InteractiveFly: GeneBrief

inactivation no afterpotential D: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Drosophila visual signal transduction, the process by which incoming light is converted to neural signals that can be passed to the brain, provides an ideal system for the molecular dissection of the process by which extracellular signals are transduced across the plasma membrane leading to neuron activation. Inactivation no afterpotential D (InaD) holds together a protein complex involved in visual signal transduction. Before more detail is provided about InaD, a brief word on the visual transduction process is in order.

In the visual signal transduction pathway, light stimulates rhodopsin, which activates an eye-specific G protein (Galphaq). Activated Galphaq triggers NORPA (No receptor potential A), a phospholipase C-beta that catalyzes the breakdown of phospholipids and generates inositol trisphosphate (IP₃) and diacylglycerol. Diacylglycerol is a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Both TRP (Transient receptor potential) and TRPL (TRP-like) are cation channels that are activated in the visual transduction process. These two proteins share homology with alpha-subunits of voltage-gated calcium and sodium channels in vertebrates. The rise in IP₃ has been thought to result in the release of Ca²⁺ from the internal Ca²⁺ stores. However, the release of Ca²⁺ has been shown not to involved the Inositol 1,4,5,-tris-phosphate receptor, leaving unanswered questions as to the source and regulation of the initial Ca²⁺ current (Acharya, 1997). It has now been shown, however, that polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. As arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999).

Inactivation no afterpotential D (InaD) was identified on the basis of an abnormal electroretinogram (Pak, 1979 ) and was later shown to display a slow deactivation of the light-induced current (Shieh, 1995). Unlike the response in wild-type flies, inaD mutants lack the prolonged depolarizing afterpotential, following stimulation by a pulse of intense blue light (Pak, 1979 and Shieh, 1995). By whole-cell patch-clamp recordings, inaD photoreceptors show a slow deactivation of light-induced currents. This abnormal visual response depends on Ca²⁺ influx; removal of extracellular Ca²⁺ masks the defective phenotype. Furthermore, inaD cells exhibit increased sensitivity toward dim light stimulation. Most of the InaD is associated with rhabdomeres, the organelles in which components of visual signaling are localized (Shieh, 1995).

Inactivation-no-afterpotential D is a photoreceptor-specific protein containing five repeated protein interaction motifs known as PDZ repeats. PDZ domains are composed of ~90 amino acid modules, identified initially in PSD-95, Discs large and ZO-1 (Drosophila homolog: Polychaetoid), which mediate protein-protein interactions by binding to the C-terminal ends of their targets. InaD lacks an SH2 domain and a guanylate kinase domain found in the MAGUK family of PDZ-containing proteins such as Discs large. InaD is an adaptor protein that homomultimerizes and has the ability to interact with multiple components of the signal transduction pathway, including Rhodopsin, NORPA, PKC, Calmodulin, TRP and TRPL (Shieh, 1997 and references and Xu, 1998a and references). The view has been widely held for many years that signaling through G protein-coupled cascades occurs via random stochastic collisions between membrane receptors and effector molecules. However, alternative proposals suggesting that signaling cascades are comprised of components that are physically coupled have been presented but have received less attention. The major conclusion from the work showing the multiple interactions of InaD is that Drosophila vision is mediated by a massive supramolecular complex and that assembly of such a complex is facilitated by homomultimerization of the scaffold protein InaD. Thus, most of the proteins critical in phototransduction appear to couple directly to InaD. The InaD supramolecular complex may not be a particle, consisting of a single InaD monomer to which a maximum of five target proteins bind. Instead, the visual cascade appears to be mediated through a more complicated higher order signaling web or complex (signalplex) consisting of an extended network of InaD homomultimers to which more than five targets bind. Most of these targets appear to bind to more than one PDZ module and several targets appear to associate with InaD via the same PDZ domains. Thus, the nature of the InaD signalplex appears to be more complicated than a single particle held together by a scaffolding protein (Xu, 1998a and references).

An intriguing question concerns the function of the complex between TRP, NORPA, and rhodopsin RH1. The release of Ca²⁺ from Ca²⁺ stores is proposed to activate TRP through a conformational coupling mechanism or via a diffusible messenger. However, this model for Drosophila phototransduction does not require a single complex containing NORPA, RH1, and TRP. An attractive possibility, which is favored, is that TRP is complexed with NORPA, RH1, and possibly INAC (eye protein kinase C) to facilitate negative feedback regulation, such as occurs in adaptation or inactivation. InaD may serve as an adapter to link TRP with NORPA and RH1 to facilitate modulation of NORPA and/or RH1 activity by TRP activity. Consistent with this proposal, disruption of the TRP-InaD interaction and mislocalization of TRP in inaD^P215 flies results in a slow deactivation of the light-induced current. Furthermore, whole cell recording studies have demonstrated that the inaD^P215 phenotype requires Ca²⁺ entry (Shieh, 1995). Under conditions in which there is no Ca²⁺ entry, the light response of inaD^P215 photoreceptor cells is indistinguishable from wild type. Further support for the proposal that TRP is associated with the signaling complex for feedback regulation of phototransduction is lent by the observation that the other proteins in the TRP signaling complex are affected by Ca²⁺. NORPA has been shown to be activated maximally at low concentrations of Ca²⁺, suggesting that the TRP-mediated Ca²⁺ flux serves a negative regulatory function. In addition, Ca²⁺ has been shown to enhance dephosphorylation of Drosophila rhodopsin and phosphorylation of Drosophila arrestin, a protein that binds to rhodopsin and plays a role in the termination mechanism of rhodopsin. Since InaD associates with calmodulin, in a Ca2+-dependent manner, InaD may be modulated by Ca2+ fluxes as well. A calcium channel, such as TRP, may be complexed with other signaling proteins, rather than at a distance in the membrane, to facilitate rapid responses to local changes in Ca2+ concentration, which might not be possible otherwise. It will be interesting to learn whether other ion channels linked to proteins containing PDZ domains are also associated with, and modulate the activities of, proteins that act upstream in signaling cascades (Chevesich, 1997).

Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors

This study reveals a novel feature of the dynamic organization of signaling components in Drosophila photoreceptors. The multi-PDZ protein INAD and its target proteins undergo light-induced recruitment to detergent-resistant membrane (DRM) rafts. Reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM fractions. Genetic analysis demonstrated that translocation of INAD-signaling complexes to DRM rafts requires activation of the entire phototransduction cascade, while constitutive activation of the light-activated channels resulted in recruitment of complexes to DRM rafts in the dark. Mutations affecting INAD and TRP showed that PDZ4 and PDZ5 domains of INAD, as well as the INAD-TRP interaction, are required for translocation of components to DRM rafts. Finally, selective recruitment of phosphorylated, and therefore activatable, eye-PKC to DRM rafts suggests that DRM domains are likely to function in signaling, rather than trafficking (Sanxaridis, 2007).

Recent studies have revealed that some phototransduction components in Drosophila and vertebrate photoreceptors undergo a light-induced translocation between the rhabdomere and cell body. The subcellular translocation of these components has been proposed to contribute to long-term light adaptation. While light appears to regulate the quantity of G_qα, TRPL, and arrestin-2 protein available for signaling in the rhabdomere, no light-induced changes in subcellular localization have been observed for any of the components of the INAD-signaling complex. This report shows instead that components of the INAD-signaling complex undergo light-regulated translocation to DRM rafts within the rhabdomeres of photoreceptors. While subcellular translocation of components out of the rhabdomere may regulate the overall level of protein available for signaling, local translocation of components to lipid raft micro-domains is likely to regulate more immediate signaling mechanisms. Although two hours of light-exposure were used in this study to induce a signal that was robust and reliable enough to be observed by the biochemical assay used, future studies, perhaps using single fluorophore tracking microscopy, will need to examine real-time translocation of components to DRM rafts (Sanxaridis, 2007).

To examine the role lipid rafts play in signaling, electroretinogram (ERG) recordings were performed on ergΔ-fed flies (ergΔ mutant yeast lack C-8 sterol isomerase and C-24 sterol methyltransferase activity, which individually and combinatorialy prevent the biosynthesis of ergosterol). No apparent differences were found from flies fed standard fly food. It is suspected, however, that signaling defects are likely to be missed in such a gross extracellular recording. Future whole-cell voltage-clamp recordings from single raft-depleted photoreceptor cells will be more informative. This, however, is not yet feasible with one-month old adult ergΔ-fed flies since whole-cell recording has only been successful with pupae or newly-eclosed flies. Given that flies are unable to develop to eclosion on the ergΔ food, an alternate ergosterol depletion method will need to be developed for these studies (Sanxaridis, 2007).

How might lipid rafts regulate signaling? The recruitment of INAD-signaling complexes to DRM raft domains may serve to further concentrate components, increasing the rate of protein-protein interactions during signaling as well as increasing the levels of local second messengers created, possibly enhancing the speed and/or amplitude of the light-response. Another possibility is that lipid rafts serve as micro-environments that protect or isolate signaling components from positive or negative regulators present in non-raft domains. For instance, translocation of PLC to lipid rafts may serve to isolate PLC from further activation by G_qα, contributing to deactivation of the light-response (Sanxaridis, 2007).

Micro-domains created by lipid rafts may also provide a special micro-environment that regulates signaling components. For example, TRP channels may have altered activation or deactivation properties in different lipid environments. Indeed, other ion channels have been shown to display different biophysical properties depending on whether they are associated with DRM rafts or not. Although it is well established that activation of the effector PLC is essential for activation of TRP and TRPL channels, it is still uncertain what element(s) downstream of PLC are responsible for directly gating the channels. Recent reports have suggested that the channels are activated by poly-unsaturated fatty acids derived from the second messenger DAG and that maintaining PIP₂ levels is necessary for sustained light responsiveness. Thus, the heterogeneous distribution of lipids present in raft and non-raft micro-domains may indeed differentially regulate TRP channels. For example, PIP₂ has been reported to accumulate in DRM raft domains of several cell types and raft disruption has been shown to mislocalize PIP₂ and affect phosphatidylinositol turnover. Such a concentrated pool of PIP₂, if present in lipid rafts of Drosophila photoreceptors, may indeed play a role in the activation of TRP channels when PLC and TRP are translocated to lipid rafts. Since some studies have reported that application of exogenous poly-unsaturated fatty acids causes the replacement of saturated fatty acids in the membrane with unsaturated ones, leading to disruption of lipid raft domains, future studies examining the gating of TRP channels by poly-unsaturated fatty acids will now need to consider the possible role of lipid rafts in signaling (Sanxaridis, 2007).

This study shows that light induces the translocation of INAD-signaling complexes to DRM rafts in the rhabdomere, highlighting another facet of the dynamic nature of signaling components in photoreceptors. While subcellular movements of proteins are likely to contribute to long-term light adaptation, local translocation to micro-domains within the rhabdomere are likely to modulate more immediate signaling mechanisms. Future studies are likely to investigate the function of lipid rafts in activation/deactivation of PLC, gating of TRP channels, as well as light-adaptation. Using Drosophila as a model system offers the opportunity to combine biochemical studies with Drosophila genetics to identify the function of lipid rafts in vivo (Sanxaridis, 2007).

REGULATION

InaD and signal transduction

The subcellular compartmentalization of signaling molecules helps to ensure the selective activation of different signal-transduction cascades within a single cell. Although there are many examples of compartmentalized signaling molecules, there are few examples of entire signaling cascades being organized as distinct signaling complexes. In Drosophila photoreceptors, the InaD protein, which consists of five PDZ domains, functions as a multivalent adaptor that brings together several components of the phototransduction cascade into a macromolecular complex. Single-photon responses have been studied in several photoreceptor mutant backgrounds. These show that the InaD macromolecular complex is the unit of signaling that underlies elementary responses. The localized activity of this signaling unit promotes reliable single-photon responses as well as rapid activation and feedback regulation. Genetic and electrophysiological tools were used to illustrate how the assembly of signaling molecules into a transduction complex limits signal amplification in vivo (Scott, 1998).

The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies have shown that the PDZ domain containing protein InaD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The InaD signaling complex also includes rhodopsin, protein kinase C (PKC), and Calmodulin, though it is not known whether these proteins bind to InaD. This study shows that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to InaD. A second ion channel, TRPL, also binds to InaD. Thus, most of the proteins involved directly in phototransduction appear to bind to InaD. Furthermore, InaD forms homopolymers and the homomultimerization occurs through two PDZ domains. Thus, it is proposed that the InaD supramolecular complex is a higher order signaling web consisting of an extended network of InaD molecules through which a G protein-coupled cascade is tethered (Xu, 1998a).

Homomultimerization of InaD

The finding that PDZ3 and PDZ4 bind rhodopsin, TRPL and PKC indicates that a single InaD molecule would not have the capacity to nucleate the entire visual transduction signaling complex unless InaD functions as a homomultimeric protein. To address this hypothesis, full-length InaD fused with MYC or FLAG epitope tags are co-expressed in 293T cells and it was found that InaD-FLAG coimmunoprecipitates with InaD-MYC. Further evidence that InaD homomultimerizes was obtained by demonstrating that 35S-InaD binds to a GST-InaD fusion immobilized on a glutathione column. The InaD homomultimerization occurs through PDZ domains (either PDZ3 or PDZ4). Furthermore, InaD PDZ3 and PDZ4 can form either homomeric or heteromeric interactions. The PDZ-PDZ interaction appears to be specific to PDZ3 and PDZ4 since neither PDZ1, PDZ2, nor PDZ5 binds to any portion of InaD, including PDZ3 or PDZ4 (Xu, 1998a).

The observation that homomeric interactions occur through either PDZ3 or PDZ4 raises the possibility that InaD may form a homopolymer rather than just a dimer. To address this possibility, a segment of InaD including just PDZ3-PDZ4 was translated in vitro and the products were fractionated by sucrose gradient sedimentation. Although a proportion of PDZ3-4 fractionates near the predicted molecular weight of the dimer (52 kD), a significant amount sediments as a much larger protein of ~200 kD. PDZ1-PDZ2 loaded onto the same gradient sediments with a single peak near its predicted monomer molecular weight of ~39 kD. Thus, PDZ1-2 does not homomultimerize or interact with PDZ3-PDZ4. The data that a proportion of PDZ3-PDZ4 sediments as a protein greater than 200 kD suggests that InaD may be capable of forming homopolymers with a subunit composition of greater than 8. In contrast to PDZ1-PDZ2, which fractionates with a single peak, four small peaks are detected with PDZ3-PDZ4 that roughly correspond to the predicted sizes of molecules with 1, 2, 4, and 6 subunits. Since the PDZ1-PDZ2 monomer and marker proteins distribute over many fractions, the PDZ3-PDZ4 peaks may be small due to a similar broad distribution of the PDZ3-PDZ4 monomer, dimer, and higher order forms (Xu, 1998a).

The findings that InaD can form homomultimers through PDZ3 and PDZ4 raises the question as to whether homomultimerization precludes InaD-target interaction or vice versa. To investigate whether homomultimerization and PDZ-target interactions can occur simultaneously, advantage was taken of the finding that PDZ3 alone is sufficient to promote homotypic interactions, whereas PDZ3L is required for binding to the opsin, TRPL, or PKC. Therefore, tests were run to determine whether PDZ3 coimmunoprecipitates with the targets after coexpressing PDZ3 and PDZ3L with either TRPL or PKC. TRPL or PKC coimmunoprecipitate with PDZ3 in the presence (but not in the absence) of PDZ3L. These results indicate that PDZ3 and PDZ3L formed a ternary complex with the target proteins and suggest that the InaD PDZ-PDZ and PDZ-target interactions are mediated via different interfaces. Consistent with this latter proposal, an NH2-terminal truncation that removes the first and second putative beta barrel from PDZ3L (PDZ3LdeltaN) disrupts interaction with PKC; however, homomeric binding still occurs. Furthermore, the COOH-terminal extension in PDZ3L is required for binding to PKC but not for the PDZ-PDZ association. The extra COOH-terminal residues in PDZ3L are not sufficient for binding to PKC since PDZ3LdeltaN does not bind PKC (Xu, 1998a).

Interaction of InaD with the major rhodopsin encoded by the ninaE locus

A suggestion that the major rhodopsin encoded by the ninaE locus may interact with InaD is that it coimmunoprecipitates with the TRP channel from wild-type but not InaDP215 mutant fly heads (Chevesich, 1997). To test whether the rhodopsin interacts with InaD in vivo, a coimmunoprecipitation experiment was performed using extracts from Drosophila heads. InaD coimmunoprecipitates with rhodopsin but not in a control reaction carried out with nonimmune serum. In addition, the proteins were co-expressed in vitro using a mammalian tissue culture system, 293T cells, and it was found that ninaE opsin and InaD coimmunoprecipitate. Evidence that the association between the opsin and InaD is direct is that in vitro-translated opsin binds to GST-InaD immobilized on a glutathione-Sepharose column but not to the GST control (Xu, 1998a).

To map the regions in InaD mediating the interactions with the opsin, TRPL, and PKC, a series of InaD constructs were generated and coexpressed with each target protein in 293T cells. Either PDZ3 or PDZ4 is sufficient to interact with the opsin, TRPL, and PKC. However, binding of each target to PDZ3 requires an extra 28 amino acids COOH-terminal to PDZ3 (PDZ3L). A requirement for additional residues for target binding to a PDZ domain is not unique since a similar COOH-terminal extension is necessary for the binding of target peptides to the PDZ domain in neuronal nitric acid synthase. The association of the targets with PDZ3L requires PDZ3 rather than just the extra 28 COOH-terminal amino acids since truncation of the NH2-terminal portion of PDZ3L-obliterates binding. Binding of the opsin, TRPL, and PKC to PDZ3L and PDZ4 appears to be specific since none of the eight other proteins or protein fragments tested bind to PDZ3L or PDZ4. These included TRPC3, Shaker B, Calmodulin, and the PLC encoded by the no receptor potential A (norpA) locus. Furthermore, consistent with a recent report that PLC binds to a GST-PDZ1 fusion protein (van Huizen, 1998), it has been found that the COOH-terminal 123 residues of PLC expressed in 293T cells coimmunoprecipitate with either PDZ1 or PDZ1-PDZ2. These data suggest that the lack of interaction between these latter two PDZ domains and either the opsin, TRPL, or PKC is not due toimproper folding of PDZ1 or PDZ1-PDZ2. Studies in other labs, using bacterial fusion proteins, indicate that PLC binds to either PDZ1 and PDZ5 (van Huizen, 1998) or PDZ5 only (Tsunoda, 1997). Although the PLC expressed in 293T cells does not bind to PDZ5, PLC interacts with PDZ5 expressed in E. coli. The lack of interaction of PLC with PDZ5 in 293T cells may be due to interference by post-translational modifications of PDZ5, endogeous proteins that bind to either PLC or PDZ5 (precluding the PLC/PDZ5 interaction), or misfolding of PDZ5 (Xu, 1998a).

Interaction of InaD with NorpA (an eye specific phospholipase C)

The Transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, Trp is found associated into a multiprotein complex with the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog can be isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel (Huber, 1996b).

Photoreceptors which use a phospholipase C-mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cbeta (PLCbeta), the light-activated Ca2+ channel TRP, and an eye-specific protein kinase C (ePKC) are clustered by the PDZ domain protein InaD. The function of ePKC was investigated by cloning the Calliphora homolog of Drosophila ePKC, by precipitating the TRP signaling complex with anti-ePKC antibodies, and by performing phosphorylation assays in isolated signaling complexes and in intact photoreceptor cells. The deduced amino acid sequence of Calliphora ePKC comprises 685 amino acids and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti-ePKC antibodies leads to the coprecipitation of PLCbeta, TRP, InaD and ePKC but not of rhodopsin. Phorbolester- and Ca2+-dependent protein phosphorylation reveals that, apart from the PDZ domain protein InaD, the Ca2+ channel TRP is a substrate of ePKC. TRP becomes phosphorylated in isolated signaling complexes. TRP phosphorylation in intact photoreceptor cells requires the presence of extracellular Ca2+ in micromolar concentrations. It is proposed that ePKC-mediated phosphorylation of TRP is part of a negative feedback loop that regulates Ca2+ influx through the TRP channel (Huber, 1998).

Drosophila InaD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in inaD alleles display mislocalizations of signaling molecules of phototransduction that include the essential effector, phospholipase C-beta (PLC-beta), also known as NORPA. The molecular and biochemical details of this functional link are unknown. InaD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). Altered NORPA proteins lacking binding sites display normal basal PLC activity but can no longer associate with InaD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of InaD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent InaD-NORPA interactions, which are essential for G protein-coupled phototransduction (van Huizen, 1998).

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (InaD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that InaD also binds to eye-specific protein kinase C (INAC) and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis the essential elements of the NORPA-InaD association have been defined and three critical residues in the C-terminal tail of NORPA, required for the interaction, have been identified. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in InaD. To evaluate the functional significance of the InaD-NORPA association in vivo, transgenic flies were derived expressing a modified NORPA that lacks the InaD interaction: NORPAC1094S. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation. Similarly, delayed activation may result from NORPAC1094S being unable to localize in close proximity to the TRP channel. It is concluded that InaD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells (Shieh, 1997).

Because PLC is activated by a G protein, the inactivation of the Galpha-GTP by the GTPase reaction may be relevant to response termination. Previous in vitro studies of mammalian PLC-beta and GAP activity used a purified recombinant M 1 muscarinic receptor reconstituted into phospholipid vesicles with G_q/11 and PLC-beta1. The addition of PLC-beta1 to the reconstituted system increases the rate at which G_q hydrolyses GTP by three orders of magnitude. Phototransduction by vertebrate photoreceptors depends on a specific GAP activity. Accordingly, genetic elimination of regulators of G-protein signaling (RGS) proteins reduces and slows down GAP activity and leads to slow termination of responses to light. Thus, it is possible that in Drosophila, the effect of the association of PLC and INAD on response termination is related to the GAP activity of PLC (Cook, 2000 and references therein).

Both vertebrate photoreceptors are able to discern single photons by a reliable and reproducible production of a unitary response (bump) upon absorption of each photon, whereas the macroscopic response to light is a superposition of multiple bumps. Generation of unitary responses is a characteristic feature of many signal transduction cascades. There is a profound difference between the mechanism of bump production in vertebrate and invertebrate species. In vertebrate phototransduction, which is characterized by signal amplification at the early stages of the cascade, the shape of the bump is, at least partially, determined at the stages of rhodopsin and G-protein action. Accordingly, in transgenic mice lacking RGS 9 and showing reduced GAP activity, the declining phase of the bump is markedly slowed down. In invertebrates, however, there is little, if any, amplification at early stages of phototransduction and the shape of the bump is determined downstream of PLC activation. The interaction of GAP activity, PLC and PLC-INAD may thus have a key role in the mechanism that ensures production of a single bump following absorption of a single photon (Cook, 2000 and references therein).

In the work reported here, Drosophila phototransduction was used as a model system to study the physiological implications of formation of a signaling complex and PLC-induced GAP activity. The functional role of the association of PLC with the INAD signaling complex was investigated. This association is required for a high rate of GTPase activity induced by the large concentration of PLC. The dual role of PLC, the target of the G protein, as an activator of the cascade and as an essential negative regulator of the G protein, ensures that every activated G protein produces a bump. Reduced levels of PLC result in accumulation of active G protein, leading to slow response termination and hence to impaired temporal and intensity resolution (Cook, 2000).

The continuous response in the PLC-deficient mutants, long after the cessation of light, suggests that reduction in PLC levels results in light-induced accumulation of a long-lived signaling component that can activate the phototransduction cascade in the dark. The most likely candidate for this component is the active form of the G_q protein (G_qalpha-GTP), since it operates directly upstream of PLC, has a long life-time and precedes bump formation. Following the previous experiments showing PLC-dependent GAP activity in a reconstituted mammalian system in vitro, a test was performed to see whether the mechanism underlying the defects in response termination is a reduced PLC-dependent GAP activity. To this end, both the light-dependent binding of the non-hydrolysable GTP analogue GTP-gamma-S, which represents the amounts of G protein available for activation by light, and GTPase activity, which reflects the amount of light-activated G protein that is turned off, were measured. The rate of turn-off reflects the level of GAP activity. Using these biochemical assays the relationship between the electrophysiological phenotype of the above mutants, their PLC levels and their GAP activity were measured. The GTP-gamma-S binding assay shows that the amounts of G protein available for activation by light are similar in the wild type and in all the PLC-defective and inaD mutants. Although G-protein activation is similar, the light-dependent GTPase activity is graded and is strongly dependent on the PLC levels in these mutants (Cook, 2000).

Since several different G proteins are present in each cell, specificity of the GTPase to the G_qalpha that operates in phototransduction was demonstrated by assaying the Galphaq1 mutant. This mutant expresses only ~1% of the normal amount of G_qalpha that is required for generating the response to light. If the GTPase that was measure reflects the activity of the G_qalpha that operates in phototransduction, it would be expected that GTP-gamma-S binding and GTPase activity will be much reduced in this mutant. As expected, in the Galphaq 1 mutant highly reduced light-activated GTP-gamma-S binding and GTPase activity is observed. The reduction in GTPase activity of the Galphaq 1 mutant does not arise from reduced levels of PLC since the mutant has normal amounts (Cook, 2000).

To demonstrate in vitro that the PLC-induced GAP activity is specifically required for inactivation of Gqalpha, a biochemical complementation approach was undertaken. GTPase activity was reconstituted by fusing head membranes of the null norpAP24 mutant with those of the Galphaq 1 hypomorphic mutant using polyethyleneglycol (PEG). The GTPase activity obtained after fusing the membranes was significantly higher than the activity observed without the complementing membranes, whereas no effect was found without PEG. Thus, the membrane fusion supplement containing PLC and G_qalpha was sufficient to reconstruct GTPase activity in mutants that otherwise show only background activity. The rescue of GTPase activity is not expected to reach wild-type level since a significant amount of membrane will not fuse and only some of each membrane type will fuse with the other type in the optimal orientation and distance. Therefore, the rescue by complementation is significant, and indicates that in Drosophila photoreceptors eye-specific PLC (NORPA) is the component required for the GAP activity of Gqalpha.

To further establish a relationship between GAP activity and amount of PLC, GTPase activity in the PLC-INAD interacting and non-interacting mutants was plotted as a function of their PLC levels. GTPase activity correlates linearly with the PLC levels. The correlation is evident in non-interacting mutants as well as in the interacting mutant norpAP57 and the transgenic Drosophila T 6, which expresses reduced amounts of normal NORPA12. The linear correlation, which applies to all mutants, suggests that PLC-INAD interaction is not required for induction of GAP activity, as was revealed in the electrophysiological experiments. Rather, it is the amount of PLC that is required for GAP activity. This conclusion is consistent with earlier biochemical data showing that the carboxy-terminal region of PLC-beta1 is sufficient to induce GAP activity in a reconstituted vesicle preparation from mammalian cells (Cook, 2000).

The ability of transduction cascades to resolve signals reliably in time depends on the turn-off of each activated component when the stimulus ceases. Negative feedback by the activated effectors fulfils this requirement and the shortest possible regulatory cycle would be the direct turnoff of the active signaling component by its target. Here it has been shown that in Drosophila phototransduction, PLC, the known target of the active G protein, induces GTPase activity and thereby inactivates the G_qalpha-GTP. This GAP activity is critical for termination of responses to light. Previous studies have shown that grouping of signaling proteins into a complex by INAD is essential for localization and for maintaining sufficiently high levels of the protein (Cook, 2000).

The results presented here suggest that all these properties are important for the ability to produce enough GAP activity to facilitate response termination and allow the target-dependent inactivation process. Therefore, the grouping of key transduction components into multiprotein structures is functionally important not only in achieving speed and efficiency of signaling by reducing diffusion distances, but also for maintaining optimal stoichiometric ratios of the participating components. When PLC levels in the signaling membranes are low relative to the amount of the active G protein, light induces production of G_qalpha-GTP at a higher rate than it is inactivated by PLC. The G_qalpha-GTP that has accumulated during illumination continues to produce bumps in the dark until all active G_qalpha-GTP molecules are hydrolysed by the scarce PLC. Hence, flies are not able to resolve light stimuli and become virtually blind at low levels of PLC. Assembly of transduction components into signaling complexes is therefore important for many facets of the physiological response, as revealed by the crucial role of PLC association with the signaling complex (Cook, 2000).

In Drosophila, phototransduction is mediated by Gq-activation of phospholipase C and is a well studied model system for understanding the kinetics of signal initiation, propagation and termination controlled by G proteins. The proper intracellular targeting and spatial arrangement of most proteins involved in fly phototransduction requires the multi-domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospholipase C-ß isozyme. The crystal structure of the N-terminal PDZ domain of InaD bound to a peptide corresponding to the C-terminus of NorpA has been determined to 1.8 Å resolution. The structure highlights an intermolecular disulfide bond necessary for high affinity interaction as determined by both in vitro and in vivo studies. Since other proteins also possess similar, cysteine-containing consensus sequences for binding PDZ domains, this disulfide-mediated 'dock-and-lock' interaction of PDZ domains with their ligands may be a relatively ubiquitous mode of coordinating signaling pathways (Kimple, 2001).

As shown by gel exclusion chromatography, NorpA forms a stable homodimer through its C-terminal domain, supporting the recent discovery that other PLC-ßs, namely mammalian PLC-ß1 and -ß2 and turkey PLC-ß, form stable dimers. As is shown in the present work, a stable CT_Dm dimer can be covalently linked to two PDZ1 molecules through intermolecular disulfide bond formation. Evidence of dimerization of NorpA and other PLC-ß isoforms, along with disulfide-mediated interaction of NorpA and InaD, necessitates a revision of the current model of Drosophila phototransduction. In this revised model, a NorpA homodimer can covalently bind two InaD molecules, each of which can homodimerize through PDZ3 and PDZ4. This increases the number and strength of connections between phototransduction components by linking several rhodopsins, eyePKCs and TRP isozymes through NorpA dimers. As in the former model, PDZ1 can also bind to the myosin NinaC, linking the entire membrane-bound signalplex to the actin cytoskeleton. Binding of PDZ1 to NinaC, which has the C-terminal sequence VDI-COO^–, requires at least the C-terminal 21 residues of NinaC, suggesting that PDZ1 may interact with NinaC in a different mode than it does with NorpA (Kimple, 2001).

The spatial localization and connectivity of relevant proteins would help explain why the Drosophila phototransduction pathway is one of the fastest G protein-coupled signaling cascades known, with activation and termination occurring within tens of milliseconds. Another factor contributing to the rapid cycling of the Drosophila phototransduction cascade is that NorpA functions not only as an effector for G_q signaling, but also as a GTPase-activating protein (GAP) for GTP-bound Galpha_q. Drosophila norpA mutations suggest that the isosteric Cys1094Ser point mutation effectively abrogates all functional PDZ1 binding to NorpA, mimicking the effects of completely deleting the PDZ1 binding site (NorpA DeltaCterm 25). The combination of well characterized norpA mutants together with structural and biochemical analyses strongly suggest that disulfide-bond formation between InaD and the penultimate residue of NorpA is critical for proper phototransduction in Drosophila. By holding NorpA in a tight, membrane-bound complex, a covalent association between InaD and NorpA aids in the rapid regulation of G_q by NorpA (Kimple, 2001).

Interaction of InaD with Calmodulin

Ca2+ influxes regulate multiple events in photoreceptor cells, including phototransduction and synaptic transmission. An important Ca2+ sensor in Drosophila vision appears to be Calmodulin, since a reduction in levels of retinal Calmodulin causes defects in adaptation and termination of the photoresponse. These functions of Calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the TRP and TRPL ion channels, NinaC and INAD. To identify additional calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, a screen was conducted for retinal Calmodulin-binding proteins. Eight additional Calmodulin-binding proteins were found that are expressed in the Drosophila retina. These included six targets that are related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein (UNC13) and a protein (CRAG) related to Rab3 GTPase exchange proteins. Two other Calmodulin-binding proteins are Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that Calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission (Xu, 1998b).

Interaction of InaD with TRP

Drosophila vision involves a G protein-coupled phospholipase C-mediated signaling pathway that leads to membrane depolarization through activation of Na+ and Ca2+ channels. inaD mutant flies have a M442K point mutation and display a slow recovery of the Ca2+ dependent current. Anti-InaD antibodies coimmunoprecipitate Trp, which is identified by its electrophoretic mobility, cross reactivity with anti-Trp antibody, and absence in a null allele trp mutant. This interaction is abolished by the inaD point mutation, both in vitro and in vivo. Interaction is localized to the 19 amino acid C-terminus of Trp by overlay assays, and to the PDZ domain of InaD, encompassing the point mutation. Flies homozygous for the InaD and trp mutations were generated and their phenotype analyzed by electroretinogram (ERG). In this extracellular recording of the compound eye, light triggers a depolarizing receptor potential and the Inad and trp mutants display characteristic responses: trp shows a receptor potential that lacks the maintained component, and Inad lacks the prolonged depolarizing afterpotential. While the InaD and trp ERG phenotypes are similar when stimulated by a pulse of intense blue light, they are distinguished using low intensity stimulation. When stimulated with a 10 second pulse of orange light, InaD displays a sustained response, whereas trp flies show the transient receptor potential response. Under the same conditions, double mutants exhibit a phenotype similar to that of trp. The finding that the response of the double mutants is qualitatively similar to that of trp alone is consistent with the interpretation that Trp and InaD act in the same sequential pathway. In the absence of Trp, InaD is not able to effect its modulatory activity, and thus, the double mutant phenotype corresponds to that of trp. Given the impaired electrophysiology of the inaD mutant, this novel interaction suggests that InaD functions as a regulatory subunit of the Trp Ca2+ channel (Shieh, 1996).

INAD, a novel protein mutated in the inactivation no afterpotential D mutation in Drosophila, is a PDZ domain protein, sharing a protein interaction domain with Drosophila proteins Discs large, Dishevelled and Canoe. InaD photoreceptor cells show a slow deactivation of light-induced current and an increased sensitivity to dim light. The store-operated Ca2+ channel, TRP, is required in photoreceptor cells for a sustained response to light. TRP forms a complex with phospholipase C-ß (No receptor potential A), rhodopsin (RH1) Calmodulin, and INAD. The current model for Drosophila phototransduction is that IP3 generated through activation of NORPA binds to the IP3 receptor, resulting in release of Ca2+ from internal Ca2+ stores. The Calmodulin binding site of TRP bears no similarity to that of TRPL, the non specific cation channel that otherwise shares sequence homology with TRP. In InaD mutant flies, TRP is no longer spatially restricted to its normal subcellular compartment, the rhabdomere. In inaD^P215 mutant flies, TRP is no longer restricted to the rhabdomeres. Instead, some TRP is detected in the cell bodies, and a large proportion of TRP is found in the extracellular central matrix. The mislocalization of TRP in the central matrix might have occurred during the normal turnover of the photoreceptor cell membrane, which involves shedding of the microvillar rhabdomeral membrane into the central matrix. The alteration in localization of TRP in inaD^P215 is specific since all other rhabdomere-specific proteins examined displayed indistinguishable expression patterns in wild type and inaD^P215. Although a significant proportion of TRP is mislocalized in inaD^P215, some TRP remains in the rhabdomeres. Since the only existing inaD allele, inaD^P215, is caused by a point mutation in the second PDZ domain, it is possible that there remains some weak interaction between TRP and INAD^P215 protein that cannot be detected in vitro. If so, it is possible that no proportion of TRP would be detected in the rhabdomeres of null inaD flies. However, the dramatic change in localization of TRP detected in inaD^P215 provides evidence that a PDZ domain protein is required in vivo for targeting or anchoring an ion channel to its normal subcellular localization. These results provide evidence that a PDZ domain protein is required, in vivo, for the anchoring of an ion channel to a signaling complex. Furthermore, disruption of this interaction results in retinal degeneration. It is proposed that the TRP channel is linked to RORPA and RH1 to facilitate feedback regulation of these upstream signaling molecules. It is suggested that TRP may be inactivated through a Ca2+-dependent mechanism mediated by Calmodulin. (Chevesich, 1997).

Phototransduction in invertebrate microvillar photoreceptors is thought to be mediated by the activation of phospholipase C (PLC), but how this leads to gating of the light-sensitive channels is unknown. Most attention has focused on inositol-1,4,5-trisphosphate, a second messenger produced by PLC from phosphatidylinositol-4,5-bisphosphate; however, PLC also generates diacylglycerol, a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Both of these fatty acids reversibly activate native light-sensitive channels [transient receptor potential (TRP) and TRP-like (TRPL)] in Drosophila photoreceptors, as well as recombinant TRPL channels expressed in Drosophila S2 cells. Recombinant channels are activated rapidly in both whole-cell recordings and inside-out patches, with a half-maximal effector concentration for linolenic acid of approximately 10 microM. Four different lipoxygenase inhibitors, which might be expected to lead to build-up of endogenous fatty acids, also activate native TRP and TRPL channels in intact photoreceptors. As arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999).

The light response in Drosophila photoreceptor cells is mediated by a series of proteins that assemble into a macromolecular complex referred to as the signalplex. The central player in the signalplex is Inactivation no afterpotential D (InaD), a protein consisting of a tandem array of five PDZ domains. At least seven proteins bind InaD, including the Transient receptor potential (Trp) channel, which depends on InaD for localization to the phototransducing organelle, the rhabdomere. However, the determinants required for localization of InaD are not known. InaD is required for retention rather than targeting of Trp to the rhabdomeres. In addition, Trp binds to InaD through the COOH terminus, and this interaction is required for localization of InaD. Two other proteins that depend on InaD for localization, phospholipase C and protein kinase C, also mislocalize. However, elimination of any other member of the signalplex has no impact on the spatial distribution of InaD. A direct interaction between Trp and InaD does not appear to have a role in the photoresponse independent of localization of multiple signaling components. Rather, the primary function of the Trp/InaD complex is to form the core unit required for localization of the signalplex to the rhabdomeres (Li, 2000).

Trp is initially localized to the rhabdomeres in young InaD^P215 and trp^delta1272 flies, whereas in older flies, the spatial distribution of Trp is severely disrupted. These data suggest that InaD may be required for retention rather than targeting of Trp to the rhabdomeres. However, an alternative interpretation of these data is that those Trp molecules synthesized in young flies are targeted through an InaD-independent mechanism, whereas InaD is required for targeting of Trp synthesized in older flies. In support of the proposal that the InaD/Trp interaction is required for retention is the observation that Trp is long lived in vivo. Trp molecules synthesized before day 1.5 decline only ~25% in concentration during the next 8 d. Thus, it appears that Trp is initially targeted to the rhabdomeres, and is subsequently mislocalized in the absence of a direct link to InaD (Li, 2000).

An intriguing question concerns the identification of proteins required for localization of InaD. The NINAC myosin III would appear to be an excellent candidate, since it binds InaD and is a putative molecular motor expressed in the photoreceptor cells. Nevertheless, NINAC is not required for localization of InaD. Other InaD-interacting proteins that function in phototransduction, such as rhodopsin, PLC, PKC, and TrpL, are also dispensable for rhabdomeral distribution of InaD. In contrast to these proteins, Trp is specifically required for proper localization of InaD. Moreover, in trp mutant flies and in transgenic flies, trp^delta1272, in which the InaD binding site is deleted, the spatial distribution of InaD is disrupted in an age-dependent manner. These data, in combination with the findings that the half-life of InaD is ~5.5 d, suggest that the mislocalization of InaD in trp mutant flies is due to a defect in retention (Li, 2000).

In addition to a requirement for the Trp/InaD interaction for localization of Trp and InaD, elimination of trp or mutation of the InaD binding site in Trp leads to an alteration in the spatial distribution of other proteins that require InaD for rhabdomere localization. These include PLC and PKC. Moreover, the mislocalization of PLC and PKC appeared to be more pronounced than that of InaD in young trp flies. One possibility is that these signaling proteins may also interact with Trp and depend on both Trp and InaD for localization. PKC appears to interact at least transiently with Trp, since Trp is a substrate for PKC. Thus, Trp and InaD appear to form the core unit that is required for localization of many of the signalplex components in the rhabdomeres (Li, 2000).

The putative tetrameric structure of Trp may contribute to the stability of the Trp/InaD core unit, since each channel would have the potential to bind four InaD proteins. Although InaD is mislocalized in trp^delta1272, there is no major impact on the localization of InaD in InaD^P215, suggesting the existence of residual interaction between Trp and InaD^P215 in vivo. Consistent with this proposal, Trp is more unstable in trp^delta1272 than in InaD^P215. The presumed tetrameric structure of Trp could enhance a weak interaction between InaD^P215 and Trp in vivo, which is not observed in pull-down assays using a monomeric Trp tail, as noted above because each channel would have the potential to bind four InaD proteins. The data presented here raise the possibility that other PDZ-containing scaffold proteins form similar core complexes in vivo with tetrameric ion channels. In support of this proposal are recent in vitro experiments indicating that there is a reciprocal requirement for localization of PSD-95 and the K⁺ channel, Kv1.4 (Li, 2000 and references therein).

A separate question that awaits further investigation concerns the mechanism underlying targeting of the signalplex to the rhabdomeres. Evidence has been presented that another PDZ-containing scaffold protein, PSD-95, is trafficked to the postsynaptic compartment after assembling on vesicles. Thus, it is plausible that the components of the signalplex may get trafficked to the rhabdomeres via vesicular transport and require the Trp/InaD core unit for retention (Li, 2000 and references therein).

The finding that Trp and InaD are long lived is surprising considering that there is very active turnover of the rhabdomeric membrane. Such turnover results in shedding of rhabdomeral membrane into the central matrix and blebbing of membrane from the base of the microvilli into the cell bodies. The association between Trp and InaD may serve to prevent removal of these proteins into the central matrix and cell bodies during membrane turnover. Interestingly, the mutual requirement for the Trp/InaD interaction for retention in the rhabdomeres is less critical in trp^delta1272 flies maintained in the dark. It is suggested that a greater proportion of Trp and InaD is retained in the dark because of less turnover of the rhabdomeral membrane in the absence of light (Li, 2000).

In contrast to InaD^P215, the electroretinogram (ERG) response in young trp^delta1272 is similar to wild-type. The only significant ERG phenotype in trp^delta1272 flies was an age-dependent decrease in the amplitude of the maintained component. This defect was presumably due to mislocalization of Trp and InaD, since the amplitude of the maintained component gradually decreases in parallel with the mislocalization of Trp and InaD in older flies. Moreover, the termination of the photoresponse appears normal even in old trp^delta1272 flies. This latter result is surprising, because PKC is mislocalized in old trp^delta1272 photoreceptor cells, and PKC is required for termination of the photoresponse. However, the rhabdomeric concentration of two substrates for PKC (Trp and InaD) is also reduced in trp^delta1272. Given that PKC, Trp, and InaD have been reported to be present in about equimolar concentrations, it is proposed that the relative stoichiometry of PKC and its substrates is important for normal termination of the photoresponse. Thus, the delay in termination resulting from a reduction in PKC concentration may be suppressed by a concomitant decrease in the levels of InaD and Trp (Li, 2000).

The defect in termination associated with InaD^P215 may not be due to perturbation of the Trp/InaD interaction, because the mutation in PDZ3 may also affect binding to other target proteins. The observation that the termination defect does not became more severe in old InaD^P215 flies suggests that the phenotype is not due to the disruption of the spatial distribution of Trp, since the mislocalization of Trp is more severe in older InaD^P215 flies (Li, 2000).

To address the specific role of the Trp/InaD interaction, the InaD binding site was mapped and transgenic flies were generated expressing a Trp derivative that does not associate with InaD. PDZ domains typically recognize COOH-terminal sequences consisting of an S/T-X-V/I motif or hydrophobic or aromatic residues. As is the case with most PDZ target proteins, it was found that the critical binding motif is at the COOH terminus. Specifically, it was found that deletion of the last four amino acids (SGWL) completely disrupts Trp/InaD binding. Furthermore, Trp^delta1272 obtained from transgenic fly head extracts fails to associate with InaD in pull-down assays. Mutation of an internal S-X-V motif (V1266D), which abolishes interaction between Trp and InaD in an overlay assay, has only minor effects. An additional deletion (amino acids 1257-1264), which eliminates the first residue of the S-X-V motif within the context of the short Trp tail (1252-1275), also retains binding with InaD in vitro (Li, 2000).

It is concluded that the primary role of the direct interaction between Trp and InaD is not to facilitate rapid signaling. The apparently normal ERG in young trp^delta1272 suggests that there is no defect in any aspect of the photoresponse. Rather, binding of Trp to InaD is critical for forming the core unit of the signalplex, which is necessary for retention of multiple signaling proteins in the rhabdomeres. This conclusion contrasts with previous reports, which have concluded that InaD functions as a regulatory subunit of the Trp channel. These analyses of trp^delta1272 indicate that the delayed termination associated with InaD^P215 is not due to disruption of the interaction with Trp. Instead, it appears that the phenotype is due to disruption of the interaction of InaD with another signaling protein that is required for proper response termination. Thus, contrary to expectations, a direct association between Trp and InaD appears to be dispensable for rapid termination (Li, 2000).

It appears that there are at least three classes of InaD binding proteins. The first class consists exclusively of Trp, because it is the only known InaD binding partner that is required for retention of InaD as well as of those InaD targets that depend on the signalplex for localization. However, there may be additional proteins that along with Trp and InaD comprise the core unit. The second group includes two proteins, PLC and PKC, which rely on InaD for localization and stability. However, there is no reciprocal requirement for these proteins for retention of any other protein in the rhabdomeres. Mutation of the InaD binding sites in PLC have been reported to cause defects in the photoresponse. However, these effects may reflect mislocalization or instability of these InaD targets rather than a direct requirement for coupling to InaD (Li, 2000).

The third class of InaD target proteins includes proteins such as rhodopsin, NINAC, and TrpL that are not dependent on InaD for localization in the rhabdomeres. It is proposed that the class I and II proteins, which depend on interaction with InaD for retention in the rhabdomeres, are constitutively bound to InaD, whereas the class III proteins may interact dynamically with InaD. As a consequence, only a subset of the class III proteins may bind to InaD at any given time. The observation that class III proteins do not depend on InaD for localization suggests that these InaD/target protein interactions have an alternative function, such as a direct role in the photoresponse. In support of this proposal, it was found that mutation of the InaD binding site in NINAC results in a pronounced delay in termination of the photoresponse. Thus, proteins that do not depend on InaD for localization may participate in the rapid activation and/or termination of the photoresponse (Li, 2000).

In Drosophila photoreceptors the multivalent PDZ protein InaD organizes the phototransduction cascade into a macromolecular signaling complex containing the effector PLC, the light-activated TRP channels, and a regulatory PKC. The subcellular localization of InaD signaling complexes is critical for signaling. How InaD complexes are anchored and assembled in photoreceptor cells has been examined. trp mutants, or transgenic flies expressing inaD alleles that disrupt the interaction between InaD and TRP, cause the mislocalization of the entire transduction complex. The InaD-TRP interaction is not required for targeting but rather for anchoring of complexes, because InaD and TRP can be targeted independently of each other. In addition to its scaffold role, InaD functions to preassemble transduction complexes. Thus the interaction of InaD with the TRP channel is required for anchoring signaling complexes in the rhabdomeres. TRP then may interact with the cytoskeleton, securing InaD and the whole complex to the membrane; ankyrin repeats on the N terminus of TRP could play a role in linking TRP to the cytoskeleton. Another possibility is that the InaD-TRP interaction reveals, or unmasks, sites on TRP or InaD that are important for membrane anchoring. Preassembly of signaling complexes helps to ensure that transduction complexes with the appropriate composition end up in the proper location. This may be a general mechanism used by cells to target different signaling machinery to the pertinent subcellular location (Tsunoda, 2001).

Common mechanisms regulating dark noise and quantum bump amplification in Drosophila photoreceptors

Absolute visual thresholds are limited by 'dark noise,' which in Drosophila photoreceptors is dominated by brief (~10 ms), small (~2 pA) inward current events, occurring at ~2/s, believed to reflect spontaneous G protein activations. These dark events were increased in rate and amplitude by a point mutation in myosin III (NINAC), which disrupts its interaction with the scaffolding protein, INAD. This phenotype mimics that previously described in null mutants of ninaC (no inactivation no afterpotential; encoding myosin III) and an associated protein, retinophilin (rtp). Dark noise was similarly increased in heterozygote mutants of diacylglycerol kinase (rdgA/+). Dark noise in ninaC, rtp, and rdgA/+ mutants was greatly suppressed by mutations of the G_q α-subunit (Gαq) and the major light-sensitive channel (trp) but not rhodopsin. ninaC, rtp, and rdgA/+ mutations also all facilitated residual light responses in Gαq and PLC hypomorphs. Raising cytosolic Ca²⁺ in the submicromolar range increased dark noise, facilitated activation of transient receptor potential (TRP) channels by exogenous agonist, and again facilitated light responses in Gαq hypomorphs. These results indicate that RTP, NINAC, INAD, and diacylglycerol kinase, together with a Ca²⁺-dependent threshold, share common roles in suppressing dark noise and regulating quantum bump generation; consequently, most spontaneous G protein activations fail to generate dark events under normal conditions. By contrast, quantum bump generation is reliable but delayed until sufficient G proteins and PLC are activated to overcome threshold, thereby ensuring generation of full-size bumps with high quantum efficiency (Chu, 2013).

Scaffolding protein INAD regulates deactivation of vision by promoting phosphorylation of transient receptor potential by eye protein kinase C in Drosophila

Drosophila visual signaling is one of the fastest G-protein-coupled transduction cascades, because effector and modulatory proteins are organized into a macromolecular complex ('transducisome'). Assembly of the complex is orchestrated by inactivation no afterpotential D (INAD), which colocalizes the transient receptor potential (TRP) Ca²⁺ channel, phospholipase Cβ, and eye protein kinase C (eye-PKC), for more efficient signal transduction. Eye-PKC is critical for deactivation of vision. Moreover, deactivation is regulated by the interaction between INAD and TRP, because abrogation of this interaction in InaD^p215 results in slow deactivation similar to that of inaC^p209 lacking eye-PKC. To elucidate the mechanisms whereby eye-PKC modulates deactivation, this study demonstrates that eye-PKC, via tethering to INAD, phosphorylates TRP in vitro. Ser⁹⁸² of TRP is phosphorylated by eye-PKC in vitro and, importantly, in the fly eye, as shown by mass spectrometry. Furthermore, transgenic expression of modified TRP bearing an Ala substitution leads to slow deactivation of the visual response similar to that of InaD^p215. These results suggest that the INAD macromolecular complex plays an essential role in termination of the light response by promoting efficient phosphorylation at Ser⁹⁸² of TRP for fast deactivation of the visual signaling (Popescu, 2006; full text of paper).

Drosophila visual transduction is a G-protein-coupled signaling pathway that provides a model system for understanding the molecular basis of signal transduction in the vertebrate nervous systems. Drosophila visual signaling is initiated with the activation of rhodopsin by light. Activated rhodopsin, via a Gq heterotrimeric protein, stimulates phospholipase Cβ (PLCβ) named no-receptor potential A (NORPA). NORPA hydrolyzes PIP₂ (phosphatidylinositol 4,5-bisphosphate) to inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG), which leads to opening of the transient receptor potential (TRP) Ca²⁺ and TRP-like channels, and depolarization of photoreceptors. The key second messenger that activates the TRP Ca²⁺ channel is thought to be either DAG or its lipid metabolites, whereas IP₃ does not appear to play a role. DAG may have a dual function, because it also activates the eye-specific protein kinase C (eye-PKC; InaC or Inactivation no afterpotential C) essential for deactivation of the light response (Popescu, 2006 and references therein).

Reversible phosphorylation modulates the dynamics of signal transduction by transiently altering activities of signaling proteins. Members of the conventional PKC family, which are activated by Ca²⁺ and DAG, are capable of phosphorylating a wide variety of protein substrates for temporal and spatial regulation of signaling processes. In Drosophila, eye-PKC is involved in the negative regulation of visual signaling, because inaC^p209 flies lacking eye-PKC display abnormal desensitization, slow deactivation, and defects in light adaptation. Eye-PKC is anchored to a macromolecular complex by tethering to INAD. Interaction with INAD enhances the stability of eye-PKC as well as targets eye-PKC to the rhabdomeres of photoreceptors, in which visual signaling occurs. Importantly, the in vivo function of eye-PKC is regulated by interaction with INAD. Previously, it was shown that eye-PKC phosphorylates TRP in vitro. The present study investigated the molecular basis of TRP phosphorylation by eye-PKC (Popescu, 2006).

To mimic eye-PKC phosphorylation of TRP in vitro, a complex-dependent kinase assay was designed. The in vitro complex-specific phosphorylation of TRP is regulated by the presence of the INAD-interacting domain in TRP, as well as the existence of INAD in the fly extracts. Extracts lacking either eye-PKC or INAD fail to support TRP phosphorylation. Similarly, extracts prepared from InaD^p215 (a modified INAD devoid of the TRP binding) are not able to promote TRP phosphorylation. Together, these findings indicate that INAD targets eye-PKC to its substrates, similar to RACK (receptor for activated C kinase). By the complex-dependent kinase assay, Ser⁹⁸² of TRP was identified as an eye-PKC phosphorylation site. Moreover, TRP isolated from flies by LC-MS was analyzed and it was found that Ser⁹⁸² of TRP is indeed phosphorylated in vivo by eye-PKC, because phosphorylated peptides encompassing Ser⁹⁸² of TRP are present in wild-type, but absent in inaC^p209 flies (Popescu, 2006).

Next, the in vivo functional contribution of phosphorylation was investigated by characterizing transgenic flies expressing a modified TRP bearing an Ala substitution at Ser⁹⁸² (trp^S982A). Remarkably, these transgenic flies displayed prolonged deactivation kinetics in response to bright light stimuli, indicating that phosphorylation of TRP at Ser⁹⁸² by eye-PKC is involved in inactivation of TRP, leading to fast deactivation. A model of the TRP regulation by eye-PKC is proposed. TRP is an integral part of the INAD complex and is opened by light. After light termination, the visual response is rapidly deactivated. Although molecular mechanisms underlying deactivation remain elusive, Ca²⁺ is known to play a vital role in response termination. The increased intracellular Ca²⁺ (primarily mediated by TRP) and DAG activate eye-PKC, which, in turn, phosphorylates TRP at Ser⁹⁸². Phosphorylation of TRP leads to a rapid inactivation of the channel on cessation of the light stimulation, without affecting the interaction between TRP and INAD. How does phosphorylation influence the TRP channel activity? Ser⁹⁸² is located within the Lys-Pro-rich region of TRP, which may function in TRP gating. It is speculated that phosphorylation at Ser⁹⁸² may induce a conformational change in the pore domain, which in turn leads to a rapid closure and inactivation of TRP. Phosphorylation has been linked directly to conformational changes that play key roles in the regulation of ion channels. It is also possible that phosphorylation of TRP at Ser⁹⁸² affects the interaction with some yet-unidentified proteins that may be important for the modulation of the TRP channel activity (Popescu, 2006).

In the absence of eye-PKC-mediated phosphorylation of TRP, deactivation of visual signaling is slower as observed in inaC^p209 or trp^S982A. It was found that inaC^p209 displays a more complex deactivation defect, whereas trp^S982A exhibits prolonged deactivation only in response to bright light. These findings suggest that, in addition to TRP, eye-PKC phosphorylates other substrates for efficient termination of the light response. Indeed, eye-PKC has been shown to phosphorylate INAD, but the functional relevance of this phosphorylation is not known. Furthermore, eye-PKC is required for the Ca²⁺-dependent inhibition of NORPA. NORPA is part of the INAD complex; however, it is not known to be phosphorylated by eye-PKC. The Ca²⁺-dependent inactivation of the light-induced current is unaltered in inaC^p209. This finding suggests the existence of a parallel Ca²⁺-dependent mechanism in inaC^p209 by which TRP is inactivated or of an upregulation of a Ca²⁺-dependent mechanism that activates other kinases to compensate for the loss of eye-PKC in inaC^p209 (Popescu, 2006).

Importantly, trp^S982A displays slow deactivation kinetics similar to that of InaD^p215. InaD^p215 was isolated based on the ina (inactivation no afterpotential) phenotype elicited by ERG. By whole-cell recordings, it was shown that InaD^p215 exhibits slow deactivation kinetics. However, a delay in latency of the quantum bump has been proposed and that activation is affected in the InaD^p215 mutant. To resolve this discrepancy, the mutant was reexamined and it was concluded that the primary defect in InaD^p215 is prolonged deactivation and not slow activation. InaD^p215 expresses INAD^M442K, which fails to associate with TRP. How does a loss of INAD–TRP interaction lead to abnormal deactivation of visual signaling? It is likely that the lack of the INAD–TRP interaction prevents the recruitment of TRP to the INAD complex and, consequently, eye-PKC-mediated regulation. Indeed, both trp^S982A and InaD^p215 exhibit similar deactivation defects, indicating that the molecular basis underlying the slow deactivation defect in InaD^p215 is attributable to a lack of negative regulation of the TRP channel by eye-PKC. Together, these findings suggest that formation of the INAD complex is essential for fast deactivation of the visual response by promoting phosphorylation of TRP by eye-PKC. Moreover, Ser⁹⁸² may be the sole eye-PKC phosphorylation site in TRP, because trp^S982A and InaD^p215 display similar deactivation defects. A loss of INAD–TRP interaction has been investigated in transgenic flies expressing modified TRP in which the INAD-interacting domain was deleted (trp^Δ1272). A reduced light response with normal deactivation kinetics in trp^Δ1272 has been reported. It has been proposed that the suppression of the delayed termination, which is attributable to a reduced eye-PKC level in trp^Δ1272 is probably masked by a concomitant decrease in TRP and INAD levels (Popescu, 2006).

To date, many proteins related to Drosophila TRP have been discovered in both invertebrates and vertebrates. These TRP ion channels are subdivided into seven subfamilies (TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, and TRPML). Drosophila TRP belongs to the TRPC subfamily. Members of the TRPC subfamily are also activated by receptor-induced activation of phospholipase C and therefore may be regulated by PKC. Indeed, phosphorylation of the TRPC channels by PKC appears important for modulating the channel activity. For example, the PKC-mediated phosphorylation of TRPC1 was shown to contribute to its SOC (store operated channel) activation, triggering Ca²⁺ entry into endothelial cells. In contrast, PKC-mediated phosphorylation was demonstrated to inhibit the activity of TRPC3 in HEK 293 cells and of TRPC6 in PC12D neuronal cells. In both cases, TRPC3 and TRPC6 are activated by DAG, whereas DAG also turns on PKC. It has been proposed that timing is important because the channels are activated by DAG more rapidly than they are inhibited by DAG-activated PKC. Heterologously expressed TRPC7 was also shown to be regulated by PKC: inhibition of PKC prolonged inactivation of the channel. Moreover, PKC phosphorylation of heterologously expressed TRPC5 resulted in desensitization of this channel, a process that was dependent on both extracellular and intracellular Ca²⁺ concentrations (Popescu, 2006).

In conclusion, this study has uncovered the molecular mechanism underlying the complex-dependent phosphorylation of TRP by eye-PKC and its role in fast deactivation of vision. Specifically, it was shown that eye-PKC phosphorylates TRP at Ser⁹⁸² in vitro and in vivo. Importantly, phosphorylation of TRP facilitates rapid inactivation of the channel because transgenic flies bearing an Ala substitution at Ser⁹⁸² display prolonged deactivation kinetics of the light response. Significantly, this slow deactivation defect is similar to that observed in InaD^p215 in which TRP fails to associate with INAD. These findings provide insights into the mechanistic basis of slow deactivation in InaD^p215, suggesting that INAD plays a critical role in targeting eye-PKC to TRP for rapid deactivation of the visual signaling. Together, these data indicate that the INAD macromolecular complex is important for deactivation of the visual response by directing eye-PKC to TRP. Furthermore, PKC-mediated phosphorylation of TRP at Ser⁹⁸² leads to fast deactivation of vision by promoting inactivation of the TRP channel (Popescu, 2006).

Light-dependent phosphorylation of the Drosophila Inactivation No Afterpotential D (INAD) scaffolding protein at Thr170 and Ser174 by eye-specific Protein kinase C

Drosophila Inactivation No Afterpotential D (INAD) is a PDZ domain-containing scaffolding protein that tethers components of the phototransduction cascade to form a supramolecular signaling complex. This study reports the identification of eight INAD phosphorylation sites using a mass spectrometry approach. PDZ1, PDZ2, and PDZ4 each harbor one phosphorylation site, three phosphorylation sites are located in the linker region between PDZ1 and 2, one site is located between PDZ2 and PDZ3, and one site is located in the N-terminal region. Using a phosphospecific antibody, it was found that INAD phosphorylated at Thr170/Ser174 is located within the rhabdomeres of the photoreceptor cells, suggesting that INAD becomes phosphorylated in this cellular compartment. INAD phosphorylation at Thr170/Ser174 depends on light, the phototransduction cascade, and on eye-Protein kinase C that is attached to INAD via one of its PDZ domains (Voolstra, 2015).

Interaction of InaD with TRPL and FKBP52

Transient receptor potential and transient receptor potential-like (TRPL) are Ca(2+)-permeable cation channels found in Drosophila photoreceptor cells associated with large multimeric signaling complexes held together by the scaffolding protein, INAD. To identify novel proteins involved in channel regulation, Drosophila INAD was used as bait in a yeast two-hybrid screen of a Drosophila head cDNA library. Sequence analysis of one identified clone showed it to be identical to the Drosophila homolog of human FK506-binding protein, FKBP52 (previously known as FKBP59). To determine the function of dFKBP59, TRPL channels and dFKBP59 were co-expressed in Sf9 cells. Expression of dFKBP59 produced an inhibition of Ca(2+) influx via TRPL in fura-2 assays. Likewise, purified recombinant dFKBP59 produced a graded inhibition of TRPL single channel activity in excised inside-out patches when added to the cytoplasmic membrane surface. Immunoprecipitations from Sf9 cell lysates using recombinant tagged dFKBP59 and TRPL showed that these proteins directly interact with each other and with INAD. Addition of FK506 prior to immunoprecipitation resulted in a temperature-dependent dissociation of dFKBP59 and TRPL. Immunoprecipitations from Drosophila S2 cells and from fly head lysates demonstrated that dFKBP59, but not dFKBP12, interacts with TRPL in vivo. Likewise, INAD immunoprecipitates with dFKBP59 from S2 cell and head lysates. Immunocytochemical evaluation of thin sections of fly heads revealed specific FKBP immunoreactivity associated with the eye. Site-directed mutagenesis showed that mutations of P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709) eliminated interaction of the TRPL with dFKBP59. These results provide strong support for the hypothesis that immunophilin dFKBP59 is part of the TRPL-INAD signaling complex and plays an important role in modulation of channel activity via interaction with conserved leucyl-prolyl dipeptides located near the cytoplasmic mouth of the channel (Goel, 2001).

Anchoring TRP to the INAD macromolecular complex requires the last 14 residues in its carboxyl terminus

Drosophila transient-receptor-potential (TRP) is a Ca2+ channel responsible for the light-dependent depolarization of photoreceptors. TRP is anchored to a macromolecular complex by tethering to inactivation-no-afterpotential D (INAD). INAD associates with the carboxyl tail of TRP via its third post-synaptic density protein 95, discs-large, zonular occludens-1 domain. This paper further explored the molecular basis of the INAD interaction and demonstrated the requirement of the last 14 residues of TRP, with the critical contribution of Gly1262, Val1266, Trp1274, and Leu1275. Oull-down assays show that the last 14 residues of TRP comprises the minimal sequence that competes with the endogenous TRP from fly extracts, leading to the co-purification of a partial INAD complex containing INAD, no-receptor-potential A, and eye-protein kinase C (PKC). Eye-PKC is critical for the negative regulation of the visual signaling and is phosphorylated TRP in vivo. To uncover the substrates of eye-PKC in the INAD complex, a complex-dependent eye-PKC assay was designed, that utilized endogenous INAD complexes isolated from flies. Activated eye-PKC was shown to phosphorylate INAD, TRP but not no-receptor-potential A. Moreover, phosphorylation of TRP is dependent on the presence of both eye-PKC and INAD. Together, these findings indicate that stable kinase-containing protein complexes may be isolated by pull-down assays, and used in this modified kinase assay to investigate phosphorylation of the proteins in the complex. It is concluded that TRP associates with INAD via its last 14 residues to facilitate its regulation by eye-PKC that fine-tunes the visual signaling (Peng, 2008).

Interaction of InaD with NINAC

Many of the proteins that are critical for Drosophila phototransduction assemble into a signaling complex, signalplex, through association with the PDZ-domain protein InaD. Some of these proteins depend on InaD for proper subcellular localization to the phototransducing organelle, the rhabdomere, making it difficult to assess any physiological function of this signaling complex independent of localization. InaD binds directly to the NINAC myosin III, yet the subcellular localization of NINAC is normal in inaD mutants. Nevertheless, the InaD binding site is sufficient to target a heterologous protein to the rhabdomeres. Disruption of the NINAC/InaD interaction delays termination of the photoreceptor response. Thus one role of this signaling complex is in rapid deactivation of the photoresponse (Wes, 1999).

Interaction of InaD with TRPL

In addition to TRP, another cation influx channel subunit, TRPL, functions in phototransduction by forming a heteromultimeric channel with TRP. To investigate whether TRPL is an INAD-interacting protein, an in vivo coimmunoprecipitation experiment was carried out: INAD was found to associate with TRPL in fly photoreceptor cells. Since TRPL heteromultimerizes with TRP, it is possible that TRPL associates with INAD through TRP. Therefore, whether TRPL and INAD coimmunoprecipitate was tested after coexpressing the two proteins in 293T cells. INAD is detected after immunoprecipitating cell extracts with TRPL antibodies but not with nonimmune serum. Furthermore, INAD is not detected after immunoprecipitating with TRPL antibodies using extracts expressing only INAD. The interactions of TRP (Shieh, 1996) and TRPL with INAD appear to be specific since a highly related member of the TRP family, human TRPC3, does not coimmunoprecipitate with INAD. Evidence that TRPL and INAD directly interactes is that 35S-labeled TRPL binds to INAD-GST fusion proteins immobilized on a column (Xu, 1998a).

Interaction of InaD with InaC (the eye specific protein kinase C)

The Calliphora homolog of the Drosophila inaD gene product was initially isolated in order to isolate and characterize key proteins of the transduction cascade in photoreceptors using the phosphoinositide signaling pathway. Drosophila inaD mutants manifest a slow deactivation of the light response. By screening a retinal cDNA library with antibodies directed against photoreceptor membrane proteins, a cDNA coding for an amino acid sequence of 665 residues has been isolated. The sequence displays 65.3% identity (77.3% similarity) with the Drosophila InaD gene product. Probing Western blots with monospecific antibodies directed against peptides comprising amino acids 272-542 [(anti-InaD-(272-542)] or amino acids 643-655 [(anti-InaD-(643-655)] of the InaD gene product reveals that the Calliphora InaD protein is specifically associated with the signal-transducing rhabdomeral photoreceptor membrane from which it can be extracted by high salt buffer containing 1.5 M NaCl. Since five out of eight consensus sequences for protein kinase C phosphorylation reside within stretches of 10-16 amino acids that are identical in the Drosophila and Calliphora InaD protein, the InaD gene product is likely to be a target of protein kinase C. Phosphorylation studies with isolated rhabdomeral photoreceptor membranes followed by InaD immunoprecipitation reveal that the InaD protein is a phosphoprotein. In vitro phosphorylation is, at least to some extent, Ca 2+ dependent and activated by phorbol 12-myristate 13-acetate. The inaC-encoded eye-specific form of a protein kinase C (eye-PKC) is co-precipitated by antibodies specific for the InaD protein from detergent extracts of rhabdomeral photoreceptor membranes, suggesting that the InaD protein and eye-PKC are interacting in these membranes. Co-precipitating with the InaD protein and eye-PKC are two other key components of the transduction pathway, namely the Trp protein, which is proposed to form a Ca2+ channel, and the norpA-encoded phospholipase C, the primary target enzyme of the transduction pathway. It is proposed that the rise of the intracellular Ca2+ concentration upon visual excitation initiates the phosphorylation of the InaD protein by eye-PKC and thereby modulates its function in the control of the light response (Huber, 1996a).

Drosophila eye-specific protein kinase C (eye-PKC) is involved in light adaptation and deactivation. eye-PKC, NORPA (phospholipase Cbeta), and transient-receptor-potential (TRP) (calcium channel) are integral components of a signal transduction complex organized by INAD, a protein containing five PDZ domains. There is a direct association between the third PDZ domain of INAD with TRP, and the carboxyl-terminal half of INAD with the last three residues of NORPA. The molecular interaction between eye-PKC and INAD is defined via the yeast two-hybrid and ligand overlay assays. The second PDZ domain of INAD interacts with the last three residues in the carboxyl-terminal tail of eye-PKC, Thr-Ile-Ile. The association between eye-PKC and INAD is disrupted by an amino acid substitution (Ile-700 to Asp) at the final residue of eye-PKC. In flies lacking endogenous eye-PKC (inaCp215), normal visual physiology is restored upon expression of wild-type eye-PKC, whereas the eye-PKCI700D mutant is completely inactive. Flies homozygous for inaCp209 and InaDp215, a mutation that causes a loss of the INAD-TRP association, were generated. These double mutants display a more severe response inactivation than either of the single mutants. Based on these findings, it is concluded that the in vivo activity of eye-PKC depends on its association with INAD and that the sensitivity of photoreceptors is cooperatively regulated by the presence of both eye-PKC and TRP in the signaling complex (Adamski, 1998).

Yeast two-hybrid and ligand overlay results both indicate that the second PDZ domain of INAD associates predominantly with eye-PKC, whereas no interaction was detected with PDZ4. This result is different from a previous report in which interaction of eye-PKC with the fourth PDZ domain of INAD was detected by affinity chromatography (Tsunoda, 1997). The current study tested a total of five constructs that contained the fourth PDZ domain: no indication of this interaction was found. These included a fusion protein that contained exactly the same region as previously tested. One possible explanation for these conflicting results is that the different assay systems are measuring different types of association between eye-PKC and INAD. INAD may bind and cluster eye-PKC to the signaling complex, and it can also act as a substrate for the kinase activity (Huber, 1996a). Amino acid substitutions made in the second PDZ domain of INAD are shown to disrupt the eye-PKC binding. None of these amino acid changes were near the serine or threonine residues that are putative PKC phosphorylation sites. Furthermore, mutations in the carboxyl-terminal tail of PKC abolish PKC binding to the second PDZ domain. Thus the interaction described for eye-PKC/INAD is a typical carboxyl-terminal tail/PDZ domain association. The basis of the reported interaction with the fourth PDZ domain remains to be determined. Another provocative explanation could be that eye-PKC may bind different PDZ domains of INAD during different physiological conditions. For example, phosphorylation of INAD may change the relative affinity of the interaction in PDZ2 and PDZ4. Clarification of the role of these two eye-PKC/INAD interactions will require analysis of transgenic flies expressing a modified InaD in which these PDZ domain are mutated (Adamski, 1998).

The COOH-terminal three residues of target proteins (often S/TXV) are essential for binding to PDZ domains. To test whether INAD interacts with its targets in a similar way, each of the last three residues in PKC (T-I-I) were changed to aspartic acid (PKCD) and the derivative was coexpressed with full-length INAD in 293T cells. Binding to INAD is not abolished as a consequence of the mutation. To directly compare whether the binding of PKCD to INAD is reduced relative to wild-type PKC, column-binding assays were performed. Although PKCD still binds to INAD, the interaction is significantly reduced (approximately eightfold). It is possible that the residual binding is due to the presence of a second INAD binding site in PKC, since another INAD-binding protein, PLC (Chevesich, 1997), contains two sites (van Huizen, 1998). Alternatively, there may be a single binding site in PKC that is close to but not at the extreme COOH terminus. If so, then mutation of the flanking COOH-terminal residues may disrupt but not obliterate binding. To differentiate between these possibilities, an attempt was made to further map the binding site(s). All of the INAD-binding capacity is contained in the COOH-terminal third of PKC, which includes most of the catalytic domain (residues 472-700). Smaller derivatives of the catalytic domain are all unstable in 293T cells, suggesting that they might have been misfolded. Thus, it was not feasible to further map the INAD binding site(s) in 293T cells or using the column-binding assay (Xu, 1998a).

Functional INAD complexes are required to mediate degeneration in photoreceptors of the Drosophila rdgA mutant

The TRP family of ion channels mediates a wide range of calcium-influx phenomena in eukaryotic cells. Many members of this family are activated downstream of phosphoinositide hydrolysis but the subsequent steps that lead to TRP channel activation in vivo remain unclear. Recently, the lipid products of phosphoinositide hydrolysis (such as diacylglycerol and its metabolites) have been implicated in activating TRP channels in both Drosophila and mammals. In Drosophila photoreceptors, lack of diacylglycerol kinase (DGK) activity (encoded by rdgA) leads to both constitutive TRP-channel activity and retinal degeneration. In this study, using a novel forward-genetic screen, InaD, a multivalent PDZ domain protein, was identified as a suppresser of retinal degeneration in rdgA mutants. InaD suppresses rdgA, and the rescue is correlated with reduced levels of phospholipase Cß (PLCß), a key enzyme for TRP channel activation. Furthermore, it was shown that light, Gq and PLCß all modulate retinal degeneration in rdgA. The results demonstrate a previously unknown requirement for a balance of PLCß and DGK activity for retinal degeneration in rdgA. They also suggest a key role for the lipid products of phosphoinositide hydrolysis in the activation of TRP channels in vivo (Georgiev, 2005).

Transmembrane signalling cascades initiated by G-protein-coupled receptors are a widely used mechanism for signalling the detection of many sensory modalities. These cascades end with the activation of plasma-membrane ion channels whose activity alters membrane potential and initiates synaptic transmission of a signal to the central nervous system. Several different families of ion channels have been implicated in this process. Historically, the oldest and best characterized are cyclic-nucleotide-gated channels, whose role in vertebrate visual and olfactory transduction is well established. More recently, members of the TRP family of ion channels have been implicated in the transduction of several sensory modalities in both vertebrate and invertebrate systems. These include light (Drosophila TRPC), pheromones (rodent TRPC2), taste (rodent TRPM), physical stimuli and temperature (Drosophila and mammal TRPV, TRPA and TRPN). Currently, a crucial factor limiting the understanding of how TRP channels encode sensory modalities is the lack of information about how these channels are activated. In several cases, only a few transduction components have been identified and the inability to perform in vivo analysis of channel activation has been a major obstacle in revealing how TRP channels are activated (Georgiev, 2005).

The Drosophila phototransduction cascade is historically the oldest and to date the best understood model for the analysis of a TRP channel involved in sensory transduction. In the fly eye, rhodopsin, a seven-transmembrane-span G-protein-coupled receptor, activates phospholipase Cß (PLCß) via Gq. This initiates a biochemical cascade that ends with the opening of two classes of calcium- and cation-selective TRPC channels, TRP and TRPL. Several key elements of the transduction cascade have been identified including Gq, PLCß and protein-kinase C. Several of these components, along with the TRP channel, are clustered into a macromolecular signalling complex by the multivalent PDZ-domain protein INAD. The INAD complex is thought to increase the speed and specificity of the light response. However, despite this wealth of detail about the components of the transduction cascade, the mechanism of activation of TRP and TRPL remains poorly understood, and is one of the outstanding problems in both sensory neurobiology and intracellular calcium signalling (Georgiev, 2005),

Although the essential role of PLCß in the activation of TRP and TRPL is well established, the biochemical events initiated by this enzyme that lead to channel activation remain unclear. Inositol-1,4,5-trisphosphate (IP₃), the best-understood second messenger generated from phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] hydrolysis by PLCß was originally postulated to be the second messenger that leads to TRP and TRPL activation. However, several recent lines of evidence strongly indicate that IP₃-induced calcium (Ca²⁺) release, or indeed a physical interaction between the IP₃ receptor (IP₃R) and the light-activated channels, is unlikely to underlie the mechanism of TRP and TRPL activation. More recently, lipid second messengers derived from PI(4,5)P₂ have been implicated in the activation of TRP and TRPL as well as their vertebrate homologues. Polyunsaturated fatty acids, potential metabolites of diacylglycerol (DAG), the primary lipid generated by PI(4,5)P₂ hydrolysis, have been shown to activate TRP and TRPL in situ, as well as in inside-out patches of TRPL channels expressed in S2 cells. In addition, both DAG and PI(4,5)P₂ have been shown to modulate TRPL channel activity in cell culture models. Analysis of TRPC2 activation in the rodent vomeronasal organs shows considerable parallels to the current understanding of the mechanism of Drosophila TRP and TRPL activation. However, despite these findings, the physiological relevance of PI(4,5)P₂-derived lipids as activators of Drosophila TRP channels in vivo remains to be established and the precise identity of the phospholipid species that is involved is unknown (Georgiev, 2005),

Recently, genetic evidence of a role for lipid messengers in the activation of TRPC channels in vivo has been obtained in Drosophila photoreceptors from an analysis of the retinal degeneration A (rdgA) mutant. The rdgA mutant was first isolated because it failed to respond to light in a behavioural assay. Analysis of retinal ultrastructure revealed that all alleles show varying degrees of photoreceptor degeneration. Biochemical analysis showed impaired diacylglycerol kinase (DGK) activity and reduced levels of light induced phosphatidic acid (PA) formation in head extracts of rdgA mutants. The gene that is defective in rdgA mutants has been cloned and found to encode an eye-enriched isoform of DGK, the principal enzyme that inactivates DAG by phosphorylation to PA. However, and most significantly, under voltage-clamp conditions, several alleles including rdgA¹, rdgA³, rdgA⁶ and rdgA^KS60 all show a small constitutively active inward current, which, on the basis of its biophysical characteristics, genetics and pharmacology, has been shown to be composed largely of TRP channels. The retinal degeneration phenotype of rdgA can be rescued by genetically removing TRP channels (i.e., the double mutant rdgA;trp), whose photoreceptors now lack their principal plasma-membrane calcium-influx channels. These results suggested a model in which excessive calcium influx through constitutively active TRP channels results in retinal degeneration in rdgA. The light response of rdgA;trp photoreceptors shows defects in deactivation suggesting that DGK might play a role in terminating the light response and recent evidence suggests that DGK plays a role in regulating signal amplification during the response to light (Hardie, 2002). Despite these recent observations that suggest a direct role for rdgA in phototransduction, previous studies have suggested a distinct mechanism underlying the retinal degeneration phenotype of rdgA. (1) Unlike most other phototransduction mutants, the retinal degeneration of rdgA is reported to be light independent; (2) norpA mutants, which lack the PLC activity essential for TRP channel activation, were reported not to suppress the retinal degeneration of rdgA; (3) several studies have suggested that a failure of rhabdomere biogenesis and protein trafficking underlies the rdgA phenotype (Georgiev, 2005),

To address these apparently conflicting results and to understand the mechanism of degeneration in rdgA, a genome-wide forward-genetic screen was undertaken for mutants that suppress or enhance the retinal-degeneration phenotype of rdgA. The goal is to identify molecules whose function might help gain an understanding of basis of the constitutive TRP-channel activity that is associated with the rdgA phenotype. This study describes the isolation and characterization of two mutants identified in the screen. Experiments are described that address the requirement for the light response in the degeneration phenotype of rdgA (Georgiev, 2005),

Therefore, although most studies agree that the hydrolysis of PI(4,5)P₂ by receptor-regulated PLCß is required for TRPC (TRP and TRPL) activation, there is little agreement about the downstream biochemical mechanisms that result in TRPC channel opening. For some members, such as TRPC3, equally compelling studies have been published showing roles in activation for IP₃/IP₃R mediated store depletion and for the lipid products of PI(4,5)P₂ hydrolysis. Recently, it has become clear that many of these conflicting results arise from several experimental factors, including the level of overexpression of the channel, the presence of endogenous TRPC members in the cell lines used and the relative promiscuity of pharmacological agents used in manipulating their activation. By contrast, in Drosophila photoreceptors, the detection of light by rhodopsin activates a signalling cascade that ends with endogenous TRPC activation. In this model, too, current debate centers around the identity of the products of PI(4,5)P₂ hydrolysis that are crucial for channel activation. Although there is substantial evidence to suggest that IP₃-mediated signalling is not essential, recent evidence suggests that the lipid products of PI(4,5)P₂ hydrolysis might be involved in activation. Analysis of photoreceptors lacking DGK activity (rdgA) has provided the first genetic evidence that suggests a role for lipid second messengers in activating TRP and TRPL in vivo. However, questions remain about the biochemical basis of the rdgA phenotype and its relevance to the normal phototransduction cascade; for example, is the constitutive channel activity the cause or the consequence of the degeneration? In addition, how can one reconcile recent findings suggesting a role for rdgA in phototransduction with long-standing observations that imply a phototransduction-independent basis for the rdgA phenotype (Georgiev, 2005)?

To address this issue, a forward-genetic screen was carried out to identify suppressors of the retinal degeneration phenotype of rdgA mutants. Such an approach is unbiased and makes no assumptions about the mechanisms underlying the degeneration process. Using a combination of deficiency mapping and bioinformatic analysis, su(1) and su(100) were identified as new alleles of InaD, a PDZ-domain protein required for the assembly of signalling complexes in Drosophila photoreceptors that is suggested to have a role in the regulation of signalling specificity and speed. InaD has not previously been reported to interact with rdgA. Trp³⁶⁵ contains a point mutation at the cytoplasmic end of S5 in the TRP channel and shows constitutive channel activity and degeneration. The finding that InaD is largely ineffective at suppressing the degeneration of Trp³⁶⁵ suggests that the mechanism of suppression is at or above the level of the channel in the transduction cascade rather than by blocking events downstream of excessive calcium influx through constitutively active TRP channels. InaD clusters several key molecules required for phototransduction, including the TRP channel; this strongly suggests that the constitutive channel activity and degeneration in rdgA are a consequence of altered phototransduction (Georgiev, 2005),

To identify the specific known (or perhaps undiscovered) protein-protein interactions of InaD that contribute to the rescue of rdgA, the InaD complex was manipulated in a manner that allowed its interaction with specific transduction components such as the TRP channel and NORPA to be individually disrupted. Wild-type TRP protein levels were found in InaD on eclosion and by analysing the effect of InaD^P215 and TRP^Delta1272 on rdgA it was found that TRP channels that could not be recruited to the INAD complex were able to mediate degeneration in rdgA just as well as wild-type channels. Thus, loss of the TRP-INAD interaction is unlikely to be a significant mechanism by which InaD rescues rdgA and the function of TRP channels within the INAD complex is not crucial to retinal degeneration (Georgiev, 2005),

By contrast, it was found that NORPA (the PLC activity) levels in su(1) and su(100) mutants are reduced on eclosion, as has been reported for InaD¹, suggesting that a reduction in PLCß activity might underlie the mechanism of suppression. InaD¹, a protein-null allele, and InaD², an allele known to disrupt the INAD-NORPA interaction, produce equivalent levels of rescue of rdgA¹, and that the levels of NORPA are inversely correlated to the extent of rescue. Thus, a major mechanism by which InaD¹, su(1) and su(100) suppress rdgA¹ is likely to involve the reduced levels of NORPA in these alleles. However, the possibility cannot be excluded that additional, unknown protein-protein interactions of PDZ5 in INAD that might also be disrupted in InaD² might also contribute to the rescue of rdgA. Given the essential role of PLCß in the activation of TRP channels, this finding implies a key role for the balance of PLCß and DGK activity in the degeneration of rdgA (Georgiev, 2005),

Although the degeneration phenotype of rdgA has been previously reported to be light independent and not suppressed by norpA mutants, the finding that InaD suppresses rdgA and that it does so by reducing levels of NORPA suggests that defects in light-induced phosphoinositide turnover might underlie the degeneration phenotype of rdgA. In the light of these findings on the suppression of rdgA by InaD, the effect was re-examined of three key elements of the phototransduction cascade that are required for activation, namely light, Galphaq and PLC. Although the absence of light could not completely suppress the degeneration of rdgA³, there was substantial suppression of degeneration in rdgA³ flies grown in complete darkness compared with those grown on a 12 hour light/12 hour dark cycle. Degeneration could also be partially suppressed but not blocked by a strong hypomorph that reduced Galphaq levels to <5% of the wild-type levels. However, most importantly, it was found that norpA mutants that lack PLCß could suppress the degeneration of rdgA in several allelic combinations for both genes. These results demonstrate a key role for activation of the phototransduction cascade in the degeneration phenotype of rdgA (Georgiev, 2005),

Although this study shows a requirement for light, Galphaq and PLC activity in the degeneration phenotype of rdgA, it was not possible to completely suppress the degeneration of even the weakest allele, rdgA³, by rearing flies in complete darkness. Indeed, reducing levels of Galphaq using the strong hypomorph Galphaq¹ (which has <5% of the wild-type Galphaq levels) was able only to slow the rate of degeneration of rdgA³. However mutants in norpA^P24 were able completely to suppress the degeneration of both rdgA³ and rdgA¹. In a recent study that measured basal PLCß activity in photoreceptors (Hardie, 2004), it was found that, similar to wild-type photoreceptors, rdgA mutants showed reduced but still substantial basal PLCß activity. This implies that, even in the dark, there is a basal turnover of PI(4,5)P₂ in rdgA photoreceptors. Thus, basal PI(4,5)P₂ hydrolysis could lead to the build up of a lipid metabolite of PI(4,5)P₂ that triggers constitutive TRP channel activity and retinal degeneration (Georgiev, 2005),

Although several studies have demonstrated the importance of INAD in targeting and stabilizing members of the phototransduction cascade to the rhabdomere, there is little agreement about the requirement, if any, for intact INAD complexes once assembled and transported to the rhabdomere to activate TRP channels. Although some studies have suggested that an intact INAD complex is crucial for generating the channel activity that underlies a quantum bump (the response to a single photon of light) others have suggested that this might not be the case. In this analysis of the mechanism by which InaD suppresses rdgA, it was found that that TRP channels not included within the INAD complex but still present in the rhabdomere are able to mediate retinal degeneration. These results support the idea that presence within the INAD macromolecular complex is not necessary for the constitutive activity of TRP channels seen in rdgA (Georgiev, 2005),

Although these data support the hypothesis that a principal mechanism by which InaD suppresses rdgA is via reduction in the levels of PLCß, they do not exclude the possibility that the disruption of INAD interactions with currently undiscovered proteins that function downstream of NORPA might play a role in constitutive TRP channel activation and degeneration in rdgA. Testing this would require the generation of an InaD allele in which the INAD-NORPA interaction is intact while disrupting the function of the other protein-protein interactions of INAD. No such allele exists, but the use of such an allele in conjunction with the rdgA mutant could be an useful approach to identifying currently undiscovered members of the INAD complex as well as the phototransduction cascade (Georgiev, 2005),

TRP channels appear to be key components of signalling cascades for the detection and coding of several sensory modalities. However, a limiting factor in advancing their role in sensory transduction is the poor understanding of their mechanism of activation. In the case of TRPC channels, this is limited by the lack of genetic model systems in which relevant components of the activation cascade can be identified. In the present study, a novel modifier screen has been described that should provide a powerful method for identifying the relevant transduction components in vivo. Starting with the rdgA mutant in which TRP channels are constitutively active and result in retinal degeneration, two new alleles of INAD, a known component of the phototransduction cascade, have been identified as suppressors of rdgA. This approach is likely to be a powerful tool to identify further components of the transduction cascade that are relevant in vivo (Georgiev, 2005),

Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors

This study reveals a novel feature of the dynamic organization of signaling components in Drosophila photoreceptors. The multi-PDZ protein INAD and its target proteins undergo light-induced recruitment to detergent-resistant membrane (DRM) rafts. Reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM fractions. Genetic analysis demonstrated that translocation of INAD-signaling complexes to DRM rafts requires activation of the entire phototransduction cascade, while constitutive activation of the light-activated channels resulted in recruitment of complexes to DRM rafts in the dark. Mutations affecting INAD and TRP showed that PDZ4 and PDZ5 domains of INAD, as well as the INAD-TRP interaction, are required for translocation of components to DRM rafts. Finally, selective recruitment of phosphorylated, and therefore activatable, eye-PKC to DRM rafts suggests that DRM domains are likely to function in signaling, rather than trafficking (Sanxaridis, 2007).

Recent studies have revealed that some phototransduction components in Drosophila and vertebrate photoreceptors undergo a light-induced translocation between the rhabdomere and cell body. The subcellular translocation of these components has been proposed to contribute to long-term light adaptation. While light appears to regulate the quantity of G_qα, TRPL, and arrestin-2 protein available for signaling in the rhabdomere, no light-induced changes in subcellular localization have been observed for any of the components of the INAD-signaling complex. This report shows instead that components of the INAD-signaling complex undergo light-regulated translocation to DRM rafts within the rhabdomeres of photoreceptors. While subcellular translocation of components out of the rhabdomere may regulate the overall level of protein available for signaling, local translocation of components to lipid raft micro-domains is likely to regulate more immediate signaling mechanisms. Although two hours of light-exposure were used in this study to induce a signal that was robust and reliable enough to be observed by the biochemical assay used, future studies, perhaps using single fluorophore tracking microscopy, will need to examine real-time translocation of components to DRM rafts (Sanxaridis, 2007).

To examine the role lipid rafts play in signaling, electroretinogram (ERG) recordings were performed on ergΔ-fed flies (ergΔ mutant yeast lack C-8 sterol isomerase and C-24 sterol methyltransferase activity, which individually and combinatorialy prevent the biosynthesis of ergosterol). No apparent differences were found from flies fed standard fly food. It is suspected, however, that signaling defects are likely to be missed in such a gross extracellular recording. Future whole-cell voltage-clamp recordings from single raft-depleted photoreceptor cells will be more informative. This, however, is not yet feasible with one-month old adult ergΔ-fed flies since whole-cell recording has only been successful with pupae or newly-eclosed flies. Given that flies are unable to develop to eclosion on the ergΔ food, an alternate ergosterol depletion method will need to be developed for these studies (Sanxaridis, 2007).

How might lipid rafts regulate signaling? The recruitment of INAD-signaling complexes to DRM raft domains may serve to further concentrate components, increasing the rate of protein-protein interactions during signaling as well as increasing the levels of local second messengers created, possibly enhancing the speed and/or amplitude of the light-response. Another possibility is that lipid rafts serve as micro-environments that protect or isolate signaling components from positive or negative regulators present in non-raft domains. For instance, translocation of PLC to lipid rafts may serve to isolate PLC from further activation by G_qα, contributing to deactivation of the light-response (Sanxaridis, 2007).

Micro-domains created by lipid rafts may also provide a special micro-environment that regulates signaling components. For example, TRP channels may have altered activation or deactivation properties in different lipid environments. Indeed, other ion channels have been shown to display different biophysical properties depending on whether they are associated with DRM rafts or not. Although it is well established that activation of the effector PLC is essential for activation of TRP and TRPL channels, it is still uncertain what element(s) downstream of PLC are responsible for directly gating the channels. Recent reports have suggested that the channels are activated by poly-unsaturated fatty acids derived from the second messenger DAG and that maintaining PIP₂ levels is necessary for sustained light responsiveness. Thus, the heterogeneous distribution of lipids present in raft and non-raft micro-domains may indeed differentially regulate TRP channels. For example, PIP₂ has been reported to accumulate in DRM raft domains of several cell types and raft disruption has been shown to mislocalize PIP₂ and affect phosphatidylinositol turnover. Such a concentrated pool of PIP₂, if present in lipid rafts of Drosophila photoreceptors, may indeed play a role in the activation of TRP channels when PLC and TRP are translocated to lipid rafts. Since some studies have reported that application of exogenous poly-unsaturated fatty acids causes the replacement of saturated fatty acids in the membrane with unsaturated ones, leading to disruption of lipid raft domains, future studies examining the gating of TRP channels by poly-unsaturated fatty acids will now need to consider the possible role of lipid rafts in signaling (Sanxaridis, 2007).

This study shows that light induces the translocation of INAD-signaling complexes to DRM rafts in the rhabdomere, highlighting another facet of the dynamic nature of signaling components in photoreceptors. While subcellular movements of proteins are likely to contribute to long-term light adaptation, local translocation to micro-domains within the rhabdomere are likely to modulate more immediate signaling mechanisms. Future studies are likely to investigate the function of lipid rafts in activation/deactivation of PLC, gating of TRP channels, as well as light-adaptation. Using Drosophila as a model system offers the opportunity to combine biochemical studies with Drosophila genetics to identify the function of lipid rafts in vivo (Sanxaridis, 2007).

Role of protein phosphatase 2A in regulating the visual signaling in Drosophila

Drosophila visual signaling, a G-protein-coupled phospholipase Cbeta (PLCbeta)-mediated mechanism, is regulated by eye-protein kinase C (PKC) that promotes light adaptation and fast deactivation, most likely via phosphorylation of inactivation no afterpotential D (INAD) and TRP (transient receptor potential). To reveal the critical phosphatases that dephosphorylate INAD, several biochemical analyses were used and protein phosphatase 2A (PP2A) was identified as a candidate. Importantly, the catalytic subunit of PP2A, Microtubule star (MTS), copurifies with INAD, and an elevated phosphorylation of INAD by eye-PKC was observed in three mts heterozygotes. To explore whether PP2A (MTS) regulates dephosphorylation of INAD by counteracting eye-PKC [INAC (inactivation no afterpotential C] in vivo, ERG recordings were performed. ina^CP209 is semidominant, because ina^CP209 heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect of ina^CP209 heterozygotes is rescued by the mts^XE2258 heterozygous background. In contrast, mts^XE2258 fails to modify the severe deactivation of norpA^P16, indicating that MTS does not modulate NORPA (no receptor potential A) (PLCbeta). Together, these results strongly indicate that dephosphorylation of INAD is catalyzed by PP2A, and a reduction of PP2A can compensate for a partial loss of function in eye-PKC, restoring the fast deactivation kinetics in vivo. Thus it is proposed that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD (Wang, 2008).

The in vitro assays show that PP2A dephosphorylates the scaffolding protein INAD, opposing the activity of eye-PKC to phosphorylate INAD. An increased level of INAD phosphorylation occured in three distinct mts heterozygotes, wherein the catalytic C subunit of PP2A has been rendered ineffective. Utilizing ERG recordings, in partial loss-of-function mutants of mts^XE2258, inaC^P209, and the combined double mutant, it was found that PP2A and eye-PKC have opposing physiological functions, and that a balance between the activities of eye-PKC and PP2A is central for the proper deactivation of the visual response. The results strongly indicate that PP2A appears to impact signaling proteins operating downstream of NORPA in the visual cascade. Integrating in vivo and in vitro findings into the current model of eye-PKC-mediated regulation of INAD, it is concluded that reversible phosphorylation of INAD is dependent on the opposing enzymatic actions of eye-PKC and PP2A and that phosphorylation of INAD is critical for fast deactivation of the visual signaling process (Wang, 2008).

The biochemical assays support PP2A as a key phosphatase responsible for the dephosphorylation of INAD. Based on both its inhibition profile with okadaic acid, and its copurification alongside the FPLC fraction with positive phosphatase activity, PP2A was identified as the prime candidate for mediating INAD dephosphorylation. After finding that a purified A/C dimer of PP2A dephosphorylates INAD in vitro, immunocomplex kinase assays were performed. As expected, a reduction in PP2A catalytic efficiency causes a significant increase in the measurable fraction of phosphorylated INAD. By examining three distinct mts heterozygotes, each carrying a C subunit mutation resulting in a partial loss of PP2A function, a significant increase was demonstrated in INAD phosphorylation levels with mts^XE2258 exhibiting the most dramatic increase. The enhanced INAD phosphorylation observed in the mts extracts strongly suggests that PP2A is closely positioned in the INAD complex to promote timely dephosphorylation of INAD. Consistently, it was demonstrated that PP2A can be coisolated with INAD, thus representing a newly identified component of the INAD macromolecular complex (Wang, 2008).

For insights into the role PP2A plays in Drosophila vision, mts^XE2258 heterozygotes were studied, and a surprisingly normal ERG waveform was found. Although a reduction in the level of active PP2A in vivo has no effect on visual function, it was found that inaC^P209 heterozygotes exhibit abnormal light adaptation, as well as delayed deactivation of visual signaling. inaC^P209 and mts^XE2258 both encode for enzymes; why would missing one functional copy of the PP2A gene not affect normal visual electrophysiology, whereas missing a functional copy of the eye-PKC gene drastically slows deactivation? inaC^P209 must be a semidominant mutation; in other words, inaC is haploinsufficient. An explanation for the haploinsufficiency of the eye-PKC gene is that the substrate repertoire of eye-PKC is defined by substrate colocalization to the INAD macromolecular complex to which eye-PKC is tethered. This hypothesis is in good agreement with the observation that the interaction with INAD is essential for the in vivo function of eye-PKC to modulate the visual response (Wang, 2008).

Although MTS is also tethered to the INAD signaling complex, unlike eye-PKC, the abundance of MTS relative to INAD in photoreceptors is likely to make it less sensitive to a reduction of its gene dosage to effect visual electrophysiology. Alternatively, it is possible that anchoring to the INAD complex by PP2A may be regulated by the interaction via its B subunit instead of the C subunit. Therefore, a reduction of MTS may not significantly modify its presence in the INAD complex (Wang, 2008).

To elucidate the functional interplay, in vivo, between eye-PKC and MTS, mts^XE2258 and inaC^P209 double heterozygotes were characterized. A significant discovery was made that only the slow deactivation defect was rescued in the mts^XE2258 heterozygous background. The selective rescue of deactivation defects by mts^XE2258, with no rescue of the abnormal light adaptation found in inaC^P209 heterozygotes, suggests that PP2A regulates proteins that lie downstream of eye-PKC, a conclusion that is also supported by the inability of mts^XE2258 to restore the slow deactivation defect in a hypomorphic allele of norp^P16 (Wang, 2008).

A concomitant reduction of the PP2A activity would lead to increased phosphorylation of multiple substrates contributing to the observed normal, fast deactivation kinetics found in the double mutant. Potential PP2A substrates may include INAD, TRP, and eye-PKC. Like other conventional PKCs, eye-PKC is most likely a phosphoprotein and hence its catalytic activity is sensitive to PP2A. It is expected that a reduction of PP2A should increase the autophosphorylation of eye-PKC, bringing about an enhanced catalytic capability. However, the presumably increased eye-PKC activity fails to restore the light adaptation abnormality, suggesting that the modulation of eye-PKC represents a lesser role of PP2A in vivo (Wang, 2008).

It is possible that PP2A dephosphorylates TRP, thus regulating deactivation kinetics. Consistently, a lack of phosphorylation at Ser⁹⁸² of TRP leads to slowed deactivation of the visual response. However, trp^P343, a null allele affecting the trp gene, displays a less severe defect in deactivation than that of inaC^P209, suggesting eye-PKC phosphorylation of TRP does not play a major role in the normal deactivation. The eye-PKC-dependent phosphorylation of INAD is currently being studied and preliminary results suggest that a loss of phosphorylation in INAD also results in slowed deactivation kinetics. This finding together with the biochemical results supports the hypothesis that the phosphatase PP2A directly regulates phosphorylation states of INAD to impact fast deactivation of the visual signaling. A light-dependent conformation change has been shown to occur in the fifth postsynaptic density-95/Discs large/zona occludens-1 (PDZ) domain of INAD, and it has been proposed that eye-PKC might orchestrate this event. These findings are in agreement with the current studies supporting a critical role of INAD phosphorylation to promote fast deactivation. It will be of great interest to elucidate how phosphorylation of INAD leads to the conformation switch in its fifth PDZ domain (Wang, 2008).

In addition to INAD, PP2A may dephosphorylate other, yet-to-be identified substrates. The observations that mts^XE2258 modified the severe deactivation defect of inaC^P209 homozygotes, suggests that a reduction of MTS increases phosphorylation of proteins, which are regulated by non-eye-PKC serine/threonine protein kinases. Several protein kinases including CaMKII, and NINAC (neither inactivation nor afterpotential C) have been shown to modulate deactivation, and may play a role in regulating deactivation in the absence of eye-PKC (Wang, 2008).

In summary, this study has demonstrates the roles of PP2A and eye-PKC in orchestrating reversible phosphorylation of INAD, and that phosphorylation of INAD is most likely involved in fast deactivation kinetics of the visual signaling in Drosophila. The biochemical findings support the critical role of PP2A to dephosphorylate INAD. Electrophysiological characterization strongly indicates that a reduction of PP2A compensates for a partial loss of function in eye-PKC leading to rescuing the slow deactivation defect (Wang, 2008).

Dependence on a retinophilin/myosin complex for stability of PKC and INAD and termination of phototransduction

Normal termination of signaling is essential to reset signaling cascades, especially those such as phototransduction that are turned on and off with great rapidity. Genetic approaches in Drosophila led to the identification of several proteins required for termination including protein kinase C (PKC), NinaC p174, which consists of fused protein kinase and myosin domains, and a PDZ scaffold protein, INAD. This study describes a mutation affecting a poorly characterized but evolutionarily conserved protein, Retinophilin (Retin), which is expressed primarily in the phototransducing compartment of photoreceptor cells, the rhabdomeres. Retin and NINAC formed a complex and were mutually dependent on each other for expression. Loss of retin resulted in an age-dependent impairment in termination of phototransduction. Mutations that affect termination of the photoresponse, typically lead to a reduction in levels of the major rhodopsin, Rh1, to attenuate signaling. Consistent with the slower termination in retin1, the mutant photoreceptor cells exhibited increased endocytosis of Rh1 and a decline in Rh1 protein. The slower termination in retin¹ was a consequence of a cascade of defects, which began with the reduction in NINAC p174 levels. The diminished p174 concentration caused a decrease in INAD. Since PKC requires interaction with INAD for protein stability, this leads to reduction in PKC levels. The decline in PKC was age-dependent, and paralleled the onset of the termination phenotype in retin¹ mutant flies. It is concluded that the slower termination of the photoresponse in retin¹ resulted from a requirement for the Retin/NINAC complex for stability of INAD and PKC (Venkatachalam, 2010).

This study describes the identification of Retin, a protein required for termination of the photoresponse. Unlike other proteins that function in termination, the retin phenotype is age-dependent. Slow termination leads to increased endocytosis and degradation of the major rhodopsin, Rh1, which serves as a negative feedback mechanism to attenuate the visual response. Consistent with a defect in termination, the age-dependent impairment in the photoresponse in retin¹ is associated with greater endocytosis of Rh1 and an age-dependent reduction in the concentration of Rh1 (Venkatachalam, 2010).

A central question concerns the basis for the age-dependent decrease in the termination rate in retin deficient flies. Retin has been reported to function in macrophages through a pathway that involves the ryanodine receptor, a store-operated channel, Orai, and the interacting protein, STIM1 (Cuttell, 2008), which is present in the endoplasmic reticulum (ER) and senses changes in ER Ca²⁺. However, Ca²⁺ release from the ER, the ryanodine receptor and the IP₃-receptor do not appear to function in Drosophila visual transduction. Furthermore, knockdown of stim1 RNA using a photoreceptor cell GAL4 in combination with UAS-stim1-RNAi transgene had no effect on phototransduction, the concentration of Retin, or other proteins reduced in retin¹ mutant eyes. The decrease in termination in retin¹ mutant flies was not due directly to loss of Retin, since the Retin protein was absent in young flies that exhibited normal termination. The retin phenotype also was not a consequence of a reduction in NINAC p174, since both 3 and 7 day-old retin¹ flies displayed similarly low levels of p174; however, only the 7 day-old flies exhibited the slow termination phenotype (Venkatachalam, 2010).

It is concluded that the age-dependent termination phenotype in retin¹ results from a reduction in PKC levels. Consistent with this proposal, the decline in PKC concentration paralleled the appearance of the termination phenotype. In young retin¹ flies, which displayed normal termination, PKC was not reduced significantly from wild-type. However, in older retin¹ flies, the PKC concentration declined two-fold. In further support of the conclusion that the 50% decrease in PKC is responsible for the termination defect in retin¹, a similar impairment in termination occurs in heterozygous flies, which are missing copy of the gene encoding the eye-enriched PKC (Venkatachalam, 2010).

The following mechanism is proposed through which Retin affects the concentration of PKC. First, Retin forms a complex with NINAC p174, and this interaction is required for the stability of p174. Both proteins co-immunoprecipitated from head extracts, and loss of Retin resulted in a lower concentration of p174. The requirement for Retin and NINAC was mutual since Retin was undetectable in flies missing p174. Second, NINAC is required for stabilizing the PDZ-containing scaffold protein INAD. NINAC and INAD interact, and it was found that a single amino acid mutation that disrupts the INAD binding site in p174 (ninaC^I1501E) causes a reduction in INAD. Third, PKC binds stoichiometrically to INAD and requires this interaction for stability. As a result, INAD and PKC displayed indistinguishable two-fold decreases in protein levels. It was found that PKC also declined to a similar extent in flies expressing NINAC^I1501E. Because INAD was reduced in ninaC^I1501E flies, but not Retin or NINAC p174, the instability of PKC was not due to non-specific effects resulting from changes in the concentrations either Retin or p174. Thus, loss of Retin causes a reduction in the level of p174, which in turn affects the concentration of INAD, leading to instability of PKC, which underlies the slower termination (Venkatachalam, 2010).

Despite the defect in termination, retin¹ flies exhibited only minor effects on retinal morphology. There are multiple examples of mutations that are associated with termination defects that display relatively minor alterations in rhabdomere morphology. These include rac2, ninaC, and stops. Of particular relevance, flies heterozygous for a mutation disrupting the eye-enriched PKC (inaC^P209/+ flies), which exhibit a termination phenotype similar to retin¹, do not undergo retinal degeneration (Venkatachalam, 2010).

Finally, both Retin and myosins with fused N-terminal protein kinase domains are found in other organisms including humans. Protein kinase/myosins (myosin IIIs) and Retin are both expressed in the mammalian retina. This raises the possibility that Retin and myosins related to NINAC may form a complex in mammalian photoreceptor cells, and are required for signaling (Venkatachalam, 2010).

InaD and signal transduction

The subcellular compartmentalization of signaling molecules helps to ensure the selective activation of different signal-transduction cascades within a single cell. Although there are many examples of compartmentalized signaling molecules, there are few examples of entire signaling cascades being organized as distinct signaling complexes. In Drosophila photoreceptors, the InaD protein, which consists of five PDZ domains, functions as a multivalent adaptor that brings together several components of the phototransduction cascade into a macromolecular complex. Single-photon responses have been studied in several photoreceptor mutant backgrounds. These show that the InaD macromolecular complex is the unit of signaling that underlies elementary responses. The localized activity of this signaling unit promotes reliable single-photon responses as well as rapid activation and feedback regulation. Genetic and electrophysiological tools were used to illustrate how the assembly of signaling molecules into a transduction complex limits signal amplification in vivo (Scott, 1998).

The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies have shown that the PDZ domain containing protein InaD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The InaD signaling complex also includes rhodopsin, protein kinase C (PKC), and Calmodulin, though it is not known whether these proteins bind to InaD. This study shows that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to InaD. A second ion channel, TRPL, also binds to InaD. Thus, most of the proteins involved directly in phototransduction appear to bind to InaD. Furthermore, InaD forms homopolymers and the homomultimerization occurs through two PDZ domains. Thus, it is proposed that the InaD supramolecular complex is a higher order signaling web consisting of an extended network of InaD molecules through which a G protein-coupled cascade is tethered (Xu, 1998a).

Homomultimerization of InaD

The finding that PDZ3 and PDZ4 bind rhodopsin, TRPL and PKC indicates that a single InaD molecule would not have the capacity to nucleate the entire visual transduction signaling complex unless InaD functions as a homomultimeric protein. To address this hypothesis, full-length InaD fused with MYC or FLAG epitope tags are co-expressed in 293T cells and it was found that InaD-FLAG coimmunoprecipitates with InaD-MYC. Further evidence that InaD homomultimerizes was obtained by demonstrating that 35S-InaD binds to a GST-InaD fusion immobilized on a glutathione column. The InaD homomultimerization occurs through PDZ domains (either PDZ3 or PDZ4). Furthermore, InaD PDZ3 and PDZ4 can form either homomeric or heteromeric interactions. The PDZ-PDZ interaction appears to be specific to PDZ3 and PDZ4 since neither PDZ1, PDZ2, nor PDZ5 binds to any portion of InaD, including PDZ3 or PDZ4 (Xu, 1998a).

The observation that homomeric interactions occur through either PDZ3 or PDZ4 raises the possibility that InaD may form a homopolymer rather than just a dimer. To address this possibility, a segment of InaD including just PDZ3-PDZ4 was translated in vitro and the products were fractionated by sucrose gradient sedimentation. Although a proportion of PDZ3-4 fractionates near the predicted molecular weight of the dimer (52 kD), a significant amount sediments as a much larger protein of ~200 kD. PDZ1-PDZ2 loaded onto the same gradient sediments with a single peak near its predicted monomer molecular weight of ~39 kD. Thus, PDZ1-2 does not homomultimerize or interact with PDZ3-PDZ4. The data that a proportion of PDZ3-PDZ4 sediments as a protein greater than 200 kD suggests that InaD may be capable of forming homopolymers with a subunit composition of greater than 8. In contrast to PDZ1-PDZ2, which fractionates with a single peak, four small peaks are detected with PDZ3-PDZ4 that roughly correspond to the predicted sizes of molecules with 1, 2, 4, and 6 subunits. Since the PDZ1-PDZ2 monomer and marker proteins distribute over many fractions, the PDZ3-PDZ4 peaks may be small due to a similar broad distribution of the PDZ3-PDZ4 monomer, dimer, and higher order forms (Xu, 1998a).

The findings that InaD can form homomultimers through PDZ3 and PDZ4 raises the question as to whether homomultimerization precludes InaD-target interaction or vice versa. To investigate whether homomultimerization and PDZ-target interactions can occur simultaneously, advantage was taken of the finding that PDZ3 alone is sufficient to promote homotypic interactions, whereas PDZ3L is required for binding to the opsin, TRPL, or PKC. Therefore, tests were run to determine whether PDZ3 coimmunoprecipitates with the targets after coexpressing PDZ3 and PDZ3L with either TRPL or PKC. TRPL or PKC coimmunoprecipitate with PDZ3 in the presence (but not in the absence) of PDZ3L. These results indicate that PDZ3 and PDZ3L formed a ternary complex with the target proteins and suggest that the InaD PDZ-PDZ and PDZ-target interactions are mediated via different interfaces. Consistent with this latter proposal, an NH2-terminal truncation that removes the first and second putative beta barrel from PDZ3L (PDZ3LdeltaN) disrupts interaction with PKC; however, homomeric binding still occurs. Furthermore, the COOH-terminal extension in PDZ3L is required for binding to PKC but not for the PDZ-PDZ association. The extra COOH-terminal residues in PDZ3L are not sufficient for binding to PKC since PDZ3LdeltaN does not bind PKC (Xu, 1998a).

Interaction of InaD with the major rhodopsin encoded by the ninaE locus

A suggestion that the major rhodopsin encoded by the ninaE locus may interact with InaD is that it coimmunoprecipitates with the TRP channel from wild-type but not InaDP215 mutant fly heads (Chevesich, 1997). To test whether the rhodopsin interacts with InaD in vivo, a coimmunoprecipitation experiment was performed using extracts from Drosophila heads. InaD coimmunoprecipitates with rhodopsin but not in a control reaction carried out with nonimmune serum. In addition, the proteins were co-expressed in vitro using a mammalian tissue culture system, 293T cells, and it was found that ninaE opsin and InaD coimmunoprecipitate. Evidence that the association between the opsin and InaD is direct is that in vitro-translated opsin binds to GST-InaD immobilized on a glutathione-Sepharose column but not to the GST control (Xu, 1998a).

To map the regions in InaD mediating the interactions with the opsin, TRPL, and PKC, a series of InaD constructs were generated and coexpressed with each target protein in 293T cells. Either PDZ3 or PDZ4 is sufficient to interact with the opsin, TRPL, and PKC. However, binding of each target to PDZ3 requires an extra 28 amino acids COOH-terminal to PDZ3 (PDZ3L). A requirement for additional residues for target binding to a PDZ domain is not unique since a similar COOH-terminal extension is necessary for the binding of target peptides to the PDZ domain in neuronal nitric acid synthase. The association of the targets with PDZ3L requires PDZ3 rather than just the extra 28 COOH-terminal amino acids since truncation of the NH2-terminal portion of PDZ3L-obliterates binding. Binding of the opsin, TRPL, and PKC to PDZ3L and PDZ4 appears to be specific since none of the eight other proteins or protein fragments tested bind to PDZ3L or PDZ4. These included TRPC3, Shaker B, Calmodulin, and the PLC encoded by the no receptor potential A (norpA) locus. Furthermore, consistent with a recent report that PLC binds to a GST-PDZ1 fusion protein (van Huizen, 1998), it has been found that the COOH-terminal 123 residues of PLC expressed in 293T cells coimmunoprecipitate with either PDZ1 or PDZ1-PDZ2. These data suggest that the lack of interaction between these latter two PDZ domains and either the opsin, TRPL, or PKC is not due toimproper folding of PDZ1 or PDZ1-PDZ2. Studies in other labs, using bacterial fusion proteins, indicate that PLC binds to either PDZ1 and PDZ5 (van Huizen, 1998) or PDZ5 only (Tsunoda, 1997). Although the PLC expressed in 293T cells does not bind to PDZ5, PLC interacts with PDZ5 expressed in E. coli. The lack of interaction of PLC with PDZ5 in 293T cells may be due to interference by post-translational modifications of PDZ5, endogeous proteins that bind to either PLC or PDZ5 (precluding the PLC/PDZ5 interaction), or misfolding of PDZ5 (Xu, 1998a).

Interaction of InaD with NorpA (an eye specific phospholipase C)

The Transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, Trp is found associated into a multiprotein complex with the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog can be isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel (Huber, 1996b).

Photoreceptors which use a phospholipase C-mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cbeta (PLCbeta), the light-activated Ca2+ channel TRP, and an eye-specific protein kinase C (ePKC) are clustered by the PDZ domain protein InaD. The function of ePKC was investigated by cloning the Calliphora homolog of Drosophila ePKC, by precipitating the TRP signaling complex with anti-ePKC antibodies, and by performing phosphorylation assays in isolated signaling complexes and in intact photoreceptor cells. The deduced amino acid sequence of Calliphora ePKC comprises 685 amino acids and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti-ePKC antibodies leads to the coprecipitation of PLCbeta, TRP, InaD and ePKC but not of rhodopsin. Phorbolester- and Ca2+-dependent protein phosphorylation reveals that, apart from the PDZ domain protein InaD, the Ca2+ channel TRP is a substrate of ePKC. TRP becomes phosphorylated in isolated signaling complexes. TRP phosphorylation in intact photoreceptor cells requires the presence of extracellular Ca2+ in micromolar concentrations. It is proposed that ePKC-mediated phosphorylation of TRP is part of a negative feedback loop that regulates Ca2+ influx through the TRP channel (Huber, 1998).

Drosophila InaD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in inaD alleles display mislocalizations of signaling molecules of phototransduction that include the essential effector, phospholipase C-beta (PLC-beta), also known as NORPA. The molecular and biochemical details of this functional link are unknown. InaD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). Altered NORPA proteins lacking binding sites display normal basal PLC activity but can no longer associate with InaD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of InaD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent InaD-NORPA interactions, which are essential for G protein-coupled phototransduction (van Huizen, 1998).

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (InaD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that InaD also binds to eye-specific protein kinase C (INAC) and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis the essential elements of the NORPA-InaD association have been defined and three critical residues in the C-terminal tail of NORPA, required for the interaction, have been identified. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in InaD. To evaluate the functional significance of the InaD-NORPA association in vivo, transgenic flies were derived expressing a modified NORPA that lacks the InaD interaction: NORPAC1094S. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation. Similarly, delayed activation may result from NORPAC1094S being unable to localize in close proximity to the TRP channel. It is concluded that InaD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells (Shieh, 1997).

Because PLC is activated by a G protein, the inactivation of the Galpha-GTP by the GTPase reaction may be relevant to response termination. Previous in vitro studies of mammalian PLC-beta and GAP activity used a purified recombinant M 1 muscarinic receptor reconstituted into phospholipid vesicles with G_q/11 and PLC-beta1. The addition of PLC-beta1 to the reconstituted system increases the rate at which G_q hydrolyses GTP by three orders of magnitude. Phototransduction by vertebrate photoreceptors depends on a specific GAP activity. Accordingly, genetic elimination of regulators of G-protein signaling (RGS) proteins reduces and slows down GAP activity and leads to slow termination of responses to light. Thus, it is possible that in Drosophila, the effect of the association of PLC and INAD on response termination is related to the GAP activity of PLC (Cook, 2000 and references therein).

Both vertebrate photoreceptors are able to discern single photons by a reliable and reproducible production of a unitary response (bump) upon absorption of each photon, whereas the macroscopic response to light is a superposition of multiple bumps. Generation of unitary responses is a characteristic feature of many signal transduction cascades. There is a profound difference between the mechanism of bump production in vertebrate and invertebrate species. In vertebrate phototransduction, which is characterized by signal amplification at the early stages of the cascade, the shape of the bump is, at least partially, determined at the stages of rhodopsin and G-protein action. Accordingly, in transgenic mice lacking RGS 9 and showing reduced GAP activity, the declining phase of the bump is markedly slowed down. In invertebrates, however, there is little, if any, amplification at early stages of phototransduction and the shape of the bump is determined downstream of PLC activation. The interaction of GAP activity, PLC and PLC-INAD may thus have a key role in the mechanism that ensures production of a single bump following absorption of a single photon (Cook, 2000 and references therein).

In the work reported here, Drosophila phototransduction was used as a model system to study the physiological implications of formation of a signaling complex and PLC-induced GAP activity. The functional role of the association of PLC with the INAD signaling complex was investigated. This association is required for a high rate of GTPase activity induced by the large concentration of PLC. The dual role of PLC, the target of the G protein, as an activator of the cascade and as an essential negative regulator of the G protein, ensures that every activated G protein produces a bump. Reduced levels of PLC result in accumulation of active G protein, leading to slow response termination and hence to impaired temporal and intensity resolution (Cook, 2000).

The continuous response in the PLC-deficient mutants, long after the cessation of light, suggests that reduction in PLC levels results in light-induced accumulation of a long-lived signaling component that can activate the phototransduction cascade in the dark. The most likely candidate for this component is the active form of the G_q protein (G_qalpha-GTP), since it operates directly upstream of PLC, has a long life-time and precedes bump formation. Following the previous experiments showing PLC-dependent GAP activity in a reconstituted mammalian system in vitro, a test was performed to see whether the mechanism underlying the defects in response termination is a reduced PLC-dependent GAP activity. To this end, both the light-dependent binding of the non-hydrolysable GTP analogue GTP-gamma-S, which represents the amounts of G protein available for activation by light, and GTPase activity, which reflects the amount of light-activated G protein that is turned off, were measured. The rate of turn-off reflects the level of GAP activity. Using these biochemical assays the relationship between the electrophysiological phenotype of the above mutants, their PLC levels and their GAP activity were measured. The GTP-gamma-S binding assay shows that the amounts of G protein available for activation by light are similar in the wild type and in all the PLC-defective and inaD mutants. Although G-protein activation is similar, the light-dependent GTPase activity is graded and is strongly dependent on the PLC levels in these mutants (Cook, 2000).

Since several different G proteins are present in each cell, specificity of the GTPase to the G_qalpha that operates in phototransduction was demonstrated by assaying the Galphaq1 mutant. This mutant expresses only ~1% of the normal amount of G_qalpha that is required for generating the response to light. If the GTPase that was measure reflects the activity of the G_qalpha that operates in phototransduction, it would be expected that GTP-gamma-S binding and GTPase activity will be much reduced in this mutant. As expected, in the Galphaq 1 mutant highly reduced light-activated GTP-gamma-S binding and GTPase activity is observed. The reduction in GTPase activity of the Galphaq 1 mutant does not arise from reduced levels of PLC since the mutant has normal amounts (Cook, 2000).

To demonstrate in vitro that the PLC-induced GAP activity is specifically required for inactivation of Gqalpha, a biochemical complementation approach was undertaken. GTPase activity was reconstituted by fusing head membranes of the null norpAP24 mutant with those of the Galphaq 1 hypomorphic mutant using polyethyleneglycol (PEG). The GTPase activity obtained after fusing the membranes was significantly higher than the activity observed without the complementing membranes, whereas no effect was found without PEG. Thus, the membrane fusion supplement containing PLC and G_qalpha was sufficient to reconstruct GTPase activity in mutants that otherwise show only background activity. The rescue of GTPase activity is not expected to reach wild-type level since a significant amount of membrane will not fuse and only some of each membrane type will fuse with the other type in the optimal orientation and distance. Therefore, the rescue by complementation is significant, and indicates that in Drosophila photoreceptors eye-specific PLC (NORPA) is the component required for the GAP activity of Gqalpha.

To further establish a relationship between GAP activity and amount of PLC, GTPase activity in the PLC-INAD interacting and non-interacting mutants was plotted as a function of their PLC levels. GTPase activity correlates linearly with the PLC levels. The correlation is evident in non-interacting mutants as well as in the interacting mutant norpAP57 and the transgenic Drosophila T 6, which expresses reduced amounts of normal NORPA12. The linear correlation, which applies to all mutants, suggests that PLC-INAD interaction is not required for induction of GAP activity, as was revealed in the electrophysiological experiments. Rather, it is the amount of PLC that is required for GAP activity. This conclusion is consistent with earlier biochemical data showing that the carboxy-terminal region of PLC-beta1 is sufficient to induce GAP activity in a reconstituted vesicle preparation from mammalian cells (Cook, 2000).

The ability of transduction cascades to resolve signals reliably in time depends on the turn-off of each activated component when the stimulus ceases. Negative feedback by the activated effectors fulfils this requirement and the shortest possible regulatory cycle would be the direct turnoff of the active signaling component by its target. Here it has been shown that in Drosophila phototransduction, PLC, the known target of the active G protein, induces GTPase activity and thereby inactivates the G_qalpha-GTP. This GAP activity is critical for termination of responses to light. Previous studies have shown that grouping of signaling proteins into a complex by INAD is essential for localization and for maintaining sufficiently high levels of the protein (Cook, 2000).

The results presented here suggest that all these properties are important for the ability to produce enough GAP activity to facilitate response termination and allow the target-dependent inactivation process. Therefore, the grouping of key transduction components into multiprotein structures is functionally important not only in achieving speed and efficiency of signaling by reducing diffusion distances, but also for maintaining optimal stoichiometric ratios of the participating components. When PLC levels in the signaling membranes are low relative to the amount of the active G protein, light induces production of G_qalpha-GTP at a higher rate than it is inactivated by PLC. The G_qalpha-GTP that has accumulated during illumination continues to produce bumps in the dark until all active G_qalpha-GTP molecules are hydrolysed by the scarce PLC. Hence, flies are not able to resolve light stimuli and become virtually blind at low levels of PLC. Assembly of transduction components into signaling complexes is therefore important for many facets of the physiological response, as revealed by the crucial role of PLC association with the signaling complex (Cook, 2000).

In Drosophila, phototransduction is mediated by Gq-activation of phospholipase C and is a well studied model system for understanding the kinetics of signal initiation, propagation and termination controlled by G proteins. The proper intracellular targeting and spatial arrangement of most proteins involved in fly phototransduction requires the multi-domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospholipase C-ß isozyme. The crystal structure of the N-terminal PDZ domain of InaD bound to a peptide corresponding to the C-terminus of NorpA has been determined to 1.8 Å resolution. The structure highlights an intermolecular disulfide bond necessary for high affinity interaction as determined by both in vitro and in vivo studies. Since other proteins also possess similar, cysteine-containing consensus sequences for binding PDZ domains, this disulfide-mediated 'dock-and-lock' interaction of PDZ domains with their ligands may be a relatively ubiquitous mode of coordinating signaling pathways (Kimple, 2001).

As shown by gel exclusion chromatography, NorpA forms a stable homodimer through its C-terminal domain, supporting the recent discovery that other PLC-ßs, namely mammalian PLC-ß1 and -ß2 and turkey PLC-ß, form stable dimers. As is shown in the present work, a stable CT_Dm dimer can be covalently linked to two PDZ1 molecules through intermolecular disulfide bond formation. Evidence of dimerization of NorpA and other PLC-ß isoforms, along with disulfide-mediated interaction of NorpA and InaD, necessitates a revision of the current model of Drosophila phototransduction. In this revised model, a NorpA homodimer can covalently bind two InaD molecules, each of which can homodimerize through PDZ3 and PDZ4. This increases the number and strength of connections between phototransduction components by linking several rhodopsins, eyePKCs and TRP isozymes through NorpA dimers. As in the former model, PDZ1 can also bind to the myosin NinaC, linking the entire membrane-bound signalplex to the actin cytoskeleton. Binding of PDZ1 to NinaC, which has the C-terminal sequence VDI-COO^–, requires at least the C-terminal 21 residues of NinaC, suggesting that PDZ1 may interact with NinaC in a different mode than it does with NorpA (Kimple, 2001).

The spatial localization and connectivity of relevant proteins would help explain why the Drosophila phototransduction pathway is one of the fastest G protein-coupled signaling cascades known, with activation and termination occurring within tens of milliseconds. Another factor contributing to the rapid cycling of the Drosophila phototransduction cascade is that NorpA functions not only as an effector for G_q signaling, but also as a GTPase-activating protein (GAP) for GTP-bound Galpha_q. Drosophila norpA mutations suggest that the isosteric Cys1094Ser point mutation effectively abrogates all functional PDZ1 binding to NorpA, mimicking the effects of completely deleting the PDZ1 binding site (NorpA DeltaCterm 25). The combination of well characterized norpA mutants together with structural and biochemical analyses strongly suggest that disulfide-bond formation between InaD and the penultimate residue of NorpA is critical for proper phototransduction in Drosophila. By holding NorpA in a tight, membrane-bound complex, a covalent association between InaD and NorpA aids in the rapid regulation of G_q by NorpA (Kimple, 2001).

Interaction of InaD with Calmodulin

Ca2+ influxes regulate multiple events in photoreceptor cells, including phototransduction and synaptic transmission. An important Ca2+ sensor in Drosophila vision appears to be Calmodulin, since a reduction in levels of retinal Calmodulin causes defects in adaptation and termination of the photoresponse. These functions of Calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the TRP and TRPL ion channels, NinaC and INAD. To identify additional calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, a screen was conducted for retinal Calmodulin-binding proteins. Eight additional Calmodulin-binding proteins were found that are expressed in the Drosophila retina. These included six targets that are related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein (UNC13) and a protein (CRAG) related to Rab3 GTPase exchange proteins. Two other Calmodulin-binding proteins are Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that Calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission (Xu, 1998b).

Interaction of InaD with TRP

Drosophila vision involves a G protein-coupled phospholipase C-mediated signaling pathway that leads to membrane depolarization through activation of Na+ and Ca2+ channels. inaD mutant flies have a M442K point mutation and display a slow recovery of the Ca2+ dependent current. Anti-InaD antibodies coimmunoprecipitate Trp, which is identified by its electrophoretic mobility, cross reactivity with anti-Trp antibody, and absence in a null allele trp mutant. This interaction is abolished by the inaD point mutation, both in vitro and in vivo. Interaction is localized to the 19 amino acid C-terminus of Trp by overlay assays, and to the PDZ domain of InaD, encompassing the point mutation. Flies homozygous for the InaD and trp mutations were generated and their phenotype analyzed by electroretinogram (ERG). In this extracellular recording of the compound eye, light triggers a depolarizing receptor potential and the Inad and trp mutants display characteristic responses: trp shows a receptor potential that lacks the maintained component, and Inad lacks the prolonged depolarizing afterpotential. While the InaD and trp ERG phenotypes are similar when stimulated by a pulse of intense blue light, they are distinguished using low intensity stimulation. When stimulated with a 10 second pulse of orange light, InaD displays a sustained response, whereas trp flies show the transient receptor potential response. Under the same conditions, double mutants exhibit a phenotype similar to that of trp. The finding that the response of the double mutants is qualitatively similar to that of trp alone is consistent with the interpretation that Trp and InaD act in the same sequential pathway. In the absence of Trp, InaD is not able to effect its modulatory activity, and thus, the double mutant phenotype corresponds to that of trp. Given the impaired electrophysiology of the inaD mutant, this novel interaction suggests that InaD functions as a regulatory subunit of the Trp Ca2+ channel (Shieh, 1996).

INAD, a novel protein mutated in the inactivation no afterpotential D mutation in Drosophila, is a PDZ domain protein, sharing a protein interaction domain with Drosophila proteins Discs large, Dishevelled and Canoe. InaD photoreceptor cells show a slow deactivation of light-induced current and an increased sensitivity to dim light. The store-operated Ca2+ channel, TRP, is required in photoreceptor cells for a sustained response to light. TRP forms a complex with phospholipase C-ß (No receptor potential A), rhodopsin (RH1) Calmodulin, and INAD. The current model for Drosophila phototransduction is that IP3 generated through activation of NORPA binds to the IP3 receptor, resulting in release of Ca2+ from internal Ca2+ stores. The Calmodulin binding site of TRP bears no similarity to that of TRPL, the non specific cation channel that otherwise shares sequence homology with TRP. In InaD mutant flies, TRP is no longer spatially restricted to its normal subcellular compartment, the rhabdomere. In inaD^P215 mutant flies, TRP is no longer restricted to the rhabdomeres. Instead, some TRP is detected in the cell bodies, and a large proportion of TRP is found in the extracellular central matrix. The mislocalization of TRP in the central matrix might have occurred during the normal turnover of the photoreceptor cell membrane, which involves shedding of the microvillar rhabdomeral membrane into the central matrix. The alteration in localization of TRP in inaD^P215 is specific since all other rhabdomere-specific proteins examined displayed indistinguishable expression patterns in wild type and inaD^P215. Although a significant proportion of TRP is mislocalized in inaD^P215, some TRP remains in the rhabdomeres. Since the only existing inaD allele, inaD^P215, is caused by a point mutation in the second PDZ domain, it is possible that there remains some weak interaction between TRP and INAD^P215 protein that cannot be detected in vitro. If so, it is possible that no proportion of TRP would be detected in the rhabdomeres of null inaD flies. However, the dramatic change in localization of TRP detected in inaD^P215 provides evidence that a PDZ domain protein is required in vivo for targeting or anchoring an ion channel to its normal subcellular localization. These results provide evidence that a PDZ domain protein is required, in vivo, for the anchoring of an ion channel to a signaling complex. Furthermore, disruption of this interaction results in retinal degeneration. It is proposed that the TRP channel is linked to RORPA and RH1 to facilitate feedback regulation of these upstream signaling molecules. It is suggested that TRP may be inactivated through a Ca2+-dependent mechanism mediated by Calmodulin. (Chevesich, 1997).

Phototransduction in invertebrate microvillar photoreceptors is thought to be mediated by the activation of phospholipase C (PLC), but how this leads to gating of the light-sensitive channels is unknown. Most attention has focused on inositol-1,4,5-trisphosphate, a second messenger produced by PLC from phosphatidylinositol-4,5-bisphosphate; however, PLC also generates diacylglycerol, a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Both of these fatty acids reversibly activate native light-sensitive channels [transient receptor potential (TRP) and TRP-like (TRPL)] in Drosophila photoreceptors, as well as recombinant TRPL channels expressed in Drosophila S2 cells. Recombinant channels are activated rapidly in both whole-cell recordings and inside-out patches, with a half-maximal effector concentration for linolenic acid of approximately 10 microM. Four different lipoxygenase inhibitors, which might be expected to lead to build-up of endogenous fatty acids, also activate native TRP and TRPL channels in intact photoreceptors. As arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999).

The light response in Drosophila photoreceptor cells is mediated by a series of proteins that assemble into a macromolecular complex referred to as the signalplex. The central player in the signalplex is Inactivation no afterpotential D (InaD), a protein consisting of a tandem array of five PDZ domains. At least seven proteins bind InaD, including the Transient receptor potential (Trp) channel, which depends on InaD for localization to the phototransducing organelle, the rhabdomere. However, the determinants required for localization of InaD are not known. InaD is required for retention rather than targeting of Trp to the rhabdomeres. In addition, Trp binds to InaD through the COOH terminus, and this interaction is required for localization of InaD. Two other proteins that depend on InaD for localization, phospholipase C and protein kinase C, also mislocalize. However, elimination of any other member of the signalplex has no impact on the spatial distribution of InaD. A direct interaction between Trp and InaD does not appear to have a role in the photoresponse independent of localization of multiple signaling components. Rather, the primary function of the Trp/InaD complex is to form the core unit required for localization of the signalplex to the rhabdomeres (Li, 2000).

Trp is initially localized to the rhabdomeres in young InaD^P215 and trp^delta1272 flies, whereas in older flies, the spatial distribution of Trp is severely disrupted. These data suggest that InaD may be required for retention rather than targeting of Trp to the rhabdomeres. However, an alternative interpretation of these data is that those Trp molecules synthesized in young flies are targeted through an InaD-independent mechanism, whereas InaD is required for targeting of Trp synthesized in older flies. In support of the proposal that the InaD/Trp interaction is required for retention is the observation that Trp is long lived in vivo. Trp molecules synthesized before day 1.5 decline only ~25% in concentration during the next 8 d. Thus, it appears that Trp is initially targeted to the rhabdomeres, and is subsequently mislocalized in the absence of a direct link to InaD (Li, 2000).

An intriguing question concerns the identification of proteins required for localization of InaD. The NINAC myosin III would appear to be an excellent candidate, since it binds InaD and is a putative molecular motor expressed in the photoreceptor cells. Nevertheless, NINAC is not required for localization of InaD. Other InaD-interacting proteins that function in phototransduction, such as rhodopsin, PLC, PKC, and TrpL, are also dispensable for rhabdomeral distribution of InaD. In contrast to these proteins, Trp is specifically required for proper localization of InaD. Moreover, in trp mutant flies and in transgenic flies, trp^delta1272, in which the InaD binding site is deleted, the spatial distribution of InaD is disrupted in an age-dependent manner. These data, in combination with the findings that the half-life of InaD is ~5.5 d, suggest that the mislocalization of InaD in trp mutant flies is due to a defect in retention (Li, 2000).

In addition to a requirement for the Trp/InaD interaction for localization of Trp and InaD, elimination of trp or mutation of the InaD binding site in Trp leads to an alteration in the spatial distribution of other proteins that require InaD for rhabdomere localization. These include PLC and PKC. Moreover, the mislocalization of PLC and PKC appeared to be more pronounced than that of InaD in young trp flies. One possibility is that these signaling proteins may also interact with Trp and depend on both Trp and InaD for localization. PKC appears to interact at least transiently with Trp, since Trp is a substrate for PKC. Thus, Trp and InaD appear to form the core unit that is required for localization of many of the signalplex components in the rhabdomeres (Li, 2000).

The putative tetrameric structure of Trp may contribute to the stability of the Trp/InaD core unit, since each channel would have the potential to bind four InaD proteins. Although InaD is mislocalized in trp^delta1272, there is no major impact on the localization of InaD in InaD^P215, suggesting the existence of residual interaction between Trp and InaD^P215 in vivo. Consistent with this proposal, Trp is more unstable in trp^delta1272 than in InaD^P215. The presumed tetrameric structure of Trp could enhance a weak interaction between InaD^P215 and Trp in vivo, which is not observed in pull-down assays using a monomeric Trp tail, as noted above because each channel would have the potential to bind four InaD proteins. The data presented here raise the possibility that other PDZ-containing scaffold proteins form similar core complexes in vivo with tetrameric ion channels. In support of this proposal are recent in vitro experiments indicating that there is a reciprocal requirement for localization of PSD-95 and the K⁺ channel, Kv1.4 (Li, 2000 and references therein).

A separate question that awaits further investigation concerns the mechanism underlying targeting of the signalplex to the rhabdomeres. Evidence has been presented that another PDZ-containing scaffold protein, PSD-95, is trafficked to the postsynaptic compartment after assembling on vesicles. Thus, it is plausible that the components of the signalplex may get trafficked to the rhabdomeres via vesicular transport and require the Trp/InaD core unit for retention (Li, 2000 and references therein).

The finding that Trp and InaD are long lived is surprising considering that there is very active turnover of the rhabdomeric membrane. Such turnover results in shedding of rhabdomeral membrane into the central matrix and blebbing of membrane from the base of the microvilli into the cell bodies. The association between Trp and InaD may serve to prevent removal of these proteins into the central matrix and cell bodies during membrane turnover. Interestingly, the mutual requirement for the Trp/InaD interaction for retention in the rhabdomeres is less critical in trp^delta1272 flies maintained in the dark. It is suggested that a greater proportion of Trp and InaD is retained in the dark because of less turnover of the rhabdomeral membrane in the absence of light (Li, 2000).

In contrast to InaD^P215, the electroretinogram (ERG) response in young trp^delta1272 is similar to wild-type. The only significant ERG phenotype in trp^delta1272 flies was an age-dependent decrease in the amplitude of the maintained component. This defect was presumably due to mislocalization of Trp and InaD, since the amplitude of the maintained component gradually decreases in parallel with the mislocalization of Trp and InaD in older flies. Moreover, the termination of the photoresponse appears normal even in old trp^delta1272 flies. This latter result is surprising, because PKC is mislocalized in old trp^delta1272 photoreceptor cells, and PKC is required for termination of the photoresponse. However, the rhabdomeric concentration of two substrates for PKC (Trp and InaD) is also reduced in trp^delta1272. Given that PKC, Trp, and InaD have been reported to be present in about equimolar concentrations, it is proposed that the relative stoichiometry of PKC and its substrates is important for normal termination of the photoresponse. Thus, the delay in termination resulting from a reduction in PKC concentration may be suppressed by a concomitant decrease in the levels of InaD and Trp (Li, 2000).

The defect in termination associated with InaD^P215 may not be due to perturbation of the Trp/InaD interaction, because the mutation in PDZ3 may also affect binding to other target proteins. The observation that the termination defect does not became more severe in old InaD^P215 flies suggests that the phenotype is not due to the disruption of the spatial distribution of Trp, since the mislocalization of Trp is more severe in older InaD^P215 flies (Li, 2000).

To address the specific role of the Trp/InaD interaction, the InaD binding site was mapped and transgenic flies were generated expressing a Trp derivative that does not associate with InaD. PDZ domains typically recognize COOH-terminal sequences consisting of an S/T-X-V/I motif or hydrophobic or aromatic residues. As is the case with most PDZ target proteins, it was found that the critical binding motif is at the COOH terminus. Specifically, it was found that deletion of the last four amino acids (SGWL) completely disrupts Trp/InaD binding. Furthermore, Trp^delta1272 obtained from transgenic fly head extracts fails to associate with InaD in pull-down assays. Mutation of an internal S-X-V motif (V1266D), which abolishes interaction between Trp and InaD in an overlay assay, has only minor effects. An additional deletion (amino acids 1257-1264), which eliminates the first residue of the S-X-V motif within the context of the short Trp tail (1252-1275), also retains binding with InaD in vitro (Li, 2000).

It is concluded that the primary role of the direct interaction between Trp and InaD is not to facilitate rapid signaling. The apparently normal ERG in young trp^delta1272 suggests that there is no defect in any aspect of the photoresponse. Rather, binding of Trp to InaD is critical for forming the core unit of the signalplex, which is necessary for retention of multiple signaling proteins in the rhabdomeres. This conclusion contrasts with previous reports, which have concluded that InaD functions as a regulatory subunit of the Trp channel. These analyses of trp^delta1272 indicate that the delayed termination associated with InaD^P215 is not due to disruption of the interaction with Trp. Instead, it appears that the phenotype is due to disruption of the interaction of InaD with another signaling protein that is required for proper response termination. Thus, contrary to expectations, a direct association between Trp and InaD appears to be dispensable for rapid termination (Li, 2000).

It appears that there are at least three classes of InaD binding proteins. The first class consists exclusively of Trp, because it is the only known InaD binding partner that is required for retention of InaD as well as of those InaD targets that depend on the signalplex for localization. However, there may be additional proteins that along with Trp and InaD comprise the core unit. The second group includes two proteins, PLC and PKC, which rely on InaD for localization and stability. However, there is no reciprocal requirement for these proteins for retention of any other protein in the rhabdomeres. Mutation of the InaD binding sites in PLC have been reported to cause defects in the photoresponse. However, these effects may reflect mislocalization or instability of these InaD targets rather than a direct requirement for coupling to InaD (Li, 2000).

The third class of InaD target proteins includes proteins such as rhodopsin, NINAC, and TrpL that are not dependent on InaD for localization in the rhabdomeres. It is proposed that the class I and II proteins, which depend on interaction with InaD for retention in the rhabdomeres, are constitutively bound to InaD, whereas the class III proteins may interact dynamically with InaD. As a consequence, only a subset of the class III proteins may bind to InaD at any given time. The observation that class III proteins do not depend on InaD for localization suggests that these InaD/target protein interactions have an alternative function, such as a direct role in the photoresponse. In support of this proposal, it was found that mutation of the InaD binding site in NINAC results in a pronounced delay in termination of the photoresponse. Thus, proteins that do not depend on InaD for localization may participate in the rapid activation and/or termination of the photoresponse (Li, 2000).

In Drosophila photoreceptors the multivalent PDZ protein InaD organizes the phototransduction cascade into a macromolecular signaling complex containing the effector PLC, the light-activated TRP channels, and a regulatory PKC. The subcellular localization of InaD signaling complexes is critical for signaling. How InaD complexes are anchored and assembled in photoreceptor cells has been examined. trp mutants, or transgenic flies expressing inaD alleles that disrupt the interaction between InaD and TRP, cause the mislocalization of the entire transduction complex. The InaD-TRP interaction is not required for targeting but rather for anchoring of complexes, because InaD and TRP can be targeted independently of each other. In addition to its scaffold role, InaD functions to preassemble transduction complexes. Thus the interaction of InaD with the TRP channel is required for anchoring signaling complexes in the rhabdomeres. TRP then may interact with the cytoskeleton, securing InaD and the whole complex to the membrane; ankyrin repeats on the N terminus of TRP could play a role in linking TRP to the cytoskeleton. Another possibility is that the InaD-TRP interaction reveals, or unmasks, sites on TRP or InaD that are important for membrane anchoring. Preassembly of signaling complexes helps to ensure that transduction complexes with the appropriate composition end up in the proper location. This may be a general mechanism used by cells to target different signaling machinery to the pertinent subcellular location (Tsunoda, 2001).

Common mechanisms regulating dark noise and quantum bump amplification in Drosophila photoreceptors

Absolute visual thresholds are limited by 'dark noise,' which in Drosophila photoreceptors is dominated by brief (~10 ms), small (~2 pA) inward current events, occurring at ~2/s, believed to reflect spontaneous G protein activations. These dark events were increased in rate and amplitude by a point mutation in myosin III (NINAC), which disrupts its interaction with the scaffolding protein, INAD. This phenotype mimics that previously described in null mutants of ninaC (no inactivation no afterpotential; encoding myosin III) and an associated protein, retinophilin (rtp). Dark noise was similarly increased in heterozygote mutants of diacylglycerol kinase (rdgA/+). Dark noise in ninaC, rtp, and rdgA/+ mutants was greatly suppressed by mutations of the G_q α-subunit (Gαq) and the major light-sensitive channel (trp) but not rhodopsin. ninaC, rtp, and rdgA/+ mutations also all facilitated residual light responses in Gαq and PLC hypomorphs. Raising cytosolic Ca²⁺ in the submicromolar range increased dark noise, facilitated activation of transient receptor potential (TRP) channels by exogenous agonist, and again facilitated light responses in Gαq hypomorphs. These results indicate that RTP, NINAC, INAD, and diacylglycerol kinase, together with a Ca²⁺-dependent threshold, share common roles in suppressing dark noise and regulating quantum bump generation; consequently, most spontaneous G protein activations fail to generate dark events under normal conditions. By contrast, quantum bump generation is reliable but delayed until sufficient G proteins and PLC are activated to overcome threshold, thereby ensuring generation of full-size bumps with high quantum efficiency (Chu, 2013).

Scaffolding protein INAD regulates deactivation of vision by promoting phosphorylation of transient receptor potential by eye protein kinase C in Drosophila

Drosophila visual signaling is one of the fastest G-protein-coupled transduction cascades, because effector and modulatory proteins are organized into a macromolecular complex ('transducisome'). Assembly of the complex is orchestrated by inactivation no afterpotential D (INAD), which colocalizes the transient receptor potential (TRP) Ca²⁺ channel, phospholipase Cβ, and eye protein kinase C (eye-PKC), for more efficient signal transduction. Eye-PKC is critical for deactivation of vision. Moreover, deactivation is regulated by the interaction between INAD and TRP, because abrogation of this interaction in InaD^p215 results in slow deactivation similar to that of inaC^p209 lacking eye-PKC. To elucidate the mechanisms whereby eye-PKC modulates deactivation, this study demonstrates that eye-PKC, via tethering to INAD, phosphorylates TRP in vitro. Ser⁹⁸² of TRP is phosphorylated by eye-PKC in vitro and, importantly, in the fly eye, as shown by mass spectrometry. Furthermore, transgenic expression of modified TRP bearing an Ala substitution leads to slow deactivation of the visual response similar to that of InaD^p215. These results suggest that the INAD macromolecular complex plays an essential role in termination of the light response by promoting efficient phosphorylation at Ser⁹⁸² of TRP for fast deactivation of the visual signaling (Popescu, 2006; full text of paper).

Drosophila visual transduction is a G-protein-coupled signaling pathway that provides a model system for understanding the molecular basis of signal transduction in the vertebrate nervous systems. Drosophila visual signaling is initiated with the activation of rhodopsin by light. Activated rhodopsin, via a Gq heterotrimeric protein, stimulates phospholipase Cβ (PLCβ) named no-receptor potential A (NORPA). NORPA hydrolyzes PIP₂ (phosphatidylinositol 4,5-bisphosphate) to inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG), which leads to opening of the transient receptor potential (TRP) Ca²⁺ and TRP-like channels, and depolarization of photoreceptors. The key second messenger that activates the TRP Ca²⁺ channel is thought to be either DAG or its lipid metabolites, whereas IP₃ does not appear to play a role. DAG may have a dual function, because it also activates the eye-specific protein kinase C (eye-PKC; InaC or Inactivation no afterpotential C) essential for deactivation of the light response (Popescu, 2006 and references therein).

Reversible phosphorylation modulates the dynamics of signal transduction by transiently altering activities of signaling proteins. Members of the conventional PKC family, which are activated by Ca²⁺ and DAG, are capable of phosphorylating a wide variety of protein substrates for temporal and spatial regulation of signaling processes. In Drosophila, eye-PKC is involved in the negative regulation of visual signaling, because inaC^p209 flies lacking eye-PKC display abnormal desensitization, slow deactivation, and defects in light adaptation. Eye-PKC is anchored to a macromolecular complex by tethering to INAD. Interaction with INAD enhances the stability of eye-PKC as well as targets eye-PKC to the rhabdomeres of photoreceptors, in which visual signaling occurs. Importantly, the in vivo function of eye-PKC is regulated by interaction with INAD. Previously, it was shown that eye-PKC phosphorylates TRP in vitro. The present study investigated the molecular basis of TRP phosphorylation by eye-PKC (Popescu, 2006).

To mimic eye-PKC phosphorylation of TRP in vitro, a complex-dependent kinase assay was designed. The in vitro complex-specific phosphorylation of TRP is regulated by the presence of the INAD-interacting domain in TRP, as well as the existence of INAD in the fly extracts. Extracts lacking either eye-PKC or INAD fail to support TRP phosphorylation. Similarly, extracts prepared from InaD^p215 (a modified INAD devoid of the TRP binding) are not able to promote TRP phosphorylation. Together, these findings indicate that INAD targets eye-PKC to its substrates, similar to RACK (receptor for activated C kinase). By the complex-dependent kinase assay, Ser⁹⁸² of TRP was identified as an eye-PKC phosphorylation site. Moreover, TRP isolated from flies by LC-MS was analyzed and it was found that Ser⁹⁸² of TRP is indeed phosphorylated in vivo by eye-PKC, because phosphorylated peptides encompassing Ser⁹⁸² of TRP are present in wild-type, but absent in inaC^p209 flies (Popescu, 2006).

Next, the in vivo functional contribution of phosphorylation was investigated by characterizing transgenic flies expressing a modified TRP bearing an Ala substitution at Ser⁹⁸² (trp^S982A). Remarkably, these transgenic flies displayed prolonged deactivation kinetics in response to bright light stimuli, indicating that phosphorylation of TRP at Ser⁹⁸² by eye-PKC is involved in inactivation of TRP, leading to fast deactivation. A model of the TRP regulation by eye-PKC is proposed. TRP is an integral part of the INAD complex and is opened by light. After light termination, the visual response is rapidly deactivated. Although molecular mechanisms underlying deactivation remain elusive, Ca²⁺ is known to play a vital role in response termination. The increased intracellular Ca²⁺ (primarily mediated by TRP) and DAG activate eye-PKC, which, in turn, phosphorylates TRP at Ser⁹⁸². Phosphorylation of TRP leads to a rapid inactivation of the channel on cessation of the light stimulation, without affecting the interaction between TRP and INAD. How does phosphorylation influence the TRP channel activity? Ser⁹⁸² is located within the Lys-Pro-rich region of TRP, which may function in TRP gating. It is speculated that phosphorylation at Ser⁹⁸² may induce a conformational change in the pore domain, which in turn leads to a rapid closure and inactivation of TRP. Phosphorylation has been linked directly to conformational changes that play key roles in the regulation of ion channels. It is also possible that phosphorylation of TRP at Ser⁹⁸² affects the interaction with some yet-unidentified proteins that may be important for the modulation of the TRP channel activity (Popescu, 2006).

In the absence of eye-PKC-mediated phosphorylation of TRP, deactivation of visual signaling is slower as observed in inaC^p209 or trp^S982A. It was found that inaC^p209 displays a more complex deactivation defect, whereas trp^S982A exhibits prolonged deactivation only in response to bright light. These findings suggest that, in addition to TRP, eye-PKC phosphorylates other substrates for efficient termination of the light response. Indeed, eye-PKC has been shown to phosphorylate INAD, but the functional relevance of this phosphorylation is not known. Furthermore, eye-PKC is required for the Ca²⁺-dependent inhibition of NORPA. NORPA is part of the INAD complex; however, it is not known to be phosphorylated by eye-PKC. The Ca²⁺-dependent inactivation of the light-induced current is unaltered in inaC^p209. This finding suggests the existence of a parallel Ca²⁺-dependent mechanism in inaC^p209 by which TRP is inactivated or of an upregulation of a Ca²⁺-dependent mechanism that activates other kinases to compensate for the loss of eye-PKC in inaC^p209 (Popescu, 2006).

Importantly, trp^S982A displays slow deactivation kinetics similar to that of InaD^p215. InaD^p215 was isolated based on the ina (inactivation no afterpotential) phenotype elicited by ERG. By whole-cell recordings, it was shown that InaD^p215 exhibits slow deactivation kinetics. However, a delay in latency of the quantum bump has been proposed and that activation is affected in the InaD^p215 mutant. To resolve this discrepancy, the mutant was reexamined and it was concluded that the primary defect in InaD^p215 is prolonged deactivation and not slow activation. InaD^p215 expresses INAD^M442K, which fails to associate with TRP. How does a loss of INAD–TRP interaction lead to abnormal deactivation of visual signaling? It is likely that the lack of the INAD–TRP interaction prevents the recruitment of TRP to the INAD complex and, consequently, eye-PKC-mediated regulation. Indeed, both trp^S982A and InaD^p215 exhibit similar deactivation defects, indicating that the molecular basis underlying the slow deactivation defect in InaD^p215 is attributable to a lack of negative regulation of the TRP channel by eye-PKC. Together, these findings suggest that formation of the INAD complex is essential for fast deactivation of the visual response by promoting phosphorylation of TRP by eye-PKC. Moreover, Ser⁹⁸² may be the sole eye-PKC phosphorylation site in TRP, because trp^S982A and InaD^p215 display similar deactivation defects. A loss of INAD–TRP interaction has been investigated in transgenic flies expressing modified TRP in which the INAD-interacting domain was deleted (trp^Δ1272). A reduced light response with normal deactivation kinetics in trp^Δ1272 has been reported. It has been proposed that the suppression of the delayed termination, which is attributable to a reduced eye-PKC level in trp^Δ1272 is probably masked by a concomitant decrease in TRP and INAD levels (Popescu, 2006).

To date, many proteins related to Drosophila TRP have been discovered in both invertebrates and vertebrates. These TRP ion channels are subdivided into seven subfamilies (TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, and TRPML). Drosophila TRP belongs to the TRPC subfamily. Members of the TRPC subfamily are also activated by receptor-induced activation of phospholipase C and therefore may be regulated by PKC. Indeed, phosphorylation of the TRPC channels by PKC appears important for modulating the channel activity. For example, the PKC-mediated phosphorylation of TRPC1 was shown to contribute to its SOC (store operated channel) activation, triggering Ca²⁺ entry into endothelial cells. In contrast, PKC-mediated phosphorylation was demonstrated to inhibit the activity of TRPC3 in HEK 293 cells and of TRPC6 in PC12D neuronal cells. In both cases, TRPC3 and TRPC6 are activated by DAG, whereas DAG also turns on PKC. It has been proposed that timing is important because the channels are activated by DAG more rapidly than they are inhibited by DAG-activated PKC. Heterologously expressed TRPC7 was also shown to be regulated by PKC: inhibition of PKC prolonged inactivation of the channel. Moreover, PKC phosphorylation of heterologously expressed TRPC5 resulted in desensitization of this channel, a process that was dependent on both extracellular and intracellular Ca²⁺ concentrations (Popescu, 2006).

In conclusion, this study has uncovered the molecular mechanism underlying the complex-dependent phosphorylation of TRP by eye-PKC and its role in fast deactivation of vision. Specifically, it was shown that eye-PKC phosphorylates TRP at Ser⁹⁸² in vitro and in vivo. Importantly, phosphorylation of TRP facilitates rapid inactivation of the channel because transgenic flies bearing an Ala substitution at Ser⁹⁸² display prolonged deactivation kinetics of the light response. Significantly, this slow deactivation defect is similar to that observed in InaD^p215 in which TRP fails to associate with INAD. These findings provide insights into the mechanistic basis of slow deactivation in InaD^p215, suggesting that INAD plays a critical role in targeting eye-PKC to TRP for rapid deactivation of the visual signaling. Together, these data indicate that the INAD macromolecular complex is important for deactivation of the visual response by directing eye-PKC to TRP. Furthermore, PKC-mediated phosphorylation of TRP at Ser⁹⁸² leads to fast deactivation of vision by promoting inactivation of the TRP channel (Popescu, 2006).

Light-dependent phosphorylation of the Drosophila Inactivation No Afterpotential D (INAD) scaffolding protein at Thr170 and Ser174 by eye-specific Protein kinase C

Drosophila Inactivation No Afterpotential D (INAD) is a PDZ domain-containing scaffolding protein that tethers components of the phototransduction cascade to form a supramolecular signaling complex. This study reports the identification of eight INAD phosphorylation sites using a mass spectrometry approach. PDZ1, PDZ2, and PDZ4 each harbor one phosphorylation site, three phosphorylation sites are located in the linker region between PDZ1 and 2, one site is located between PDZ2 and PDZ3, and one site is located in the N-terminal region. Using a phosphospecific antibody, it was found that INAD phosphorylated at Thr170/Ser174 is located within the rhabdomeres of the photoreceptor cells, suggesting that INAD becomes phosphorylated in this cellular compartment. INAD phosphorylation at Thr170/Ser174 depends on light, the phototransduction cascade, and on eye-Protein kinase C that is attached to INAD via one of its PDZ domains (Voolstra, 2015).

Interaction of InaD with TRPL and FKBP52

Transient receptor potential and transient receptor potential-like (TRPL) are Ca(2+)-permeable cation channels found in Drosophila photoreceptor cells associated with large multimeric signaling complexes held together by the scaffolding protein, INAD. To identify novel proteins involved in channel regulation, Drosophila INAD was used as bait in a yeast two-hybrid screen of a Drosophila head cDNA library. Sequence analysis of one identified clone showed it to be identical to the Drosophila homolog of human FK506-binding protein, FKBP52 (previously known as FKBP59). To determine the function of dFKBP59, TRPL channels and dFKBP59 were co-expressed in Sf9 cells. Expression of dFKBP59 produced an inhibition of Ca(2+) influx via TRPL in fura-2 assays. Likewise, purified recombinant dFKBP59 produced a graded inhibition of TRPL single channel activity in excised inside-out patches when added to the cytoplasmic membrane surface. Immunoprecipitations from Sf9 cell lysates using recombinant tagged dFKBP59 and TRPL showed that these proteins directly interact with each other and with INAD. Addition of FK506 prior to immunoprecipitation resulted in a temperature-dependent dissociation of dFKBP59 and TRPL. Immunoprecipitations from Drosophila S2 cells and from fly head lysates demonstrated that dFKBP59, but not dFKBP12, interacts with TRPL in vivo. Likewise, INAD immunoprecipitates with dFKBP59 from S2 cell and head lysates. Immunocytochemical evaluation of thin sections of fly heads revealed specific FKBP immunoreactivity associated with the eye. Site-directed mutagenesis showed that mutations of P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709) eliminated interaction of the TRPL with dFKBP59. These results provide strong support for the hypothesis that immunophilin dFKBP59 is part of the TRPL-INAD signaling complex and plays an important role in modulation of channel activity via interaction with conserved leucyl-prolyl dipeptides located near the cytoplasmic mouth of the channel (Goel, 2001).

Anchoring TRP to the INAD macromolecular complex requires the last 14 residues in its carboxyl terminus

Drosophila transient-receptor-potential (TRP) is a Ca2+ channel responsible for the light-dependent depolarization of photoreceptors. TRP is anchored to a macromolecular complex by tethering to inactivation-no-afterpotential D (INAD). INAD associates with the carboxyl tail of TRP via its third post-synaptic density protein 95, discs-large, zonular occludens-1 domain. This paper further explored the molecular basis of the INAD interaction and demonstrated the requirement of the last 14 residues of TRP, with the critical contribution of Gly1262, Val1266, Trp1274, and Leu1275. Oull-down assays show that the last 14 residues of TRP comprises the minimal sequence that competes with the endogenous TRP from fly extracts, leading to the co-purification of a partial INAD complex containing INAD, no-receptor-potential A, and eye-protein kinase C (PKC). Eye-PKC is critical for the negative regulation of the visual signaling and is phosphorylated TRP in vivo. To uncover the substrates of eye-PKC in the INAD complex, a complex-dependent eye-PKC assay was designed, that utilized endogenous INAD complexes isolated from flies. Activated eye-PKC was shown to phosphorylate INAD, TRP but not no-receptor-potential A. Moreover, phosphorylation of TRP is dependent on the presence of both eye-PKC and INAD. Together, these findings indicate that stable kinase-containing protein complexes may be isolated by pull-down assays, and used in this modified kinase assay to investigate phosphorylation of the proteins in the complex. It is concluded that TRP associates with INAD via its last 14 residues to facilitate its regulation by eye-PKC that fine-tunes the visual signaling (Peng, 2008).

Interaction of InaD with NINAC

Many of the proteins that are critical for Drosophila phototransduction assemble into a signaling complex, signalplex, through association with the PDZ-domain protein InaD. Some of these proteins depend on InaD for proper subcellular localization to the phototransducing organelle, the rhabdomere, making it difficult to assess any physiological function of this signaling complex independent of localization. InaD binds directly to the NINAC myosin III, yet the subcellular localization of NINAC is normal in inaD mutants. Nevertheless, the InaD binding site is sufficient to target a heterologous protein to the rhabdomeres. Disruption of the NINAC/InaD interaction delays termination of the photoreceptor response. Thus one role of this signaling complex is in rapid deactivation of the photoresponse (Wes, 1999).

Interaction of InaD with TRPL

In addition to TRP, another cation influx channel subunit, TRPL, functions in phototransduction by forming a heteromultimeric channel with TRP. To investigate whether TRPL is an INAD-interacting protein, an in vivo coimmunoprecipitation experiment was carried out: INAD was found to associate with TRPL in fly photoreceptor cells. Since TRPL heteromultimerizes with TRP, it is possible that TRPL associates with INAD through TRP. Therefore, whether TRPL and INAD coimmunoprecipitate was tested after coexpressing the two proteins in 293T cells. INAD is detected after immunoprecipitating cell extracts with TRPL antibodies but not with nonimmune serum. Furthermore, INAD is not detected after immunoprecipitating with TRPL antibodies using extracts expressing only INAD. The interactions of TRP (Shieh, 1996) and TRPL with INAD appear to be specific since a highly related member of the TRP family, human TRPC3, does not coimmunoprecipitate with INAD. Evidence that TRPL and INAD directly interactes is that 35S-labeled TRPL binds to INAD-GST fusion proteins immobilized on a column (Xu, 1998a).

Interaction of InaD with InaC (the eye specific protein kinase C)

The Calliphora homolog of the Drosophila inaD gene product was initially isolated in order to isolate and characterize key proteins of the transduction cascade in photoreceptors using the phosphoinositide signaling pathway. Drosophila inaD mutants manifest a slow deactivation of the light response. By screening a retinal cDNA library with antibodies directed against photoreceptor membrane proteins, a cDNA coding for an amino acid sequence of 665 residues has been isolated. The sequence displays 65.3% identity (77.3% similarity) with the Drosophila InaD gene product. Probing Western blots with monospecific antibodies directed against peptides comprising amino acids 272-542 [(anti-InaD-(272-542)] or amino acids 643-655 [(anti-InaD-(643-655)] of the InaD gene product reveals that the Calliphora InaD protein is specifically associated with the signal-transducing rhabdomeral photoreceptor membrane from which it can be extracted by high salt buffer containing 1.5 M NaCl. Since five out of eight consensus sequences for protein kinase C phosphorylation reside within stretches of 10-16 amino acids that are identical in the Drosophila and Calliphora InaD protein, the InaD gene product is likely to be a target of protein kinase C. Phosphorylation studies with isolated rhabdomeral photoreceptor membranes followed by InaD immunoprecipitation reveal that the InaD protein is a phosphoprotein. In vitro phosphorylation is, at least to some extent, Ca 2+ dependent and activated by phorbol 12-myristate 13-acetate. The inaC-encoded eye-specific form of a protein kinase C (eye-PKC) is co-precipitated by antibodies specific for the InaD protein from detergent extracts of rhabdomeral photoreceptor membranes, suggesting that the InaD protein and eye-PKC are interacting in these membranes. Co-precipitating with the InaD protein and eye-PKC are two other key components of the transduction pathway, namely the Trp protein, which is proposed to form a Ca2+ channel, and the norpA-encoded phospholipase C, the primary target enzyme of the transduction pathway. It is proposed that the rise of the intracellular Ca2+ concentration upon visual excitation initiates the phosphorylation of the InaD protein by eye-PKC and thereby modulates its function in the control of the light response (Huber, 1996a).

Drosophila eye-specific protein kinase C (eye-PKC) is involved in light adaptation and deactivation. eye-PKC, NORPA (phospholipase Cbeta), and transient-receptor-potential (TRP) (calcium channel) are integral components of a signal transduction complex organized by INAD, a protein containing five PDZ domains. There is a direct association between the third PDZ domain of INAD with TRP, and the carboxyl-terminal half of INAD with the last three residues of NORPA. The molecular interaction between eye-PKC and INAD is defined via the yeast two-hybrid and ligand overlay assays. The second PDZ domain of INAD interacts with the last three residues in the carboxyl-terminal tail of eye-PKC, Thr-Ile-Ile. The association between eye-PKC and INAD is disrupted by an amino acid substitution (Ile-700 to Asp) at the final residue of eye-PKC. In flies lacking endogenous eye-PKC (inaCp215), normal visual physiology is restored upon expression of wild-type eye-PKC, whereas the eye-PKCI700D mutant is completely inactive. Flies homozygous for inaCp209 and InaDp215, a mutation that causes a loss of the INAD-TRP association, were generated. These double mutants display a more severe response inactivation than either of the single mutants. Based on these findings, it is concluded that the in vivo activity of eye-PKC depends on its association with INAD and that the sensitivity of photoreceptors is cooperatively regulated by the presence of both eye-PKC and TRP in the signaling complex (Adamski, 1998).

Yeast two-hybrid and ligand overlay results both indicate that the second PDZ domain of INAD associates predominantly with eye-PKC, whereas no interaction was detected with PDZ4. This result is different from a previous report in which interaction of eye-PKC with the fourth PDZ domain of INAD was detected by affinity chromatography (Tsunoda, 1997). The current study tested a total of five constructs that contained the fourth PDZ domain: no indication of this interaction was found. These included a fusion protein that contained exactly the same region as previously tested. One possible explanation for these conflicting results is that the different assay systems are measuring different types of association between eye-PKC and INAD. INAD may bind and cluster eye-PKC to the signaling complex, and it can also act as a substrate for the kinase activity (Huber, 1996a). Amino acid substitutions made in the second PDZ domain of INAD are shown to disrupt the eye-PKC binding. None of these amino acid changes were near the serine or threonine residues that are putative PKC phosphorylation sites. Furthermore, mutations in the carboxyl-terminal tail of PKC abolish PKC binding to the second PDZ domain. Thus the interaction described for eye-PKC/INAD is a typical carboxyl-terminal tail/PDZ domain association. The basis of the reported interaction with the fourth PDZ domain remains to be determined. Another provocative explanation could be that eye-PKC may bind different PDZ domains of INAD during different physiological conditions. For example, phosphorylation of INAD may change the relative affinity of the interaction in PDZ2 and PDZ4. Clarification of the role of these two eye-PKC/INAD interactions will require analysis of transgenic flies expressing a modified InaD in which these PDZ domain are mutated (Adamski, 1998).

The COOH-terminal three residues of target proteins (often S/TXV) are essential for binding to PDZ domains. To test whether INAD interacts with its targets in a similar way, each of the last three residues in PKC (T-I-I) were changed to aspartic acid (PKCD) and the derivative was coexpressed with full-length INAD in 293T cells. Binding to INAD is not abolished as a consequence of the mutation. To directly compare whether the binding of PKCD to INAD is reduced relative to wild-type PKC, column-binding assays were performed. Although PKCD still binds to INAD, the interaction is significantly reduced (approximately eightfold). It is possible that the residual binding is due to the presence of a second INAD binding site in PKC, since another INAD-binding protein, PLC (Chevesich, 1997), contains two sites (van Huizen, 1998). Alternatively, there may be a single binding site in PKC that is close to but not at the extreme COOH terminus. If so, then mutation of the flanking COOH-terminal residues may disrupt but not obliterate binding. To differentiate between these possibilities, an attempt was made to further map the binding site(s). All of the INAD-binding capacity is contained in the COOH-terminal third of PKC, which includes most of the catalytic domain (residues 472-700). Smaller derivatives of the catalytic domain are all unstable in 293T cells, suggesting that they might have been misfolded. Thus, it was not feasible to further map the INAD binding site(s) in 293T cells or using the column-binding assay (Xu, 1998a).

Functional INAD complexes are required to mediate degeneration in photoreceptors of the Drosophila rdgA mutant

The TRP family of ion channels mediates a wide range of calcium-influx phenomena in eukaryotic cells. Many members of this family are activated downstream of phosphoinositide hydrolysis but the subsequent steps that lead to TRP channel activation in vivo remain unclear. Recently, the lipid products of phosphoinositide hydrolysis (such as diacylglycerol and its metabolites) have been implicated in activating TRP channels in both Drosophila and mammals. In Drosophila photoreceptors, lack of diacylglycerol kinase (DGK) activity (encoded by rdgA) leads to both constitutive TRP-channel activity and retinal degeneration. In this study, using a novel forward-genetic screen, InaD, a multivalent PDZ domain protein, was identified as a suppresser of retinal degeneration in rdgA mutants. InaD suppresses rdgA, and the rescue is correlated with reduced levels of phospholipase Cß (PLCß), a key enzyme for TRP channel activation. Furthermore, it was shown that light, Gq and PLCß all modulate retinal degeneration in rdgA. The results demonstrate a previously unknown requirement for a balance of PLCß and DGK activity for retinal degeneration in rdgA. They also suggest a key role for the lipid products of phosphoinositide hydrolysis in the activation of TRP channels in vivo (Georgiev, 2005).

Transmembrane signalling cascades initiated by G-protein-coupled receptors are a widely used mechanism for signalling the detection of many sensory modalities. These cascades end with the activation of plasma-membrane ion channels whose activity alters membrane potential and initiates synaptic transmission of a signal to the central nervous system. Several different families of ion channels have been implicated in this process. Historically, the oldest and best characterized are cyclic-nucleotide-gated channels, whose role in vertebrate visual and olfactory transduction is well established. More recently, members of the TRP family of ion channels have been implicated in the transduction of several sensory modalities in both vertebrate and invertebrate systems. These include light (Drosophila TRPC), pheromones (rodent TRPC2), taste (rodent TRPM), physical stimuli and temperature (Drosophila and mammal TRPV, TRPA and TRPN). Currently, a crucial factor limiting the understanding of how TRP channels encode sensory modalities is the lack of information about how these channels are activated. In several cases, only a few transduction components have been identified and the inability to perform in vivo analysis of channel activation has been a major obstacle in revealing how TRP channels are activated (Georgiev, 2005).

The Drosophila phototransduction cascade is historically the oldest and to date the best understood model for the analysis of a TRP channel involved in sensory transduction. In the fly eye, rhodopsin, a seven-transmembrane-span G-protein-coupled receptor, activates phospholipase Cß (PLCß) via Gq. This initiates a biochemical cascade that ends with the opening of two classes of calcium- and cation-selective TRPC channels, TRP and TRPL. Several key elements of the transduction cascade have been identified including Gq, PLCß and protein-kinase C. Several of these components, along with the TRP channel, are clustered into a macromolecular signalling complex by the multivalent PDZ-domain protein INAD. The INAD complex is thought to increase the speed and specificity of the light response. However, despite this wealth of detail about the components of the transduction cascade, the mechanism of activation of TRP and TRPL remains poorly understood, and is one of the outstanding problems in both sensory neurobiology and intracellular calcium signalling (Georgiev, 2005),

Although the essential role of PLCß in the activation of TRP and TRPL is well established, the biochemical events initiated by this enzyme that lead to channel activation remain unclear. Inositol-1,4,5-trisphosphate (IP₃), the best-understood second messenger generated from phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] hydrolysis by PLCß was originally postulated to be the second messenger that leads to TRP and TRPL activation. However, several recent lines of evidence strongly indicate that IP₃-induced calcium (Ca²⁺) release, or indeed a physical interaction between the IP₃ receptor (IP₃R) and the light-activated channels, is unlikely to underlie the mechanism of TRP and TRPL activation. More recently, lipid second messengers derived from PI(4,5)P₂ have been implicated in the activation of TRP and TRPL as well as their vertebrate homologues. Polyunsaturated fatty acids, potential metabolites of diacylglycerol (DAG), the primary lipid generated by PI(4,5)P₂ hydrolysis, have been shown to activate TRP and TRPL in situ, as well as in inside-out patches of TRPL channels expressed in S2 cells. In addition, both DAG and PI(4,5)P₂ have been shown to modulate TRPL channel activity in cell culture models. Analysis of TRPC2 activation in the rodent vomeronasal organs shows considerable parallels to the current understanding of the mechanism of Drosophila TRP and TRPL activation. However, despite these findings, the physiological relevance of PI(4,5)P₂-derived lipids as activators of Drosophila TRP channels in vivo remains to be established and the precise identity of the phospholipid species that is involved is unknown (Georgiev, 2005),

Recently, genetic evidence of a role for lipid messengers in the activation of TRPC channels in vivo has been obtained in Drosophila photoreceptors from an analysis of the retinal degeneration A (rdgA) mutant. The rdgA mutant was first isolated because it failed to respond to light in a behavioural assay. Analysis of retinal ultrastructure revealed that all alleles show varying degrees of photoreceptor degeneration. Biochemical analysis showed impaired diacylglycerol kinase (DGK) activity and reduced levels of light induced phosphatidic acid (PA) formation in head extracts of rdgA mutants. The gene that is defective in rdgA mutants has been cloned and found to encode an eye-enriched isoform of DGK, the principal enzyme that inactivates DAG by phosphorylation to PA. However, and most significantly, under voltage-clamp conditions, several alleles including rdgA¹, rdgA³, rdgA⁶ and rdgA^KS60 all show a small constitutively active inward current, which, on the basis of its biophysical characteristics, genetics and pharmacology, has been shown to be composed largely of TRP channels. The retinal degeneration phenotype of rdgA can be rescued by genetically removing TRP channels (i.e., the double mutant rdgA;trp), whose photoreceptors now lack their principal plasma-membrane calcium-influx channels. These results suggested a model in which excessive calcium influx through constitutively active TRP channels results in retinal degeneration in rdgA. The light response of rdgA;trp photoreceptors shows defects in deactivation suggesting that DGK might play a role in terminating the light response and recent evidence suggests that DGK plays a role in regulating signal amplification during the response to light (Hardie, 2002). Despite these recent observations that suggest a direct role for rdgA in phototransduction, previous studies have suggested a distinct mechanism underlying the retinal degeneration phenotype of rdgA. (1) Unlike most other phototransduction mutants, the retinal degeneration of rdgA is reported to be light independent; (2) norpA mutants, which lack the PLC activity essential for TRP channel activation, were reported not to suppress the retinal degeneration of rdgA; (3) several studies have suggested that a failure of rhabdomere biogenesis and protein trafficking underlies the rdgA phenotype (Georgiev, 2005),

To address these apparently conflicting results and to understand the mechanism of degeneration in rdgA, a genome-wide forward-genetic screen was undertaken for mutants that suppress or enhance the retinal-degeneration phenotype of rdgA. The goal is to identify molecules whose function might help gain an understanding of basis of the constitutive TRP-channel activity that is associated with the rdgA phenotype. This study describes the isolation and characterization of two mutants identified in the screen. Experiments are described that address the requirement for the light response in the degeneration phenotype of rdgA (Georgiev, 2005),

Therefore, although most studies agree that the hydrolysis of PI(4,5)P₂ by receptor-regulated PLCß is required for TRPC (TRP and TRPL) activation, there is little agreement about the downstream biochemical mechanisms that result in TRPC channel opening. For some members, such as TRPC3, equally compelling studies have been published showing roles in activation for IP₃/IP₃R mediated store depletion and for the lipid products of PI(4,5)P₂ hydrolysis. Recently, it has become clear that many of these conflicting results arise from several experimental factors, including the level of overexpression of the channel, the presence of endogenous TRPC members in the cell lines used and the relative promiscuity of pharmacological agents used in manipulating their activation. By contrast, in Drosophila photoreceptors, the detection of light by rhodopsin activates a signalling cascade that ends with endogenous TRPC activation. In this model, too, current debate centers around the identity of the products of PI(4,5)P₂ hydrolysis that are crucial for channel activation. Although there is substantial evidence to suggest that IP₃-mediated signalling is not essential, recent evidence suggests that the lipid products of PI(4,5)P₂ hydrolysis might be involved in activation. Analysis of photoreceptors lacking DGK activity (rdgA) has provided the first genetic evidence that suggests a role for lipid second messengers in activating TRP and TRPL in vivo. However, questions remain about the biochemical basis of the rdgA phenotype and its relevance to the normal phototransduction cascade; for example, is the constitutive channel activity the cause or the consequence of the degeneration? In addition, how can one reconcile recent findings suggesting a role for rdgA in phototransduction with long-standing observations that imply a phototransduction-independent basis for the rdgA phenotype (Georgiev, 2005)?

To address this issue, a forward-genetic screen was carried out to identify suppressors of the retinal degeneration phenotype of rdgA mutants. Such an approach is unbiased and makes no assumptions about the mechanisms underlying the degeneration process. Using a combination of deficiency mapping and bioinformatic analysis, su(1) and su(100) were identified as new alleles of InaD, a PDZ-domain protein required for the assembly of signalling complexes in Drosophila photoreceptors that is suggested to have a role in the regulation of signalling specificity and speed. InaD has not previously been reported to interact with rdgA. Trp³⁶⁵ contains a point mutation at the cytoplasmic end of S5 in the TRP channel and shows constitutive channel activity and degeneration. The finding that InaD is largely ineffective at suppressing the degeneration of Trp³⁶⁵ suggests that the mechanism of suppression is at or above the level of the channel in the transduction cascade rather than by blocking events downstream of excessive calcium influx through constitutively active TRP channels. InaD clusters several key molecules required for phototransduction, including the TRP channel; this strongly suggests that the constitutive channel activity and degeneration in rdgA are a consequence of altered phototransduction (Georgiev, 2005),

To identify the specific known (or perhaps undiscovered) protein-protein interactions of InaD that contribute to the rescue of rdgA, the InaD complex was manipulated in a manner that allowed its interaction with specific transduction components such as the TRP channel and NORPA to be individually disrupted. Wild-type TRP protein levels were found in InaD on eclosion and by analysing the effect of InaD^P215 and TRP^Delta1272 on rdgA it was found that TRP channels that could not be recruited to the INAD complex were able to mediate degeneration in rdgA just as well as wild-type channels. Thus, loss of the TRP-INAD interaction is unlikely to be a significant mechanism by which InaD rescues rdgA and the function of TRP channels within the INAD complex is not crucial to retinal degeneration (Georgiev, 2005),

By contrast, it was found that NORPA (the PLC activity) levels in su(1) and su(100) mutants are reduced on eclosion, as has been reported for InaD¹, suggesting that a reduction in PLCß activity might underlie the mechanism of suppression. InaD¹, a protein-null allele, and InaD², an allele known to disrupt the INAD-NORPA interaction, produce equivalent levels of rescue of rdgA¹, and that the levels of NORPA are inversely correlated to the extent of rescue. Thus, a major mechanism by which InaD¹, su(1) and su(100) suppress rdgA¹ is likely to involve the reduced levels of NORPA in these alleles. However, the possibility cannot be excluded that additional, unknown protein-protein interactions of PDZ5 in INAD that might also be disrupted in InaD² might also contribute to the rescue of rdgA. Given the essential role of PLCß in the activation of TRP channels, this finding implies a key role for the balance of PLCß and DGK activity in the degeneration of rdgA (Georgiev, 2005),

Although the degeneration phenotype of rdgA has been previously reported to be light independent and not suppressed by norpA mutants, the finding that InaD suppresses rdgA and that it does so by reducing levels of NORPA suggests that defects in light-induced phosphoinositide turnover might underlie the degeneration phenotype of rdgA. In the light of these findings on the suppression of rdgA by InaD, the effect was re-examined of three key elements of the phototransduction cascade that are required for activation, namely light, Galphaq and PLC. Although the absence of light could not completely suppress the degeneration of rdgA³, there was substantial suppression of degeneration in rdgA³ flies grown in complete darkness compared with those grown on a 12 hour light/12 hour dark cycle. Degeneration could also be partially suppressed but not blocked by a strong hypomorph that reduced Galphaq levels to <5% of the wild-type levels. However, most importantly, it was found that norpA mutants that lack PLCß could suppress the degeneration of rdgA in several allelic combinations for both genes. These results demonstrate a key role for activation of the phototransduction cascade in the degeneration phenotype of rdgA (Georgiev, 2005),

Although this study shows a requirement for light, Galphaq and PLC activity in the degeneration phenotype of rdgA, it was not possible to completely suppress the degeneration of even the weakest allele, rdgA³, by rearing flies in complete darkness. Indeed, reducing levels of Galphaq using the strong hypomorph Galphaq¹ (which has <5% of the wild-type Galphaq levels) was able only to slow the rate of degeneration of rdgA³. However mutants in norpA^P24 were able completely to suppress the degeneration of both rdgA³ and rdgA¹. In a recent study that measured basal PLCß activity in photoreceptors (Hardie, 2004), it was found that, similar to wild-type photoreceptors, rdgA mutants showed reduced but still substantial basal PLCß activity. This implies that, even in the dark, there is a basal turnover of PI(4,5)P₂ in rdgA photoreceptors. Thus, basal PI(4,5)P₂ hydrolysis could lead to the build up of a lipid metabolite of PI(4,5)P₂ that triggers constitutive TRP channel activity and retinal degeneration (Georgiev, 2005),

Although several studies have demonstrated the importance of INAD in targeting and stabilizing members of the phototransduction cascade to the rhabdomere, there is little agreement about the requirement, if any, for intact INAD complexes once assembled and transported to the rhabdomere to activate TRP channels. Although some studies have suggested that an intact INAD complex is crucial for generating the channel activity that underlies a quantum bump (the response to a single photon of light) others have suggested that this might not be the case. In this analysis of the mechanism by which InaD suppresses rdgA, it was found that that TRP channels not included within the INAD complex but still present in the rhabdomere are able to mediate retinal degeneration. These results support the idea that presence within the INAD macromolecular complex is not necessary for the constitutive activity of TRP channels seen in rdgA (Georgiev, 2005),

Although these data support the hypothesis that a principal mechanism by which InaD suppresses rdgA is via reduction in the levels of PLCß, they do not exclude the possibility that the disruption of INAD interactions with currently undiscovered proteins that function downstream of NORPA might play a role in constitutive TRP channel activation and degeneration in rdgA. Testing this would require the generation of an InaD allele in which the INAD-NORPA interaction is intact while disrupting the function of the other protein-protein interactions of INAD. No such allele exists, but the use of such an allele in conjunction with the rdgA mutant could be an useful approach to identifying currently undiscovered members of the INAD complex as well as the phototransduction cascade (Georgiev, 2005),

TRP channels appear to be key components of signalling cascades for the detection and coding of several sensory modalities. However, a limiting factor in advancing their role in sensory transduction is the poor understanding of their mechanism of activation. In the case of TRPC channels, this is limited by the lack of genetic model systems in which relevant components of the activation cascade can be identified. In the present study, a novel modifier screen has been described that should provide a powerful method for identifying the relevant transduction components in vivo. Starting with the rdgA mutant in which TRP channels are constitutively active and result in retinal degeneration, two new alleles of INAD, a known component of the phototransduction cascade, have been identified as suppressors of rdgA. This approach is likely to be a powerful tool to identify further components of the transduction cascade that are relevant in vivo (Georgiev, 2005),

Role of protein phosphatase 2A in regulating the visual signaling in Drosophila

Drosophila visual signaling, a G-protein-coupled phospholipase Cbeta (PLCbeta)-mediated mechanism, is regulated by eye-protein kinase C (PKC) that promotes light adaptation and fast deactivation, most likely via phosphorylation of inactivation no afterpotential D (INAD) and TRP (transient receptor potential). To reveal the critical phosphatases that dephosphorylate INAD, several biochemical analyses were used and protein phosphatase 2A (PP2A) was identified as a candidate. Importantly, the catalytic subunit of PP2A, Microtubule star (MTS), copurifies with INAD, and an elevated phosphorylation of INAD by eye-PKC was observed in three mts heterozygotes. To explore whether PP2A (MTS) regulates dephosphorylation of INAD by counteracting eye-PKC [INAC (inactivation no afterpotential C] in vivo, ERG recordings were performed. ina^CP209 is semidominant, because ina^CP209 heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect of ina^CP209 heterozygotes is rescued by the mts^XE2258 heterozygous background. In contrast, mts^XE2258 fails to modify the severe deactivation of norpA^P16, indicating that MTS does not modulate NORPA (no receptor potential A) (PLCbeta). Together, these results strongly indicate that dephosphorylation of INAD is catalyzed by PP2A, and a reduction of PP2A can compensate for a partial loss of function in eye-PKC, restoring the fast deactivation kinetics in vivo. Thus it is proposed that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD (Wang, 2008).

The in vitro assays show that PP2A dephosphorylates the scaffolding protein INAD, opposing the activity of eye-PKC to phosphorylate INAD. An increased level of INAD phosphorylation occured in three distinct mts heterozygotes, wherein the catalytic C subunit of PP2A has been rendered ineffective. Utilizing ERG recordings, in partial loss-of-function mutants of mts^XE2258, inaC^P209, and the combined double mutant, it was found that PP2A and eye-PKC have opposing physiological functions, and that a balance between the activities of eye-PKC and PP2A is central for the proper deactivation of the visual response. The results strongly indicate that PP2A appears to impact signaling proteins operating downstream of NORPA in the visual cascade. Integrating in vivo and in vitro findings into the current model of eye-PKC-mediated regulation of INAD, it is concluded that reversible phosphorylation of INAD is dependent on the opposing enzymatic actions of eye-PKC and PP2A and that phosphorylation of INAD is critical for fast deactivation of the visual signaling process (Wang, 2008).

The biochemical assays support PP2A as a key phosphatase responsible for the dephosphorylation of INAD. Based on both its inhibition profile with okadaic acid, and its copurification alongside the FPLC fraction with positive phosphatase activity, PP2A was identified as the prime candidate for mediating INAD dephosphorylation. After finding that a purified A/C dimer of PP2A dephosphorylates INAD in vitro, immunocomplex kinase assays were performed. As expected, a reduction in PP2A catalytic efficiency causes a significant increase in the measurable fraction of phosphorylated INAD. By examining three distinct mts heterozygotes, each carrying a C subunit mutation resulting in a partial loss of PP2A function, a significant increase was demonstrated in INAD phosphorylation levels with mts^XE2258 exhibiting the most dramatic increase. The enhanced INAD phosphorylation observed in the mts extracts strongly suggests that PP2A is closely positioned in the INAD complex to promote timely dephosphorylation of INAD. Consistently, it was demonstrated that PP2A can be coisolated with INAD, thus representing a newly identified component of the INAD macromolecular complex (Wang, 2008).

For insights into the role PP2A plays in Drosophila vision, mts^XE2258 heterozygotes were studied, and a surprisingly normal ERG waveform was found. Although a reduction in the level of active PP2A in vivo has no effect on visual function, it was found that inaC^P209 heterozygotes exhibit abnormal light adaptation, as well as delayed deactivation of visual signaling. inaC^P209 and mts^XE2258 both encode for enzymes; why would missing one functional copy of the PP2A gene not affect normal visual electrophysiology, whereas missing a functional copy of the eye-PKC gene drastically slows deactivation? inaC^P209 must be a semidominant mutation; in other words, inaC is haploinsufficient. An explanation for the haploinsufficiency of the eye-PKC gene is that the substrate repertoire of eye-PKC is defined by substrate colocalization to the INAD macromolecular complex to which eye-PKC is tethered. This hypothesis is in good agreement with the observation that the interaction with INAD is essential for the in vivo function of eye-PKC to modulate the visual response (Wang, 2008).

Although MTS is also tethered to the INAD signaling complex, unlike eye-PKC, the abundance of MTS relative to INAD in photoreceptors is likely to make it less sensitive to a reduction of its gene dosage to effect visual electrophysiology. Alternatively, it is possible that anchoring to the INAD complex by PP2A may be regulated by the interaction via its B subunit instead of the C subunit. Therefore, a reduction of MTS may not significantly modify its presence in the INAD complex (Wang, 2008).

To elucidate the functional interplay, in vivo, between eye-PKC and MTS, mts^XE2258 and inaC^P209 double heterozygotes were characterized. A significant discovery was made that only the slow deactivation defect was rescued in the mts^XE2258 heterozygous background. The selective rescue of deactivation defects by mts^XE2258, with no rescue of the abnormal light adaptation found in inaC^P209 heterozygotes, suggests that PP2A regulates proteins that lie downstream of eye-PKC, a conclusion that is also supported by the inability of mts^XE2258 to restore the slow deactivation defect in a hypomorphic allele of norp^P16 (Wang, 2008).

A concomitant reduction of the PP2A activity would lead to increased phosphorylation of multiple substrates contributing to the observed normal, fast deactivation kinetics found in the double mutant. Potential PP2A substrates may include INAD, TRP, and eye-PKC. Like other conventional PKCs, eye-PKC is most likely a phosphoprotein and hence its catalytic activity is sensitive to PP2A. It is expected that a reduction of PP2A should increase the autophosphorylation of eye-PKC, bringing about an enhanced catalytic capability. However, the presumably increased eye-PKC activity fails to restore the light adaptation abnormality, suggesting that the modulation of eye-PKC represents a lesser role of PP2A in vivo (Wang, 2008).

It is possible that PP2A dephosphorylates TRP, thus regulating deactivation kinetics. Consistently, a lack of phosphorylation at Ser⁹⁸² of TRP leads to slowed deactivation of the visual response. However, trp^P343, a null allele affecting the trp gene, displays a less severe defect in deactivation than that of inaC^P209, suggesting eye-PKC phosphorylation of TRP does not play a major role in the normal deactivation. The eye-PKC-dependent phosphorylation of INAD is currently being studied and preliminary results suggest that a loss of phosphorylation in INAD also results in slowed deactivation kinetics. This finding together with the biochemical results supports the hypothesis that the phosphatase PP2A directly regulates phosphorylation states of INAD to impact fast deactivation of the visual signaling. A light-dependent conformation change has been shown to occur in the fifth postsynaptic density-95/Discs large/zona occludens-1 (PDZ) domain of INAD, and it has been proposed that eye-PKC might orchestrate this event. These findings are in agreement with the current studies supporting a critical role of INAD phosphorylation to promote fast deactivation. It will be of great interest to elucidate how phosphorylation of INAD leads to the conformation switch in its fifth PDZ domain (Wang, 2008).

In addition to INAD, PP2A may dephosphorylate other, yet-to-be identified substrates. The observations that mts^XE2258 modified the severe deactivation defect of inaC^P209 homozygotes, suggests that a reduction of MTS increases phosphorylation of proteins, which are regulated by non-eye-PKC serine/threonine protein kinases. Several protein kinases including CaMKII, and NINAC (neither inactivation nor afterpotential C) have been shown to modulate deactivation, and may play a role in regulating deactivation in the absence of eye-PKC (Wang, 2008).

In summary, this study has demonstrates the roles of PP2A and eye-PKC in orchestrating reversible phosphorylation of INAD, and that phosphorylation of INAD is most likely involved in fast deactivation kinetics of the visual signaling in Drosophila. The biochemical findings support the critical role of PP2A to dephosphorylate INAD. Electrophysiological characterization strongly indicates that a reduction of PP2A compensates for a partial loss of function in eye-PKC leading to rescuing the slow deactivation defect (Wang, 2008).

Dependence on a retinophilin/myosin complex for stability of PKC and INAD and termination of phototransduction

Normal termination of signaling is essential to reset signaling cascades, especially those such as phototransduction that are turned on and off with great rapidity. Genetic approaches in Drosophila led to the identification of several proteins required for termination including protein kinase C (PKC), NinaC p174, which consists of fused protein kinase and myosin domains, and a PDZ scaffold protein, INAD. This study describes a mutation affecting a poorly characterized but evolutionarily conserved protein, Retinophilin (Retin), which is expressed primarily in the phototransducing compartment of photoreceptor cells, the rhabdomeres. Retin and NINAC formed a complex and were mutually dependent on each other for expression. Loss of retin resulted in an age-dependent impairment in termination of phototransduction. Mutations that affect termination of the photoresponse, typically lead to a reduction in levels of the major rhodopsin, Rh1, to attenuate signaling. Consistent with the slower termination in retin1, the mutant photoreceptor cells exhibited increased endocytosis of Rh1 and a decline in Rh1 protein. The slower termination in retin¹ was a consequence of a cascade of defects, which began with the reduction in NINAC p174 levels. The diminished p174 concentration caused a decrease in INAD. Since PKC requires interaction with INAD for protein stability, this leads to reduction in PKC levels. The decline in PKC was age-dependent, and paralleled the onset of the termination phenotype in retin¹ mutant flies. It is concluded that the slower termination of the photoresponse in retin¹ resulted from a requirement for the Retin/NINAC complex for stability of INAD and PKC (Venkatachalam, 2010).

This study describes the identification of Retin, a protein required for termination of the photoresponse. Unlike other proteins that function in termination, the retin phenotype is age-dependent. Slow termination leads to increased endocytosis and degradation of the major rhodopsin, Rh1, which serves as a negative feedback mechanism to attenuate the visual response. Consistent with a defect in termination, the age-dependent impairment in the photoresponse in retin¹ is associated with greater endocytosis of Rh1 and an age-dependent reduction in the concentration of Rh1 (Venkatachalam, 2010).

A central question concerns the basis for the age-dependent decrease in the termination rate in retin deficient flies. Retin has been reported to function in macrophages through a pathway that involves the ryanodine receptor, a store-operated channel, Orai, and the interacting protein, STIM1 (Cuttell, 2008), which is present in the endoplasmic reticulum (ER) and senses changes in ER Ca²⁺. However, Ca²⁺ release from the ER, the ryanodine receptor and the IP₃-receptor do not appear to function in Drosophila visual transduction. Furthermore, knockdown of stim1 RNA using a photoreceptor cell GAL4 in combination with UAS-stim1-RNAi transgene had no effect on phototransduction, the concentration of Retin, or other proteins reduced in retin¹ mutant eyes. The decrease in termination in retin¹ mutant flies was not due directly to loss of Retin, since the Retin protein was absent in young flies that exhibited normal termination. The retin phenotype also was not a consequence of a reduction in NINAC p174, since both 3 and 7 day-old retin¹ flies displayed similarly low levels of p174; however, only the 7 day-old flies exhibited the slow termination phenotype (Venkatachalam, 2010).

It is concluded that the age-dependent termination phenotype in retin¹ results from a reduction in PKC levels. Consistent with this proposal, the decline in PKC concentration paralleled the appearance of the termination phenotype. In young retin¹ flies, which displayed normal termination, PKC was not reduced significantly from wild-type. However, in older retin¹ flies, the PKC concentration declined two-fold. In further support of the conclusion that the 50% decrease in PKC is responsible for the termination defect in retin¹, a similar impairment in termination occurs in heterozygous flies, which are missing copy of the gene encoding the eye-enriched PKC (Venkatachalam, 2010).

The following mechanism is proposed through which Retin affects the concentration of PKC. First, Retin forms a complex with NINAC p174, and this interaction is required for the stability of p174. Both proteins co-immunoprecipitated from head extracts, and loss of Retin resulted in a lower concentration of p174. The requirement for Retin and NINAC was mutual since Retin was undetectable in flies missing p174. Second, NINAC is required for stabilizing the PDZ-containing scaffold protein INAD. NINAC and INAD interact, and it was found that a single amino acid mutation that disrupts the INAD binding site in p174 (ninaC^I1501E) causes a reduction in INAD. Third, PKC binds stoichiometrically to INAD and requires this interaction for stability. As a result, INAD and PKC displayed indistinguishable two-fold decreases in protein levels. It was found that PKC also declined to a similar extent in flies expressing NINAC^I1501E. Because INAD was reduced in ninaC^I1501E flies, but not Retin or NINAC p174, the instability of PKC was not due to non-specific effects resulting from changes in the concentrations either Retin or p174. Thus, loss of Retin causes a reduction in the level of p174, which in turn affects the concentration of INAD, leading to instability of PKC, which underlies the slower termination (Venkatachalam, 2010).

Despite the defect in termination, retin¹ flies exhibited only minor effects on retinal morphology. There are multiple examples of mutations that are associated with termination defects that display relatively minor alterations in rhabdomere morphology. These include rac2, ninaC, and stops. Of particular relevance, flies heterozygous for a mutation disrupting the eye-enriched PKC (inaC^P209/+ flies), which exhibit a termination phenotype similar to retin¹, do not undergo retinal degeneration (Venkatachalam, 2010).

Finally, both Retin and myosins with fused N-terminal protein kinase domains are found in other organisms including humans. Protein kinase/myosins (myosin IIIs) and Retin are both expressed in the mammalian retina. This raises the possibility that Retin and myosins related to NINAC may form a complex in mammalian photoreceptor cells, and are required for signaling (Venkatachalam, 2010).

DEVELOPMENTAL BIOLOGY

Adult

A 2 kb InaD transcript is detected in wild-type flies, which is absent in the eyes absent mutant that lacks compound eyes. The INAD mRNA is also present in the InaD mutant. Anti-InaD antisera recognize the presence of InaD exclusively in the retina of the eye. The antisera also stain three ocelli, simple eyes located at the apex of the head. InaD is closely associated with rhabdomeres, which are visual organelles consisting of densely packed microvilli (Shieh, 1995).

REFERENCES

Search PubMed for articles about Drosophila inactivation no after potential D

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date revised: 22 February 2022

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