constant of ~260 ms, this rapid phase was ~40× faster than the overall translocation (τ ~10 s) itself the fastest protein translocation reported in a photoreceptor to date, and probably diffusionally limited. It therefore seems unlikely that the fast phase represents a 40x faster, ninaC-dependent movement of Arr2 from cell body into the rhabdomere. Instead we suggest that it represents a change in the fluorescence efficiency of Arr2-GFP as it is released (via Ca²⁺ influx) from the rhabdomeric p174 NINAC isoform. A lower fluorescence when bound to NINAC might reflect crowding of the GFP-fluorophore, or could be due to some other feature of the nano-environment of the fluorophore when Arr2 is bound to NINAC in the microvilli. This interpretation was supported by the ability to eliminate the rapid phase by pre-illumination with long wavelength light, which induces Ca²⁺ influx without net change in M. The rapid phase then re-emerged with a time constant of ∼3 s in the dark, presumably representing rapid rebinding of Arr2-GFP to NINAC (Hardie, 2012).

After more than a few minutes in the dark, Arr2 translocation into the rhabdomere became progressively slower, with clear functional consequences in a parallel slowing of the decay of the ERG. This gradual slowing was considerably more pronounced in ninaC-null mutants, where it si proposed that the slowing represents binding or sequestration of Arr2 via one or more NINAC-independent target(s) or compartment(s). Release from such sites also requires activation of the phototransduction cascade, and translocation could be accelerated back to levels typical of short dark-adaptation times by pre-illumination with bright orange light, which itself does not generate a net increase in M. It seems likely that the rise in Ca²⁺ is also responsible for release from this site; however, the involvement of other products of the phototransduction cascade cannot be excluded. The identity of this second site or compartment remains a subject for future investigation. Given previous reports that Arr2 can bind to phosphoinositides, negatively charged phosphoinositide species on endomembranes, which could be screened by Ca²⁺, might represent promising candidates. Drosophila Arr2 is an unusually basic (positively charged) protein and may thus have a strong tendency to bind to such sites. The finding that the slowing of translocation with dark adaptation was more pronounced in ninaC mutants suggests that one of the functions of cytosolic NINAC may be to prevent immobilization of Arr2 by this alternative potential sink. Because the Ca²⁺-dependent release of Arr2 from NINAC occurs on a subsecond timescale, this then allows more rapid translocation (and hence recovery of the electrical response) after a period in the dark (Hardie, 2012).

These results demonstrate that Arr2 translocation is accelerated by Ca²⁺ influx, and suggest that this is mediated by a disinhibitory mechanism, whereby NINAC p132 binds to Arr2 under low Ca²⁺ conditions in the dark, rapidly releasing it in response to Ca²⁺ influx associated with the photoresponse. Although inferred from essentially independent experiments, this mechanism is strikingly similar to one previously proposed for the rapid, Ca²⁺-dependent inactivation of M during the light response itself. In that study the time constant of M inactivation by Arr2 was found to be accelerated from ~200 ms under Ca²⁺-free conditions to ~20 ms following Ca²⁺ influx. This Ca²⁺ dependence was eliminated in both ninaC-null mutants, and in ninaC^Δ174 mutants lacking the rhabdomeric p174 (but not in ninaC^Δ132 mutants lacking cytosolic p132). The results also indicated a disinhibitory mechanism, leading to the proposal that Arr2 in the microvilli was bound to rhabdomeric NINAC p174 under low Ca²⁺ conditions in the dark, thus hindering its diffusional access to activated M. Ca²⁺ influx via the first activated TRP channels, then rapidly releases Arr2, allowing it to diffuse, bind to, and inactivate M (Hardie, 2012).

NINAC p132 and p174 share a common CaM binding site (CBS) and although p174 has a second CBS not found in p132, the pronounced slowing of translocation with dark adaptation in null ninaC^P235 mutants was recapitulated in mutants lacking the common CBS. It is therefore suggested that essentially the same mechanism underlies the Ca²⁺-dependent rapid translocation of Arr2, but now acting via NINAC p132 rather than p174 and working over much larger distances (several micrometers as opposed to the nanometer dimensions of single microvilli) and hence slower timescales (Hardie, 2012).

The proposed interaction between NINAC and Arr2 finds some support from biochemical data reporting coimmunoprecipitation of Arr2 and NINAC in extracts from whole heads (Lee, 2004). However, that study also reported that both Arr2 and NINAC had significant in vitro affinity for phosphoinositides. It was proposed that the NINAC/Arr2 association was indirect and mediated by both Arr2 and NINAC binding to phosphoinositide-rich membrane. Although the current results clearly indicate that Ca²⁺-dependent modulation of Arr2 binding to M and Ca²⁺-dependent translocation of Arr2 are both dependent upon NINAC, the possibility cannot be excluded that the interaction is mediated indirectly via a NINAC-dependent target. Ultimate verification will require direct demonstration of NINAC/Arr2 binding and its dependence upon Ca²⁺/CaM (Hardie, 2012).

The regulated multisink model proposed in this study differs fundamentally from an earlier model in which NINAC was proposed as a molecular motor transporting Arr2 in phosphoinositide-rich vesicles. By contrast it shows strong parallels with current models for arrestin translocation in vertebrate rods. Here, phosphorylated rhodopsin represents the light-activated sink in the outer segments, while microtubules have been proposed as the cytosolic sink in the inner segments. There is also evidence indicating light-regulated acceleration of translocation in vertebrate rods. The mechanism is unclear; however, intriguingly a recent study has implicated roles for PLC and protein kinase C possibly stimulating release of arrestin from its cytosolic sink (Hardie, 2012 and references therein).

Such regulated-sink models have the advantage of simplicity: directed translocation requires no more than diffusion coupled with regulated binding, can rapidly transport virtually unlimited quantities of protein, and per se consumes essentially no energy. While it can be conveniently studied in photoreceptors with their distinctive polarized morphologies and high concentrations of transduction machinery, translocation according to the same general principles may represent a general and elegant solution to the problem of directed movements of signaling proteins (Hardie, 2012).

Role of rhodopsin and arrestin phosphorylation in retinal degeneration of Drosophila

Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. Enhanced green fluorescent protein-tagged arrestin 2 (Arr2) was used to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. It was demonstrated that during degeneration of norpA^P24 photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpA^P24 is the failure to activate CaMKII (Ca²⁺/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca²⁺ entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. How reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration was investigated. To uncover the consequence underlying a lack of CaMKII activation, ala¹ flies were characterized in which CaMKII was suppressed by an inhibitory peptide, and it was shown that morphology of rhabdomeres was not affected. In contrast, it was found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpA^P24 photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, it is concluded that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration (Kristaponyte, 2012).

EFFECTS OF MUTATION

Arrestins have been implicated in the regulation of many G protein-coupled receptor signaling cascades. Mutations in two Drosophila photoreceptor-specific arrestin genes, arrestin 1 and arrestin 2, were generated. Analysis of the light response in these mutants shows that the Arr1 and Arr2 proteins are mediators of rhodopsin inactivation and are essential for the termination of the phototransduction cascade in vivo. The saturation of arrestin function by an excess of activated rhodopsin is responsible for a continuously activated state of the photoreceptors known as the prolonged depolarized afterpotential. In the absence of arrestins, photoreceptors undergo light-dependent retinal degeneration as a result of the continued activity of the phototransduction cascade. These results demonstrate the fundamental requirement for members of the arrestin protein family in the regulation of G protein-coupled receptors and signaling cascades in vivo (Dolph, 1993).

Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration; ceramidase rescues retinal degeneration in arrestin and phospholipase C mutants

Mutations in proteins of the Drosophila phototransduction cascade, a prototypic guanine nucleotide-binding protein-coupled receptor signaling system, lead to retinal degeneration and have been used as models to understand human degenerative disorders. In this study, modulating the sphingolipid biosynthetic pathway rescued retinal degeneration in Drosophila mutants. Targeted expression of Drosophila neutral ceramidase rescues retinal degeneration in arrestin and phospholipase C mutants. Decreasing flux through the de novo sphingolipid biosynthetic pathway also suppresses degeneration in these mutants. Both genetic backgrounds modulate the endocytic machinery because they suppressed defects in a dynamin mutant. Suppression of degeneration in arrestin mutant flies expressing ceramidase correlates with a decrease in ceramide levels. Thus, enzymes of sphingolipid metabolism may be suitable targets in the therapeutic management of retinal degeneration (Acharya, 2003).

Sphingolipids are integral components of eukaryotic cell membranes and also a rich source of second messengers for several signal transduction cascades. Sphingolipid metabolism generates and interconverts various metabolites including ceramide, sphingosine, and sphingosine 1-phosphate, which are second messengers in diverse signaling pathways that affect cell cycle, apoptosis, and angiogenesis, among others. Serine palmitoyl-CoA transferase (SPT) catalyzes the rate-limiting first step in the de novo biosynthesis of sphingolipids including ceramide. Ceramidases hydrolyze ceramide to sphingosine, and neutral or alkaline ceramidase is proposed to function in signaling. Mutant analyses in yeast have implicated enzymes of sphingolipid metabolism in endocytic membrane trafficking events. This study modulated the sphingolipid biosynthetic pathway in vivo in Drosophila and examined its effects on mutants with endocytic defects in photoreceptors (Acharya, 2003).

Each of the 800 ommatidia of a Drosophila compound eye consists of eight photoreceptor cells (R1 to R8). Each cell has a rhabdomere, a specialized microvillar structure derived from the plasma membrane that houses the phototransduction machinery. Rhabdomere architecture is sensitive to perturbations in the phototransduction cascade and has been used to monitor photoreceptor degeneration. Drosophila phototransduction is a prototypic GTP-binding protein-coupled receptor (GPCR) cascade that is initiated by light activation of rhodopsin. Association of arrestin 2 with phosphorylated rhodopsin leads to deactivation of rhodopsin. Drosophila arrestin 2 also acts as a clathrin adaptor, mediating endocytosis of arrestin-rhodopsin complexes. arr2³ mutants (Val⁵² to Asp) make less than 1% of the protein, are defective in endocytosis, accumulate abnormal multivesicular bodies, show extensive retinal degeneration, and undergo necrotic cell death. These changes also result in a precipitous drop in rhodopsin levels in these photoreceptors. Thus, arr2³ photoreceptors provide a sensitive background for examining the in vivo effects of modulating the sphingolipid pathway in endocytosis (Acharya, 2003).

The ceramidase gene was cloned into an UAS vector, and it was expressed in the Drosophila eye with the use of a glass multimer reporter (GMR)-Gal4 driver. Extracts from these fly heads showed increased neutral ceramidase activity, confirming that the protein was a bona fide neutral ceramidase. Expression of GMR-Gal4; UAS-ceramidase in R1 to R6 did not affect photoreceptor integrity. Expression of ceramidase in arr2³ rescued photoreceptor degeneration. In transmission electron micrographs (TEMs), rhabdomeres were intact, and multivacuolar bodies and degenerating photoreceptors, characteristic of arr2³ mutants, were completely absent in a 3-day arr2³ fly expressing ceramidase. A near-wild-type level of rhodopsin was seen in rescued flies, reflecting photoreceptor integrity. Thus, expression of ceramidase in arr2³ preserved rhabdomere structure and organization. Although defective, newly eclosed arr2³ flies transduce light signals. As they age, they undergo progressive degeneration and lose their ability to transmit signals. To test whether rescued flies retain their functional ability to signal, electroretinogram recordings (ERG) were carried out from 7-day-old flies exposed to light. Because of extensive degeneration, ERGs of arr2³ flies had a very small amplitude, whereas arr2³ flies expressing ceramidase showed a robust response. However, the slow inactivation kinetics characteristic of arr2³ still persisted in the rescued flies. Rescued arrestin mutant flies transduced signals even on aging because the structural integrity of these photoreceptors was preserved (Acharya, 2003).

Because expression of ceramidase in arrestin mutant flies suppressed retinal degeneration and because ceramidase hydrolyzed ceramide, it was reasoned that the rescue would be accompanied by a decrease in ceramide levels in these photoreceptors. Electrospray ionization tandem mass spectrometry (ESI/MS/MS) was used to estimate ceramide levels in lipid extracts of membranes prepared from fly heads of control, ceramidase expressor, arrestin mutant, and arrestin mutant expressing ceramidase. Ceramide molecular species containing tetradecasphingenine and their 2-hydroxy counterparts were identified by negative ion ESI/MS/MS with neutral loss of 200.2 and 271.2 mass units, respectively. As expected, expression of ceramidase reduces the ceramide levels in control animals. Lipid extracts from arrestin mutants showed an increase in ceramide levels, probably reflecting changes accompanying the severely degenerating photoreceptors. Expression of ceramidase in arrestin mutants decreased ceramide levels by 50% in all species measured. Thus, rescue of degeneration correlated with a decrease in ceramide levels in the mutant flies. Because sphingosine is a product of the ceramidase reaction, whether increased sphingosine could suppress retinal degeneration in arrestin mutants was evaluated. Viability of certain lace alleles (LCB2 subunit of SPT, a.k.a. Serine palmitoyltransferase subunit II), which are deficient in de novo sphingosine biosynthesis, is increased when flies are raised in food supplemented with sphingosine. Arrestin mutant flies were raised under similar conditions and their photoreceptors were examined by electron microscopy. The rhabdomeres of R1 to R6 cells showed no suppression; instead, they showed enhanced degeneration of these photoreceptors. Thus, it is believed that sphingosine on its own is not a likely candidate for suppression of degeneration in the present study; instead, suppression correlated with decreased ceramide levels in rescued mutant flies (Acharya, 2003).

Ceramidase suppressed degeneration in arr2³ mutants with chronically active rhodopsin and defects in clathrin-dependent endocytosis. It is possible that ceramidase suppressed degeneration by altering the balance of the endocytic pathway, thereby alleviating cytotoxicity arising from defective endocytosis. To test this, ceramidase was expressed in a dynamin mutant background in the eye. Dynamin is a guanosine triphosphatase essential for clathrin-mediated endocytosis. In Drosophila, a temperature-sensitive mutant of dynamin, shibire (shi^ts1), has a general defect in endocytosis. These results were recapitulated in mammalian cells when a similar mutant dynamin was overexpressed. A temperature-sensitive dominant-negative mutant, UAS-shi^ts1, under the control of a GMR-Gal4 driver, was used to preferentially express the mutant protein in the eye. These photoreceptors showed profound retinal degeneration characterized by loss of rhabdomere and accumulation of multivesicular bodies and vacuoles in R1 to R6, whereas R7 was largely unaffected. Ceramidase expression suppressed degeneration in UAS-shi^ts1 photoreceptors. Rhabdomeres were largely intact, vacuolated cells were fewer, and trapezoidal arrangement of rhabdomeres was retained. As in other degenerating mutants, rhodopsin levels were low in shi^ts1 mutants compared with those of the wild type but were restored upon ceramidase expression (Acharya, 2003).

Whether SPT, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, could affect the degeneration observed in these mutants was tested. In Drosophila, the Lace gene encodes the LCB2 subunit of SPT. The P-lacW-inserted lace allele l(2)k05305 is an insertion of a P-element 8 to 9 base pairs upstream of the transcription start site of lace and is homozygous lethal. arr2³ and UAS-shi^ts1 mutants were crossed into the lace heterozygous background and photoreceptors were examined by transmission electron microscopy. lace heterozygotes had intact photoreceptors. lace partially suppressed retinal degeneration in arr2³ mutants and in UAS-shi^ts1 mutants (Acharya, 2003).

Finally, whether ceramidase and lace suppressed degeneration in a phospholipase C mutant, where endocytosis has been implicated in the degenerative process, was examined. Norp A encodes an eye-specific phospholipase C that activates GPCR signaling by generating inositol trisphosphate and diacylglycerol. norp A mutant flies do not show light-induced receptor potential and are blind. Although norp A mutants degenerated slowly, these changes were obvious even in 3-day-old flies. Expression of ceramidase in a norp A mutant suppressed retinal degeneration. Lace heterozygotes also suppressed norp A degeneration. arr2³ mutants undergo necrotic cell death, whereas norp A mutants accumulate rhodopsin-arrestin complexes and undergo apoptotic cell death. Thus, regardless of the mode of cell death ceramidase expression and lace mutant rescued degeneration. Because they also suppressed degeneration in a dynamin mutant, it is inferred that the sphingolipid pathway exerts its beneficial effect by altering the dynamics of the endocytic process. This is supported by observations that a sphingoid base is required for yeast endocytosis and that in mammalian cells ceramide analogs modulate fluid-phase and receptor-mediated endocytosis (Acharya, 2003).

The molecular details of suppression of retinal degeneration by ceramidase overexpression and lace mutant remain to be elucidated. A common denominator in both situations is the likely decrease in concentrations of ceramide, which could be responsible for activating a cascade that suppresses degeneration (Acharya, 2003).

A large volume of work suggests that receptor desensitization, endocytosis, and recycling play a crucial role in GPCR signaling in higher organisms. In light of the current finding, it will be interesting to study sphingolipid metabolism in GPCR-mediated processes. Several inherited forms of human retinal degenerations result from mutations in rhodopsin, arrestin, and phosphodiesterase, among others. Individuals with Oguchi disease have mutations in visual arrestin and a form of degenerative night blindness. Rescue of degeneration in Drosophila visual mutants provides a strong basis for exploring strategies that manipulate sphingolipid enzymes for therapeutic management of retinal degeneration in higher organisms (Acharya, 2003).

Neutral ceramidase, a key enzyme of sphingolipid metabolism (see Long-chain base synthesis resulting in ceramide formation from Shayman, 2000), hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. This study isolated and characterized functional null mutants of Drosophila ceramidase. Secreted ceramidase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. In the absence of ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of rhodopsin, and do not respond to light stimulus. Consistent with a cell-nonautonomous function, overexpression of ceramidase in tissues distant from photoreceptors suppresses photoreceptor degeneration in an arrestin mutant and facilitates membrane turnover in a rhodopsin null mutant. Furthermore, the results show that secreted ceramidase is internalized and localizes to endosomes. These findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function (Acharya, 2008).

EVOLUTIONARY HOMOLOGS

Arrestin structure and interaction with rhodopsin

Visual arrestin plays a crucial role in the termination of the light response in vertebrate photoreceptors by binding selectively to light-activated, phosphorylated rhodopsin. Arrestin localizes predominantly to the inner segments and perinuclear region of dark-adapted rod photoreceptors, whereas light induces redistribution of arrestin to the rod outer segments. The mechanism by which arrestin redistributes in response to light is not known, but it is thought to be associated with the ability of arrestin to bind photolyzed, phosphorylated rhodopsin in the outer segment. In this study, it was shown that light-driven translocation of arrestin is unaffected in two different mouse models in which rhodopsin phosphorylation is lacking. It was further shown that arrestin movement is initiated by rhodopsin but does not require transducin signaling. These results exclude passive diffusion and point toward active transport as the mechanism for light-dependent arrestin movement in rod photoreceptor cells (Mendez, 2003).

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, it was shown that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin (Kisselev, 2004).

The phosphorylated carboxyl terminus of rhodopsin is required for the stable binding of visual arrestin to the full length rhodopsin molecule. Phosphorylation of the carboxyl terminus has been shown to induce conformational changes in arrestin, which promote its binding to the cytoplasmic loops of rhodopsin. However, it has not been determined whether phosphorylation is also responsible for the direct binding of the rhodopsin carboxyl terminus to arrestin. To further investigate the role of rhodopsin phosphorylation on arrestin binding, surface plasmon resonance was used to measure the interaction between a synthetic phosphopeptide corresponding to the carboxyl terminus of rhodopsin and visual arrestin in real time. Synthetic peptides were generated that correspond to the phosphorylated and nonphosphorylated carboxyl terminus of bovine rhodopsin. These peptides were immobilized on a biosensor chip and their interaction with purified visual arrestin was monitored by surface plasmon resonance on a BIAcore 2000 or 3000. A synthetic peptide phosphorylated on residues corresponding to Ser-338, Thr-340, Thr-342 and Ser-343 of bovine rhodopsin is sufficient for direct binding to visual arrestin. In contrast, a second phosphopeptide phosphorylated on Thr-340 and Thr-342 and a nonphosphorylated synthetic peptide are not able to bind arrestin. A peptide fully substituted at all serine and threonine residues with glutamic acid is unable to substitute for phosphorylation. It is concluded that surface plasmon resonance is a sensitive method for detecting small differences in affinity. This technique was successful in detecting differences in the affinity of phosphorylated and nonphosphorylated rhodopsin peptides for visual arrestin. The data suggest that these are low-affinity interactions and indicate that phosphorylation is responsible for the direct binding of the rhodopsin carboxyl terminus to visual arrestin. Four phosphorylated residues are sufficient for this interaction. Because the affinity of the synthetic phosphopeptide for arrestin is substantially lower than the full length rhodopsin molecule, the cytoplasmic loops and rhodopsin carboxyl terminus appear to interact in a cooperative manner to stably bind arrestin (Liu, 2004).

The binding of visual arrestin to phosphorylated, activated rhodopsin serves as a model for studying the inactivation process of a large class of G-protein coupled receptor systems. In this study, the use of insertional mutagenesis, fluorescence labeling, and scanning alanine mutagenesis was combined to identify the surface of interaction between arrestin and rhodopsinThe ten amino acid myc tag (EQKLISEEDL) was inserted in eleven loop structures that connect ßstrands and the tagged arrestins were heterologously expressed in yeast. Binding competition assays were performed with these proteins, using an anti-myc monoclonal antibody. Site specific cysteines were also substituted in selected loop structures in arrestin. These cysteines were labeled with a fluorescent reporter to assess the proximity of the introduced cysteine with rhodopsin in the bound complex. Competitive inhibition of arrestin binding to light activated, phosphorylated rhodopsin with an anti-myc antibody showed that all competitive sites lay along a single surface encompassing the N- and C-terminal domains. Fluorescence labeling of these loop structures and subsequent interaction with rhodopsin indicates close apposition of loops 68-78 and 248-253 to rhodopsin in the receptor bound state. Scanning mutagenesis of loop 248-253 implicates Ser-251 and/or Ser-252 as a potential interaction point with rhodopsin. These results clearly suggest a surface of arrestin to which rhodopsin binds upon light activation and phosphorylation. This surface encompasses elements from both the N- and C-terminal domains of arrestin (Smith, 2004).

Arrestins selectively bind to phosphorylated activated forms of their cognate G protein-coupled receptors. Arrestin binding prevents further G protein activation and often redirects signaling to other pathways. The comparison of the high-resolution crystal structures of arrestin2, visual arrestin, and rhodopsin as well as earlier mutagenesis and peptide inhibition data collectively suggest that the elements on the concave sides of both arrestin domains most likely participate in receptor binding directly, thereby dictating its receptor preference. Using comparative binding of visual arrestin/arrestin2 chimeras to the preferred target of visual arrestin, light-activated phosphorylated rhodopsin (PRh*), and to the arrestin2 target, phosphorylated activated m2 muscarinic receptor (P-m2 mAChR*), the elements that determine the receptor specificity of arrestins were determined. It was found that residues 49-90 (ß-strands V and VI and adjacent loops in the N-domain) and 237-268 (ß-strands XV and XVI in the C-domain) in visual arrestin and homologous regions in arrestin2 are largely responsible for their receptor preference. Only 35 amino acids (22 of which are nonconservative substitutions) in the two elements are different. Simultaneous exchange of both elements between visual arrestin and arrestin2 fully reverses their receptor specificity, demonstrating that these two elements in the two domains of arrestin are necessary and sufficient to determine their preferred receptor targets (Vishnivetskiy, 2004).

The visual arrestins in rhabdomeral photoreceptors are multifunctional phosphoproteins. They are rapidly phosphorylated in response to light, but the functional relevance of this phosphorylation is not yet fully understood. The phosphorylation of Limulus visual arrestin is particularly complex in that it becomes phosphorylated on three sites, and one or more of these site are phosphorylated even in the dark. The purpose of this study was to examine in detail the light-stimulated phosphorylation of each of the three sites in Limulus visual arrestin in intact photoreceptors. It was found that light increases the phosphorylation of all three sites (S377, S381, and S396), that S381 is a preferred phosphorylation site, and that S377 and S381 are highly phosphorylated in the dark. The major effect of light was to increase the phosphorylation of S396, the site located closest to the C-terminal and very close to the adaptin binding motif. It is speculated that the phosphorylation of this site may be particularly important for regulating the light-driven endocytosis of rhabdomeral membrane (Sineshchekova, 2004).

The mechanism of visual arrestin release from light-activated rhodopsin was addressed using fluorescently labeled arrestin mutants. Two mutants, I72C and S251C, when labeled with the small, solvent-sensitive fluorophore monobromobimane, exhibit spectral changes only upon binding light-activated, phosphorylated rhodopsin. This analysis indicates that these changes are probably due to a burying of the probes at these sites in the rhodopsin-arrestin or phospholipid-arrestin interface. Using a fluorescence approach based on this observation, it was demonstrated that arrestin and retinal release are linked and are described by similar activation energies. However, at physiological temperatures, it was found that arrestin slows the rate of retinal release approximately 2-fold and abolishes the pH dependence of retinal release. Using fluorescence, EPR, and biochemical approaches, intriguing evidence was found that arrestin binds to a post-Meta II photodecay product, possibly Meta III. It is speculated that arrestin regulates levels of free retinal in the rod cell to help limit the formation of damaging oxidative retinal adducts. Such adducts may contribute to diseases like atrophic age-related macular degeneration (AMD). Thus, arrestin may serve to both attenuate rhodopsin signaling and protect the cell from excessive retinal levels under bright light conditions (Sommer, 2005).

G-protein coupled receptor signaling is terminated by arrestin proteins which preferentially bind to the activated phosphorylated form of the receptor. Arrestins also bind the active unphosphorylated and inactive phosphorylated receptors. Binding to the non-preferred forms of the receptor is important for visual arrestin translocation in rod photoreceptors and the regulation of receptor signaling and trafficking by non-visual arrestins. Given the importance of arrestin interactions with the various functional forms of the receptor, an extensive analysis was performed of the receptor-binding surface of arrestin using site-directed mutagenesis. The data indicate that a large number of surface charges are important for arrestin interaction with all forms of the receptor. Arrestin elements involved in receptor binding are differentially engaged by the various functional forms of the receptor, each requiring a unique subset of arrestin residues in a specific spatial configuration. Several additional phosphate binding elements were identified in the N-domain, and the active receptor was demonstrated to preferentially engage the arrestin C-domain. It was also found that the inter-domain contact surface is important for arrestin interaction with the non-preferred forms of the receptor and that residues in this region play a role in arrestin transition into its high-affinity receptor-binding state (Hanson, 2005).

Arrestins play a fundamental role in the regulation and signal transduction of G protein-coupled receptors. This paper described the crystal structure of cone arrestin at 2.3Å resolution. The overall structure of cone visual arrestin is similar to the crystal structures of rod visual and the non-visual arrestin-2, consisting of two domains, each containing ten ß-sheets. However, at the tertiary structure level, there are two major differences, in particular on the concave surfaces of the two domains implicated in receptor binding and in the loop between ß-strands I and II. Functional analysis shows that cone arrestin, in sharp contrast to its rod counterpart, binds cone pigments and non-visual receptors. Conversely, non-visual arrestin-2 binds cone pigments, suggesting that it may also regulate phototransduction and/or photopigment trafficking in cone photoreceptors. These findings indicate that cone arrestin displays structural and functional features intermediate between the specialized rod arrestin and the non-visual arrestins, which have broad receptor specificity. A unique functional feature of cone arrestin is the low affinity for its cognate receptor, resulting in an unusually rapid dissociation of the complex. Transient arrestin binding to the photopigment in cones may be responsible for the extremely rapid regeneration and reuse of the photopigment that is essential for cone function at high levels of illumination (Sutton, 2005).

A ß-arrestin binding determinant common to the second-intracellular loops of Rhodopsin-family G protein-coupled receptors

Beta-arrestins have been shown to competitively inhibit G protein-dependent signaling and mediate endocytosis for many of the hundreds of non-visual rhodopsin-family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology when considered in toto can all recruit and activate the two highly conserved ß-arrestin proteins as part of their signaling/desensitization process. Though the serine and threonine residues that form GPCR kinase (GRK) phosphorylation sites are common ß-arrestin associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. This study identified a ß-arrestin binding determinant common to rhodopsin-family GPCRs formed from the proximal ten residues of the second intracellular loop. It was demonstrated by both gain and loss of function studies for the serotonin 2C, ß2-adrenergic, alpha2a-adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids proline or alanine, naturally occurring in rhodopsin-family receptors six residues distal to the highly conserved second-loop DRY motif, regulate ß-arrestin binding and ß-arrestin mediated internalization. In particular, as demonstrated for the b2AR, this occurs independently of changes in GRK phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning ß-arrestin from its inactive form to its active receptor binding state (Marion, 2005).

Arrestin-mediated ERK and JNK activation

ß-arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. c-Jun amino-terminal kinase 3 (JNK3) has been identified as a binding partner of ß-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with ß-arrestin 2. Cellular transfection of ß-arrestin 2 causes cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activates JNK3 and triggers the colocalization of ß-arrestin 2 and active JNK3 to intracellular vesicles. Thus, ß-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR (McDonald, 2000).

In budding yeast, Ste5 is a scaffold protein that forms a multicomponent complex with the Fus3 (Kss1) MAPK, Ste7 MAPKK, and Ste11 MAPKKK to facilitate the specific and efficient activation of the mating pheromone pathway. Also in yeast, Pbs2, which itself is a MAPKK, has been proposed as a possible scaffold protein in the HOG (high-osmolarity glycerol response) signal transduction pathway. An intriguing similarity between Ste5 and ß-arrestin 2 is that both are recruited to the plasma membrane as a consequence of agonist stimulation of a GPCR. In mammalian cells, a group of JNK-interacting proteins (JIP1, -2 and -3) have been identified as scaffolding proteins for specific JNK signaling modules (McDonald, 2000 and references therein).

Like members of the JIP family, ß-arrestin 2 associates with all three kinase components of a single MAPK cascade, thus enhancing signaling efficiency. However, unlike JIPs, the ß-arrestin 2-MAPK module is regulated by agonist stimulation of GPCRs. It is likely that individual JNK isoforms exhibit distinct patterns of regulation. For example, as demonstrated in this study, JNK3 activity appears to be specifically enhanced by ß-arrestin 2, whereas JNK1 activity is unaffected. Thus, formation of similar complexes may prevent inappropriate cross talk between the various MAPK pathways (McDonald, 2000).

JNK1 is activated in response to several GPCRs. The association of JNK3 with ß-arrestin 2 provides a mechanism whereby ß-arrestin 2 might localize JNK3 to specific subcellular compartments and/or target JNK3 to specific substrates in response to GPCR agonists. The results reported here add to a growing list of functions subserved by ß-arrestins in regulating signaling through heptahelical receptors. By acting as a scaffold for a specific MAPK pathway, ß-arrestin 2 provides a mechanism for bringing this signaling module under the control of such receptors. Other evidence (suggests that ß-arrestins may also play roles in organizing pathways leading from GPCRs to activation of the ERKs (McDonald, 2000).

Activation of 7TM receptors typically causes their phosphorylation with consequent arrestin binding and desensitization. Arrestins also act as scaffolds, mediating signaling to Raf and ERK and, for some receptors, inhibiting nuclear translocation of ERK. GnRH receptors act via Gq/11 to stimulate the PLC/Ca2+/PKC cascade and the Raf/MEK/ERK cassette. Uniquely, type I mammalian GnRHRs lack the C-tails that are found in other 7TM receptors (including non-mammalian GnRHRs) and are implicated in arrestin binding. This study compares ERK signaling by human (h) and Xenopus (X) GnRHRs. In HeLa cells XGnRHRs undergo rapid and arrestin-dependent internalization and cause arrestin/GFP translocation to the membrane and endosomes, whereas hGnRHRs do not. Internalized XGnRHRs co-localize with arrestin/GFP, whereas hGnRHRs do not. Both receptors mediate transient ERK phosphorylation and nuclear translocation (revealed by immunohistochemistry or by imaging of co-transfected ERK2/GFP) and for both, ERK phosphorylation is reduced by PKC inhibition but not by inhibiting EGF receptor autophosphorylation. In the presence of PKC inhibitor, Darrestin(319-418) blocks XGnRHR-mediated, but not hGnRHR-mediated, ERK phosphorylation. When receptor number is varied, hGnRHRs activate PLC and ERK more efficiently than XGnRHRs, but are less efficient at causing ERK2/GFP translocation. At high receptor number, XGnRHRs and hGnRHRs both cause ERK2/GFP translocation to the nucleus but at low receptor number XGnRHRs cause ERK2/GFP translocation whereas hGnRHRs do not. Thus, experiments with XGnRHRs have revealed the first direct evidence of arrestin-mediated (likely G protein-independent) GnRHR signalling, whereas those with hGnRHRs imply that scaffolds other than arrestins can determine GnRHR effects on ERK compartmentalization (Caunt, 2005).

Beta-arrestin mediates desensitization and internalization of ß-adrenergic receptors (ßARs), but also acts as a scaffold protein in extracellular signal-regulated kinase (ERK) cascade. Thus, the role of ß-arrestin2 was examined in the ßAR-mediated ERK signaling pathways. Isoproterenol stimulation equally activates cytoplasmic and nuclear ERK in COS-7 cells expressing ß1AR or ß2AR. However, the activity of nuclear ERK is enhanced by co-expression of ß-arrestin2 in ß2AR- (but not ß1AR-) expressing cells. Pertussis toxin treatment and blockade of Gßgamma action inhibits ß-arrestin2-enhanced nuclear activation of ERK, suggesting that ß-arrestin2 promotes nuclear ERK localization in a Gßgamma dependent mechanism upon receptor stimulation. ß2AR containing the carboxyl terminal region of ß1AR has lost the ß-arrestin2-promoted nuclear translocation. Since the carboxyl terminal region is important for ß-arrestin binding, these results demonstrate that recruitment of ß-arrestin2 to carboxyl terminal region of ß2AR is important for ERK localization to the nucleus (Kobayashi, 2005).

Physiological effects of ß adrenergic receptor (ß2AR) stimulation have been classically shown to result from G_s-dependent adenylyl cyclase activation. A novel signaling mechanism has been demonstrated wherein ß-arrestins mediate ß2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the ß2AR expressed in HEK-293 cells has been resolved into two components dependent, respectively, on G_s-G_i/protein kinase A (PKA) or ß-arrestins. G protein-dependent activity is rapid, peaking within 2-5 min, is quite transient, is blocked by pertussis toxin (G_i inhibitor) and H-89 (PKA inhibitor), and is insensitive to depletion of endogenous ß-arrestins by siRNA. ß-Arrestin-dependent activation is slower in onset (peak 5-10 min), less robust, but more sustained and shows little decrement over 30 min. It is insensitive to pertussis toxin and H-89 and sensitive to depletion of either ß-arrestin1 or -2 by small interfering RNA. In G_s knock-out mouse embryonic fibroblasts, wild-type ß2AR recruited ß-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel ß2AR mutant (ß2AR^{T68F,Y132G,Y219A} or ß2AR^TYY), rationally designed based on Evolutionary Trace analysis, is incapable of G protein activation but can recruit ß-arrestins, undergo ß-arrestin-dependent internalization, and activate ß-arrestin-dependent ERK. Interestingly, overexpression of GRK-5 or -6 increases mutant receptor phosphorylation and ß-arrestin recruitment, leads to the formation of stable receptor-ß-arrestin complexes on endosomes, and increases agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gßgamma release upon G protein activation, is ineffective unless it is constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the ß2AR can signal to ERK via a GRK5/6-ß-arrestin-dependent pathway, which is independent of G protein coupling (Shenoy, 2006).

ß-arrestin interaction with GPCRs

Beta-arrestins are multifunctional adaptor proteins that mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and ß-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in ß-arrestins upon binding to the activated phosphorylated receptor. This study characterizes the conformational changes in ß-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and MALDI-TOF MS profiles of ß-arrestin 2 in the presence of a phosphopeptide (V₂R-pp) derived from the C terminus of the vasopressin type II receptor (V₂R) or the corresponding nonphosphopeptide (V₂R-np). V₂R-pp binds to ß-arrestin 2 specifically, whereas V₂R-np does not. Activation of ß-arrestin 2 upon V₂R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg(394), and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg(8). Interestingly, binding of the polyanion heparin also leads to release of the C terminus of ß-arrestin 2; however, heparin and V₂R-pp have different binding site(s) and/or induce different conformational changes in ß-arrestin 2. Release of the C terminus from the rest of ß-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to ß-arrestin 2 by 10-fold. Thus, the V₂R-pp can activate ß-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of ß-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-ß-arrestin interactions can be modeled in vitro (Xiao, 2004).

ß-arrestin-mediated internalization of GPCRs

Expression levels of the chemokine receptor, CC chemokine receptor 5 (CCR5), at the cell surface determine cell susceptibility to HIV entry and infection. Cellular activation by CCR5 itself, but also by unrelated receptors, leads to cross-phosphorylation and cross-internalization of CCR5. This study addresses the underlying molecular mechanisms of homologous and heterologous CCR5 regulation. As shown by bioluminescence resonance energy transfer experiments, CCR5 forms constitutive homo- as well as hetero-oligomeric complexes together with C5aR but not with the unrelated AT(1a)R in living cells. Stimulation with CCL5 of RBL cells that co-express CCR5 together with an N-terminally truncated CCR5-DeltaNT mutant, results in both protein kinase C (PKC)- and G protein-coupled receptor (GPCR) kinase (GRK)-mediated cross-phosphorylation of the mutant unligated receptor, as determined by phosphosite-specific monoclonal antibody. Similarly, both PKC and GRK cross-phosphorylates CCR5 in a heterologous manner after C5a stimulation of RBL-CCR5/C5aR cells, whereas AT(1a)R stimulation results only in classical PKC-mediated CCR5 phosphorylation. Co-expression of CCR5-DeltaNT together with a phosphorylation-deficient CCR5 mutant that neither binds ß-arrestin nor undergoes internalization partially restores the CCL5-induced association of ß-arrestin with the homo-oligomeric receptor complex and augmented cellular uptake of (125)I-CCL5. Co-expression of C5aR, but not of AT(1a)R, promotes CCR5 co-internalization upon agonist stimulation by a mechanism independent of CCR5 phosphorylation. Co-internalization of phosphorylated CCR5 is also observed in C5a-stimulated macrophages. Finally, co-expression of a constitutively internalized C5aR-US28(CT) mutant leads to intracellular accumulation of CCR5 in the absence of ligand stimulation. These results show that GRKs and ß-arrestin are involved in heterologous receptor regulation by cross-phosphorylating and co-internalizing unligated receptors within homo- or hetero-oligomeric protein complexes (Huttenrauch, 2005).

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to ß-arrestin after agonist exposure. To define the importance of receptor phosphorylation and ß-arrestin binding in desensitization, and to determine whether ß-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, TRH receptors were expressed in fibroblasts from mice lacking ß-arrestin-1 and/or ß-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing ß-arrestins, including a ß-arrestin mutant that did not permit receptor internalization. TRH causes extensive receptor endocytosis in the presence of ß-arrestins, but receptors remain primarily on the plasma membrane without ß-arrestin. ß-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous ß-arrestins reduced TRH-stimulated inositol phosphate production by 48% (ß-arrestin-1), 71% (ß-arrestin-2), and 84% (ß-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, is unaffected by ß-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without ß-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that ß-arrestin binding is critical for desensitization and internalization (Jones, 2005).

The neuronal Na(+)/H(+) exchanger NHE5 isoform not only resides in the plasma membrane but also accumulates in recycling vesicles by means of clathrin-mediated endocytosis. To further investigate the underlying molecular mechanisms, a human brain cDNA library was screened for proteins that interact with the cytoplasmic C-terminal region of NHE5 by using yeast two-hybrid methodology. One candidate cDNA identified by this procedure encoded ß-arrestin2, a specialized adaptor/scaffolding protein required for internalization and signaling of members of the G protein-coupled receptor superfamily. Direct interaction between the two proteins was demonstrated in vitro by GST fusion protein pull-down assays. Sequences within the N-terminal receptor activation-recognition domain and the C-terminal secondary receptor-binding domain of ß-arrestin2 confer strong binding to the C terminus of NHE5. Full-length NHE5 and ß-arrestin2 also associated in intact cells, as revealed by their coimmunoprecipitation from extracts of transfected CHO cells. Moreover, ectopic expression of both proteins causes a redistribution of ß-arrestin2 from the cytoplasm to vesicles containing NHE5, and significantly decreases the abundance of the transporter at the cell surface. Comparable results were also obtained for the ß-arrestin1 isoform. These data reveal a broader role for arrestins in the trafficking of integral plasma membrane proteins than previously recognized (Szabo, 2005).

There is considerable evidence for the role of carboxyl terminal serines 355,356,364 in GRK-mediated phosphorylation and desensitization of ß₂-adrenergic receptors (ß₂ARs). Receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) were used to determine the role of these sites in ß-arrestin-dependent ß₂AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant does not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nM isoproterenol for 5 minutes induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with PKA site mediated desensitization. When exposed to 1 muM isoproterenol to trigger GRK site mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phosphosite-specific antibody, it was determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist induced internalization of the SD3 and SA3 mutants were 44% and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited EGFP-ß-arrestin2 to the plasma membrane while the SA3 mutant did not. EGFP-ß-arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent ß-arrestin1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355,356 in wild-type receptors. These data demonstrate (1) that the lack of GRK sites does not impair PKA site phosphorylation (2) that the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced ß-arrestin binding and receptor internalization, and (3) that serines 355,356,364 play a pivotal role in the GRK-mediated desensitization, ß-arrestin binding and internalization of ß₂ARs (Vaughan, 2006).

ß-arrestin and degradation of GRKs

G-protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of G-protein-coupled receptors (GPCRs). GRK2 expression is altered in several pathological conditions, but the molecular mechanisms that modulate GRK2 cellular levels are largely unknown. GRK2 is degraded rapidly by the proteasome pathway. This process is enhanced by GPCR stimulation and is severely impaired in a GRK2 mutant that lacks kinase activity (GRK2-K220R). ß-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of ß-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with ß-arrestin mutants that display an impaired c-Src interaction. The presence of an inactive c-Src mutant or of tyrosine kinase inhibitors strongly inhibits co-transfected or endogenous GRK2 turnover, respectively, and a GRK2 mutant with impaired phosphorylation by c-Src shows a markedly retarded degradation. This pathway for the modulation of GRK2 protein stability puts forward a new feedback mechanism for regulating GRK2 levels and GPCR signaling (Penela, 2001).

These results are consistent with the notion that GRK2-dependent binding of ß-arrestin to GPCRs allows the recruitment of c-Src to the receptor signaling complex at the plasma membrane, leading to phosphorylation of GRK2 on tyrosine residues and its targeting for degradation. This model is in agreement with the rapid ß-arrestin and c-Src recruitment following ß2AR stimulation, and with the agonist-stimulated phosphorylation of GRK2 by c-Src. Under basal conditions, ß-arrestin recruitment to the plasma membrane would be promoted by the activated state of different endogenous GPCRs and/or by the reported basal activity of overexpressed ß2AR. In the presence of GPCR agonists, an acceleration of the GRK2 degradation rate is detected, consistent with a more efficient ß-arrestin and c-Src translocation to the receptor complex. Although detailed knowledge of the sequential assembly of these proteins in a multimolecular complex is lacking, and other molecular interactions may participate in c-Src binding to the receptor complex and GRK2 tyrosine phosphorylation, the proposed model is consistent with the co-immunoprecipitation of ß-arrestin and c-Src, of GRK2 and ß-arrestin and of GRK2 and c-Src. Disruption of the ß-arrestin-c-Src interaction with specific mutants or inhibition of the phosphorylation step by dominant-negative Src or a GRK2 mutant lacking critical phosphorylation sites results, as predicted by this model, in a marked reduction in GRK2 degradation (Penela, 2001).

Translocation of arrestins

Light sensitivity and adaptation, general characteristics of rod photoreceptor cell vision, allow rods to modulate their response depending on the lighting environment to which they are exposed. In dim light, rods are maximally sensitive, whereas, in bright light, rods are essentially inactive. In the retinas of dark-adapted mice, arrestin (an inhibitory protein) is located in the rod inner segment (RIS), and transducin (an activating protein) is located in the rod outer segment (ROS). In light-adapted retinas, the proteins have reciprocal localizations. In this study, the data demonstrate that the temporal and spatial changes in the subcellular localization of arrestin and beta-transducin are correlated with the amount of light to which the animals are exposed. By using the frog Xenopus laevis and immunofluorescence confocal microscopy, the results also show that in the dark-adapted retina some arrestin remains in the ROS. The data most dramatically demonstrate that this residual arrestin is highly concentrated in the connecting cilium, the axoneme, and the microtubules associated with the disc incisures. These data suggest a structure-function relationship between the light-dependent positional status of arrestin and the elements of the rod photoreceptor cytoskeleton. The massive, rapid, light-induced reciprocal changes in the subcellular concentrations of these proteins must directly affect phototransduction and appear to be a general phenomenon by which photoreceptor cells rapidly and transiently regulate the trafficking and subcellular concentration of a variety of signal transduction proteins within the RIS and ROS. Hereditary mutations in the components of the movement mechanism should lead to defects in vision and possibly blindness (McGinnis, 2002).

The light-dependent redistribution of phototransduction components in photoreceptor cells plays a role in light adaptation. Upon illumination, rod and cone arrestins (Arr and cArr) translocate from the inner to the outer segments while transducin subunits (Talpha, Tbetagamma) translocate in the opposite direction. The underlying translocation mechanisms are unclear. This study examines these translocations in mice with defective phototransduction. The distribution of Arr, cArr, Talpha, and Tbetagamma was examined using immunoblotting and immunocytochemistry in dark- and light-adapted single knockout mice lacking G-protein coupled receptor kinase 1 (Grk1^-/-) and double knockout mice lacking GRK1 and transducin alpha subunit (Grk1^-/-/Gnat1^-/-), or lacking GRK1 and arrestin (Grk1^-/-/Arr^-/-). Arr redistributed in the light to the outer segments was studied in Grk1^-/- mice as well as in Grk1^-/-/Gnat1^-/- double knockout retinas. Immunoblotting revealed that approximately 25-50% of Arr associates with the membrane in light-adapted wild-type, Grk1^-/- and Gnat1^-/-/Grk1^-/- mouse retinas. In contrast, cArr does not stably associate with light-adapted membranes in either wild-type or Grk1^-/- retinas under these experimental conditions, but redistributes to the cone outer segments in a light-dependent manner. The redistribution of transducin subunits to the inner segments in light occurs in both wild-type and Grk1^-/-/Arr^-/- double knockout photoreceptors. However, Tbetagamma subunits do not redistribute in the absence of Talpha, suggesting that transducin only translocates as an intact heterotrimer. It is concluded that in rods, Arr redistribution requires neither rhodopsin phosphorylation nor phototransduction, suggesting the presence of another light-dependent pathway to trigger translocation. In cones, the light-dependent movement of cArr appears to be independent of stable association with the cone pigments. The light-dependent translocations of Arr and transducin subunits in opposite directions appear to be based on independent mechanisms (Zhang, 2003).

Microtubles kinesin are involved in transport of arrestin

To test whether kinesin-II is important for transport in the mammalian photoreceptor cilium, and to identify its potential cargoes, Cre-loxP mutagenesis was used to remove the kinesin-II subunit, KIF3A, specifically from photoreceptors. Complete loss of KIF3A caused large accumulations of opsin, arrestin, and membranes within the photoreceptor inner segment, while the localization of alpha-transducin is unaffected. Other membrane, organelle, and transport markers, as well as opsin processing appeared normal. Loss of KIF3A ultimately causes apoptotic photoreceptor cell death similar to a known opsin transport mutant. The data suggest that kinesin-II is required to transport opsin and arrestin from the inner to the outer segment and that blocks in this transport pathway lead to photoreceptor cell death as found in retinitis pigmentosa (Marszalek, 2000).

Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slows only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slows the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. These results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin (Peterson, 2005).

Direct binding of visual arrestin to microtubules

Proper function of visual arrestin is indispensable for rapid signal shut-off in rod photoreceptors. Dramatic light-dependent changes in arrestin subcellular localization are believed to play an important role in light adaptation of photoreceptor cells. This study shows that visual arrestin binds microtubules. The truncated splice variant of visual arrestin, p44, demonstrates dramatically higher affinity for microtubules than the full-length protein (p48). Enhanced microtubule binding of p44 underlies its earlier reported preferential localization to detergent-resistant membranes, where it is anchored via membrane-associated microtubules in a rhodopsin-independent fashion. Experiments with purified proteins demonstrate that arrestin interaction with microtubules is direct and does not require any additional protein partners. Most important, arrestin interactions with microtubules and light-activated phosphorylated rhodopsin are mutually exclusive, suggesting that microtubule interaction may play a role in keeping p44 arrestin away from rhodopsin in dark-adapted photoreceptors (Nair, 2004).

Ubiquitinization, trafficking and stability of ß-arrestin

Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein ß-arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the ß₂-adrenergic receptor (ß₂AR) this requires ubiquitination of ß-arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of ß-arrestin, G protein-coupled receptors are categorized into two classes. For class A receptors (e.g., ß₂AR), which recycle rapidly, ß-arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors (e.g., V2 vasopressin receptors), which recycle slowly, ß-arrestin internalizes with the receptor into endosomes. In COS-7 and human embryonic kidney (HEK)-293 cells, stimulation of the ß₂AR or V2 vasopressin receptor leads, respectively, to transient or stable ß-arrestin ubiquitination. The time course of ubiquitination and deubiquitination of ß-arrestin correlates with its association with and dissociation from each type of receptor. Chimeric receptors, constructed by switching the cytoplasmic tails of the two classes of receptors (ß₂AR and V2 vasopressin receptors), demonstrate reversal of the patterns of both ß-arrestin trafficking and ß-arrestin ubiquitination. To explore the functional consequences of ß-arrestin ubiquitination a yellow fluorescent protein-tagged ß-arrestin2-ubiquitin chimera was constructed that cannot be deubiquitinated by cellular deubiquitinases. This 'permanently ubiquitinated' ß-arrestin does not dissociate from the ß₂AR but rather internalizes with it into endosomes, thus transforming this class A receptor into a class B receptor with respect to its trafficking pattern. Overexpression of this ß-arrestin ubiquitin chimera in HEK-293 cells also results in enhancement of ß₂AR internalization and degradation. In the presence of N-ethylmaleimide (an inhibitor of deubiquitinating enzymes), coimmunoprecipitation of the receptor and ß-arrestin is increased dramatically, suggesting that deubiquitination of ß-arrestin triggers its dissociation from the receptor. Thus the ubiquitination status of ß-arrestin determines the stability of the receptor-ß-arrestin complex as well as the trafficking pattern of ß-arrestin (Shenoy, 2003).

Angiotensin II type 1a (AT1a), vasopressin V2, and neurokinin 1 (NK1) receptors are seven-transmembrane receptors (7TMRs) that bind and co-internalize with the multifunctional adaptor protein, ß-arrestin. These receptors also lead to robust and persistent activation of extracellular-signal regulated kinase 1/2 (ERK1/2) localized on endosomes. The co-trafficking of receptor-ß-arrestin complexes to endosomes requires stable ß-arrestin ubiquitination. Lysines at positions 11 and 12 in ß-arrestin2 are specific and required sites for AngII-mediated sustained ubiquitination of arrestin. Thus, upon AngII stimulation the mutant ß-arrestin2(K11,12R) is only transiently ubiquitinated, does not form stable endocytic complexes with the AT1aR, and is impaired in scaffolding-activated ERK1/2. Fusion of a ubiquitin moiety in-frame to ß-arrestin2(K11,12R) restores AngII-mediated trafficking and signaling. Wild type ß-arrestin2 and ß-arrestin2(K11R,K12R)-Ub, but not ß-arrestin2(K11R,K12R), prevent nuclear translocation of pERK. These findings imply that sustained ß-arrestin ubiquitination not only directs co-trafficking of receptor-ß-arrestin complexes but also orchestrates the targeting of '7TMR signalosomes' to microcompartments within the cell. Surprisingly, binding of ß-arrestin2(K11R,K12R) to V2R and NK1R is indistinguishable from that of wild type ß-arrestin2. Moreover, ubiquitination patterns and ERK scaffolding of ß-arrestin2(K11,12R) are unimpaired with respect to V2R stimulation. In contrast, a quintuple lysine mutant [ß-arrestin2(K18R,K107R,K108R,K207R,K296R)] is impaired in endosomal trafficking in response to V2R but not AT1aR stimulation. These findings delineate a novel regulatory mechanism for 7TMR signaling, dictated by the ubiquitination of ß-arrestin on specific lysines that become accessible for modification due to the specific receptor-bound conformational states of ß-arrestin2 (Shenoy, 2005).

Oligomerization of ß-arrestins

Arrestins are important proteins that regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous mammalian ß-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Oligomerization status of ß-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged ß-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, ß-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of ß-arrestin2 and ß-arrestin1 prevents ß-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. These data clearly indicate that ß-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions (Storez, 2005).

Dishevelled recruits ß-arrestin

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane. Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells is dependent on added Wnt5A protein and is accomplished by the multifunctional adaptor protein ß-arrestin 2 (ßarr2), which is recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of ß-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling (Chen, 2003).

Transcriptional regulation of mammalian visual arrestin

The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, a new member of the otd family, Crx (Cone rod homeobox) has been isolated. Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, ß-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases (Chen, 1997).

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10, attempts were made to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, interactions were observed between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 binds sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. The data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells (Dorval, 2006).

Nuclear function of ß-arrestin

Chromatin modification is considered to be a fundamental mechanism of regulating gene expression to generate coordinated responses to environmental changes, however, whether it could be directly regulated by signals mediated by G protein-coupled receptors (GPCRs), the largest surface receptor family, is not known. This study shows that stimulation of delta-opioid receptor, a member of the GPCR family, induces nuclear translocation of ß-arrestin 1 (ßarr1), which was previously known as a cytosolic regulator and scaffold of GPCR signaling. In response to receptor activation, ßarr1 translocates to the nucleus and is selectively enriched at specific promoters such as that of p27 and c-fos, where it facilitates the recruitment of histone acetyltransferase p300, resulting in enhanced local histone H4 acetylation and transcription of these genes. These results reveal a novel function of ßarr1 as a cytoplasm-nucleus messenger in GPCR signaling and elucidate an epigenetic mechanism for direct GPCR signaling from cell membrane to the nucleus through signal-dependent histone modification (Kang, 2005)

C. elegans arrestin regulates neural G protein signaling and olfactory adaptation and recovery

Although regulation of G protein-coupled receptor signaling by receptor kinases and arrestins is a well established biochemical process, the physiological significance of such regulation remains poorly understood. To better understand the in vivo consequences of arrestin function, the function of the sole arrestin in Caenorhabditis elegans, ARR-1, were studied. ARR-1 is primarily expressed in the nervous system, including the HSN neuron and various chemosensory neurons involved in detecting soluble and volatile odorants. arr-1 null mutants exhibit normal chemotaxis but have significant defects in olfactory adaptation and recovery to volatile odorants. In contrast, adaptation is enhanced in animals overexpressing ARR-1. Both the adaptation and recovery defects of arr-1 mutants are rescued by transgenic expression of wild-type ARR-1, whereas expression of a C-terminally truncated ARR-1 effectively rescues only the adaptation defect. A potential mechanistic basis for these findings is revealed by in vitro studies demonstrating that wild-type ARR-1 binds proteins of the endocytic machinery and promotes receptor endocytosis, whereas C-terminally truncated ARR-1 does not. These results demonstrate that ARR-1 functions to regulate chemosensory signaling, enabling organisms to adapt to a variety of environmental cues, and provide an in vivo link between arrestin, receptor endocytosis, and temporal recovery from adaptation (Palmitessa, 2005).

Mammalian visual arrestin and photoreceptor cell death

A study was performed to determine whether constitutive signal flow arising from defective rhodopsin shut-off causes photoreceptor cell death in arrestin knockout mice. The retinas of cyclic-light-reared, pigmented arrestin knockout mice and wild-type littermate control mice were examined histologically for photoreceptor cell loss from 100 days to 1 year of age. In separate experiments, to determine whether constant light would accelerate the degeneration in arrestin knockout mice, these animals and wild-type control mice were exposed for 1, 2, or 3 weeks to fluorescent light at an intensity of 115 to 150 fc. The degree of photoreceptor cell loss was quantified histologically by obtaining a mean outer nuclear layer thickness for each animal. In arrestin knockout mice maintained in cyclic light, photoreceptor loss was evident at 100 days of age, and it became progressively more severe, with less than 50% of photoreceptors surviving at 1 year of age. The photoreceptor degeneration appeared to be caused by light, because when these mice were reared in the dark, the retinal structure was indistinguishable from normal. When exposed to constant light, the retinas of wild-type pigmented mice showed no light-induced damage, regardless of exposure duration. By contrast, the retinas of arrestin knockout mice showed rapid degeneration in constant light, with a loss of 30% of photoreceptors after 1 week of exposure and greater than 60% after 3 weeks of exposure. The results indicate that constitutive signal flow due to arrestin knockout leads to photoreceptor degeneration. Excessive light accelerates the cell death process in pigmented arrestin knockout mice. Human patients with naturally occurring mutations that lead to nonfunctional arrestin and rhodopsin kinase have Oguchi disease, a form of stationary night blindness. The present findings suggest that such patients may be at greater risk of the damaging effects of light than those with other forms of retinal degeneration, and they provide an impetus to restrict excessive light exposure as a protective measure in patients with constitutive signal flow in phototransduction (Chen, 1999).

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, it is proposed that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations (Chuang, 2004).

Search PubMed for articles about Drosophila Arrestin2

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date revised: 1 September 2020

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