Inactivation No Afterpotential D Research Articles

Purification of Endogenous Drosophila Transient Receptor Potential Channels.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase Cβ/No receptor potential A (PLCβ/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Calmodulin binds to Drosophila TRP with an unexpected mode

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca2+ modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca2+-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.

Calmodulin Enhances Cryptochrome Binding to INAD in Drosophila Photoreceptors.

Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. We have previously shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. Here, we predict and demonstrate a novel Ca2+-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses we were able to identify and characterize a CaM binding motif in the dCRY C-terminus. Similarly, we also detailed the CaM binding site of the scaffold protein INAD and demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. Our results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM.

An Exquisitely Specific PDZ/Target Recognition Revealed by the Structure of INAD PDZ3 in Complex with TRP Channel Tail

The vast majority of PDZ domains are known to bind to a few C-terminal tail residues of target proteins with modest binding affinities and specificities. Such promiscuous PDZ/target interactions are not compatible with highly specific physiological functions of PDZ domain proteins and their targets. Here, we report an unexpected PDZ/target binding occurring between the scaffold protein inactivation no afterpotential D (INAD) and transient receptor potential (TRP) channel in Drosophila photoreceptors. The C-terminal 15 residues of TRP are required for the specific interaction with INAD PDZ3. The INAD PDZ3/TRP peptide complex structure reveals that only the extreme C-terminal Leu of TRP binds to the canonical αB/βB groove of INAD PDZ3. The rest of the TRP peptide, by forming a β hairpin structure, binds to a surface away from the αB/βB groove of PDZ3 and contributes to the majority of the binding energy. Thus, the INAD PDZ3/TRP channel interaction is exquisitely specific and represents a new mode of PDZ/target recognitions.

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. Here, we report 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, we found that INAD phosphorylated at Thr170/Ser174 was 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.

Fly cryptochrome and the visual system

Cryptochromes are flavoproteins, structurally and evolutionarily related to photolyases, that are involved in the development, magnetoreception, and temporal organization of a variety of organisms. Drosophila CRYPTOCHROME (dCRY) is involved in light synchronization of the master circadian clock, and its C terminus plays an important role in modulating light sensitivity and activity of the protein. The activation of dCRY by light requires a conformational change, but it has been suggested that activation could be mediated also by specific "regulators" that bind the C terminus of the protein. This C-terminal region harbors several protein-protein interaction motifs, likely relevant for signal transduction regulation. Here, we show that some functional linear motifs are evolutionarily conserved in the C terminus of cryptochromes and that class III PDZ-binding sites are selectively maintained in animals. A coimmunoprecipitation assay followed by mass spectrometry analysis revealed that dCRY interacts with Retinal Degeneration A (RDGA) and with Neither Inactivation Nor Afterpotential C (NINAC) proteins. Both proteins belong to a multiprotein complex (the Signalplex) that includes visual-signaling molecules. Using bioinformatic and molecular approaches, dCRY was found to interact with Neither Inactivation Nor Afterpotential C through Inactivation No Afterpotential D (INAD) in a light-dependent manner and that the CRY-Inactivation No Afterpotential D interaction is mediated by specific domains of the two proteins and involves the CRY C terminus. Moreover, an impairment of the visual behavior was observed in fly mutants for dCRY, indicative of a role, direct or indirect, for this photoreceptor in fly vision.

Structural mechanism of disulphide bond-mediated redox switches

The oxidation of cysteine sulphydryl in proteins produces sulphenic acid that can form a reversible disulphide bond with another cysteine. The disulphide bond formation often triggers switches in protein structure and activity, especially when the distance between the two cysteine sulphur atoms is longer than the resulting disulphide bond distance. As an early example for the reversible disulphide bond-mediated functional switches, the reduced and oxidized forms of the bacterial transcription factor OxyR were characterized by X-ray crystallography. Recently, the Drosophila vision signalling protein, the association of inactivation-no-afterpotential D (INAD) was analysed by structural and functional methods. The two conserved cysteines of INAD were found to cycle between reduced and oxidized states during the light signal processing in Drosophila eyes, which was achieved by conformation dependent modulation of the disulphide bond redox potential. The production of the hypertension control peptide angiotensins was also shown to be controlled by the reversible disulphide bond in the precursor protein angiotensinogen. The crystal structure of the complex of angiotensiongen with its processing enzyme renin elucidated the role of the disulphide bond in stabilizing the precursor-enzyme complex facilitating the production of angiotensins. The increasing importance of the disulphide bond-mediated redox switches in normal and diseased states has implications in the development of novel antioxidant-based therapeutic approaches.

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 (neither inactivation nor afterpotential C) p174, which consists of fused protein kinase and myosin domains, and a PDZ (postsynaptic density-95/Discs Large/zona occludens-1) scaffold protein, INAD (inactivation no afterpotential D). Here, we describe 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 retin(1), the mutant photoreceptor cells exhibited increased endocytosis of Rh1 and a decline in Rh1 protein. The slower termination in retin(1) 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. Because 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 retin(1) mutant flies. We conclude that the slower termination of the photoresponse in retin(1) resulted from a requirement for the Retin/NINAC complex for stability of INAD and PKC.

Role of Protein Phosphatase 2A in Regulating the Visual Signaling inDrosophila

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, we used several biochemical analyses and identified protein phosphatase 2A (PP2A) as a candidate. Importantly, the catalytic subunit of PP2A, microtubule star (MTS), is copurified 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, we performed ERG recordings. We discovered that inaC(P209) was semidominant, because inaC(P209) heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect of inaC(P209) heterozygotes was rescued by the mts(XE2258) heterozygous background. In contrast, mts(XE2258) failed to modify the severe deactivation of norpA(P16), indicating that MTS does not modulate NORPA (no receptor potential A) (PLCbeta). Together, our 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. We thus propose that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD.

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). We previously reported that INAD associated with the carboxyl tail of TRP via its third post-synaptic density protein 95, discs-large, zonular occludens-1 domain. In this paper, we 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. We also revealed by pull-down assays that the last 14 residues of TRP comprised 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 was shown to phosphorylate TRP in vivo. To uncover the substrates of eye-PKC in the INAD complex, we designed a complex-dependent eye-PKC assay, which utilized endogenous INAD complexes isolated from flies. We demonstrate that activated eye-PKC phosphorylates 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. We conclude that TRP associates with INAD via its last 14 residues to facilitate its regulation by eye-PKC that fine-tunes the visual signaling.

An Active Player

Scaffolding proteins that bind to and organize components of signaling pathways into macromolecular complexes are crucial to the efficiency, specificity, and speed of various signaling pathways. For instance, INAD (inactivation, no after-potential D), a PDZ domain-containing scaffolding protein that, together with three of its binding partners, constitutes the core complex of the Drosophila phototransduction cascade, is critical to the electrical response to light (see Montell). Using x-ray crystallography, Mishra et al . found that, in the oxidized state, the fifth INAD PDZ protein interaction domain (PDZ5) assumed a distorted conformation inconsistent with canonical ligand binding. This conformation depended on an intramolecular disulfide bond and, in the reduced state, PDZ5 structurally resembled other PDZ domains. In transgenic flies expressing wild-type INAD, light exposure led to formation of the disulfide bond (and thus the oxidized conformation), whereas subsequent darkness was associated with return to the reduced form; development of the oxidized form was attenuated in flies lacking the major isoform of rhodopsin. Electrophysiological analysis of isolated photoreceptor cells expressing a mutant form of INAD in which PDZ5 only existed in the reduced conformation indicated that the sensitivity of the mutant form to light was normal but that loss of a refractory period led to defective termination of the response to bright light. Moreover, flies with the mutant INAD showed a defective escape response to a brief exposure to darkness (mimicking the shadow of a predator). Thus, rather than performing a purely passive function in organizing signaling proteins, INAD appears to play a dynamic role in the Drosophila phototransduction pathway. P. Mishra, M. Socolich, M. A. Wall, J. Graves, Z. Wang, R. Ranganathan, Dynamic scaffolding in a G protein-coupled signaling system. Cell 131 , 80-92 (2007). [PubMed] C. Montell, Dynamic regulation of the INAD signaling scaffold becomes crystal clear. Cell 131 , 19-21 (2007). [PubMed]

Dynamic Scaffolding in a G Protein-Coupled Signaling System

The INAD scaffold organizes a multiprotein complex that is essential for proper visual signaling in Drosophila photoreceptor cells. Here we show that one of the INAD PDZ domains (PDZ5) exists in a redox-dependent equilibrium between two conformations--a reduced form that is similar to the structure of other PDZ domains, and an oxidized form in which the ligand-binding site is distorted through formation of a strong intramolecular disulfide bond. We demonstrate transient light-dependent formation of this disulfide bond in vivo and find that transgenic flies expressing a mutant INAD in which PDZ5 is locked in the reduced state display severe defects in termination of visual responses and visually mediated reflex behavior. These studies demonstrate a conformational switch mechanism for PDZ domain function and suggest that INAD behaves more like a dynamic machine rather than a passive scaffold, regulating signal transduction at the millisecond timescale through cycles of conformational change.

Scaffolding Protein INAD Regulates Deactivation of Vision by Promoting Phosphorylation of Transient Receptor Potential by Eye Protein Kinase C inDrosophila

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 Cbeta, 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, here we demonstrate that eye-PKC, via tethering to INAD, phosphorylates TRP in vitro. We reveal that 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.

Dissecting independent channel and scaffolding roles of the Drosophila transient receptor potential channel

Drosophila transient receptor potential (TRP) serves dual roles as a cation channel and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering TRP channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study we describe the isolation of a set of new trp alleles, including trp 14 with an amino acid substitution juxtaposed to the TRP domain. The trp 14 flies stably express TRP and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp Δ 1272, had minor effects on retinal morphology whereas disruption of channel function caused profound light-induced cell death. This retinal degeneration was greatly suppressed by elimination of the Na+/Ca2+ exchanger, CalX, indicating that the cell death was due primarily to deficient Ca2+ entry rather than disruption of the TRP-anchoring function.

Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor.

The coupling mechanism between depletion of Ca(2+) stores in the endoplasmic reticulum and plasma membrane store-operated ion channels is fundamental to Ca(2+) signaling in many cell types and has yet to be completely elucidated. Using Ca(2+) release-activated Ca(2+) (CRAC) channels in RBL-2H3 cells as a model system, we have shown that CRAC channels are maintained in the closed state by an inhibitory factor rather than being opened by the inositol 1,4,5-trisphosphate receptor. This inhibitory role can be fulfilled by the Drosophila protein INAD (inactivation-no after potential D). The action of INAD requires Ca(2+) and can be reversed by a diffusible Ca(2+) influx factor. Thus the coupling between the depletion of Ca(2+) stores and the activation of CRAC channels may involve a mammalian homologue of INAD and a low-molecular-weight, diffusible store-depletion signal.

The Calliphora rpa mutant lacks the PDZ domain-assembled INAD signalling complex.

The visual transduction cascade of fly photoreceptors is a G protein-coupled phospholipase C-signalling pathway which is assembled into a supramolecular signalling complex by the PDZ (postsynaptic density protein-95, discs large, Z0-1) domain protein INAD (inactivation no afterpotential D). The norpA-encoded phospholipase Cbeta, the light-activated transient receptor potential (TRP) Ca2+ channel and an eye-specific protein kinase C are bound to INAD and together form the core of the signalling complex. In the present study we show that the Calliphora rpa mutant, which has previously been hypothesized to represent an equivalent of Drosophila norpA mutants, has normal amounts of norpA mRNA but fails to express inaD mRNA. Electrophysiological recordings from the eyes of the rpa mutant reveal that the electroretinogram is reduced (about 12% of wild type) but not completely absent, and that it exhibits markedly prolonged deactivation kinetics. Furthermore, rpa mutants display a slow, light-dependent degeneration of the photoreceptor cells. With respect to the INAD signalling complex, the rpa mutant is similar to the Drosophila inaD null mutant: not only INAD itself, but also the other core components of the INAD signalling complex, are reduced or absent in photoreceptor membranes of rpa flies. Residual TRP is localized throughout the plasma membrane of the photoreceptor cell, rather than being restricted to the microvillar photoreceptor membrane. [35S]methionine-labelling of newly synthesized retinal proteins reveals that TRP is synthesized in the rpa mutant at wild-type level, but is transported to or incorporated into the microvillar photoreceptor membrane at a much lower rate. We thus suggest, that the formation of the INAD signalling complex is required for specifically targeting its components to the photoreceptor membrane.

A novel capacitative calcium entry channel expressed in excitable cells.

In addition to voltage-gated calcium influx, capacitative calcium entry (CCE) represents a major pathway for calcium entry into the cell. Here we report the structure, expression and functional properties of a novel CCE channel, TRP5. This channel is a member of a new subfamily of mammalian homologues of the Drosophila transient receptor potential (TRP) protein, now comprising TRP5 (also CCE2) and the structurally related CCE1 (also TRP4). Like TRP4, TRP5 forms ion channels mainly permeable for Ca2+ which are not active under resting conditions but can be activated by manoeuvres known to deplete intracellular calcium stores. Accordingly, dialysis of TRP5-expressing cells with inositol-(1,4,5)-trisphosphate evokes inward rectifying currents which reversed polarity at potentials more positive than +30 mV. Ca2+ store depletion with thapsigargin induced TRP5-mediated calcium entry dependent on the concentration of extracellular calcium, as seen by dual wavelength fura-2 fluorescence ratio measurements. TRP5 transcripts are expressed almost exclusively in brain, where they are present in mitral cells of the olfactory bulb, in lateral cerebellar nuclei and, together with TRP4 transcripts, in CA1 pyramidal neurons of the hippocampus, indicating the presence of CCE channels in excitable cells and their participation in neuronal calcium homeostasis.

Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo.

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 and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA-INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. 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, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. 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, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells.