no receptor potential A

Promoter Structure

The 5'-flanking region of the Drosophila melanogaster norpA gene has been sequenced and its promoter characterized. The potential promoter region, which was deduced from the determination of the transcription start point (tsp), lacks a distinct TATA box sequence. Deletion analysis of the promoter region suggests that the minimal promoter necessary for efficient expression of the gene is located between -138 (PstI) and +278 relative to the tsp. Within this minimal promoter region, at least two downstream regulatory elements responsible for the stimulation of gene expression seem to exist in the DNA fragments between +44 and +121 and between +214 and +278. Among these, the DNA fragment between +44 and +121 affects promoter activity more dramatically (about 6-7 fold). This DNA fragment contains the consensus promoter element previously reported to be important for photoreceptor cell-specific expression, and this promoter element seems to be working in the norpA gene expression (Doh, 1997).

Enzymatic activity of NorpA

To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in an extract from normal Drosophila heads, which is absent in an extract from norpA mutant heads, was isolated and purified and its partial amino acid sequences were determined. The purified enzyme was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) are quite different from those of partially purified enzyme. The maximal activity for PIP2 hydrolysis is observed at calcium concentrations between 10(-7) and 10(-5) M for both the crude and partially purified enzymes. In contrast, the activity for PI hydrolysis of the crude enzyme increases with increasing calcium concentrations, while that of the partially purified enzyme reaches a maximum at calcium concentrations between 10(-6) and 10(-4) M, and decreases at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme are similar. The crude enzyme hydrolyzes PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increases with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA, which was suggested to be norpA gene (Toyoshima, 1990).

Fatty acid and InsP3 signaling downstream of NorpA

Light-sensitive channels encoded by the Drosophila transient receptor potential-like gene (trpl) are activated in situ by an unknown mechanism requiring activation of Gq and phospholipase C (PLC). Recent studies have variously concluded that heterologously expressed TRPL channels are activated by direct Gq-protein interaction, InsP3 or Ca2+. In an attempt to resolve this confusion an exploration was carried out of the mechanism for the activation of TRPL channels co-expressed with a PLC-specific muscarinic receptor in a Drosophila cell line (S2 cells). Simultaneous whole-cell recordings and ratiometric Indo-1 Ca2+ measurements indicate that agonist (CCh)-induced activation of TRPL channels is not always associated with a rise in Ca2+. Internal perfusion with BAPTA (10 mM) reduces, but does not block, the response to agonist. In most cases, releasing caged Ca2+ facilitates the level of spontaneous channel activity, but similar concentrations (200-500 nM) can also inhibit TRPL activity. Releasing caged InsP3 invariably releases Ca2+ from internal stores but has only a minor influence on TRPL activity and none at all when Ca2+ release is buffered with BAPTA. Caged InsP3 also fails to activate any light-sensitive channels in situ in Drosophila photoreceptors. Two phospholipase C inhibitors (U-73122 4 microM and bromo-phenacyl bromide 50 microM) reduce both spontaneous and agonist-induced TRPL activity in S2 cells. The results suggest that, as in situ, TRPL activation involves G-protein and PLC; that Ca2+ can both facilitate and in some cases inhibit TRPL channels, but that neither Ca2+ nor InsP3 is the primary activator of the channel (Hardie, 1998).

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. Since 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).

Drosophila phototransduction is a G protein-coupled, calcium-regulated signaling cascade that serves as a model system for the dissection of phospholipase C (PLC) signaling in vivo. The Drosophila light-activated conductance is constituted in part by the Transient receptor potential (Trp) ion channel, yet trp mutants still display a robust response, which demonstrates the presence of additional channels. The transient receptor potential-like (trpl) gene encodes a protein displaying 40% amino acid identity with TRP. Mammalian homologs of TRP and TRPL recently have been isolated and postulated to encode components of the elusive I(crac) conductance. TRP and TRPL localize to the membrane of the transducing organelle, together with rhodopsin and PLC, consistent with a role in PLC signaling during phototransduction. To determine the function of TRPL in vivo, trpl mutants were isolated and characterized physiologically and genetically. The light-activated conductance is composed of TRP and TRPL ion channels and each can be activated on its own. Genetic and electrophysiological tools were used to study the contribution of each channel type to the light response and show that TRP and TRPL can serve partially overlapping functions (Niemeyer, 1996).

Membrane association of NorpA

Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. The predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA. Biochemical assays as well as antisera generated against the major gene product of norpA were used to examine PLC subcellular distribution before and during phototransduction. Both phospholipase C activity and the NorpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as NorpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the NorpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C gamma, is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C (McKay, 1994b).

Ca2+ and the phosphoinositide pathway

Photoreceptors of dissociated Drosophila retinae were loaded with the fluorescent Ca2+ indicators, fluo-3 and Calcium Green-5N. In fluo-3-loaded, wild-type photoreceptors, a rapid increase in fluorescence (Ca2+ signal) accompanies the light-evoked inward current. Removal of extracellular Ca2+ greatly reduces the Ca2+ signal, indicating Ca2+ influx as its major cause. In Calcium Green-5N-loaded trp mutants, which lack a large fraction of the Ca2+ permeability underlying the light-evoked inward current, the Ca2+ signal is smaller relative to wild-type photoreceptors. Fluo-3-loaded norpA mutant photoreceptors, which lack a light-activated phospholipase C, generate no light-evoked inward current and no Ca2+ signal. The phosphoinositide pathway therefore appears necessary for both excitation and changes in cytosolic free Ca2+ concentration (Peretz, 1994).

Light stimulates phosphatidylinositol bisphosphate phospholipase C (PLC) activity in Drosophila photoreceptors. The mechanism of this reaction was investigated by assaying PLC activity in Drosophila head membranes using exogenous phospholipid substrates. PLC activation depends on the photoconversion of rhodopsin to metarhodopsin and is reduced in norpAEE5 PLC and ninaEP332 rhodopsin mutants. NorpA PLC is stimulated by light at free Ca2+ concentrations between 10 nM and 1 microM. This finding is consistent with a Ca(2+)-mediated positive feedback mechanism that contributes to the rapid temporal response of invertebrate photoreceptor cells. The guanyl nucleotide dependence of light-stimulated PLC activity indicates that a G protein regulates NorpA. This was confirmed by the observation that light stimulation of PLC activity is deficient in mutants that lack the eye-specific G protein beta subunit G beta e. These results indicate that G beta e functions as the beta subunit of the G protein coupling rhodopsin to NorpA PLC (Running Deer, 1995).

Drosophila phototransduction is a phosphoinositide-mediated and Ca(2+)-regulated signaling cascade ideal for the dissection of feedback regulatory mechanisms. To study the roles of intracellular Ca2+ ([Ca2+]i) in this process, novel techniques were developed for the measurement of [Ca2+]i in intact photoreceptors. Flies were genetically engineered to express a UV-specific rhodopsin in place of the normal rhodopsin, so that long wavelength light can be used to image [Ca2+]i changes while minimally exciting the photoreceptor cells. Activation with UV generates [Ca2+]i increases that are spatially localized to the rhabdomeres and that are entirely dependent on the influx of extracellular Ca2+. Application of intracellular Ca2+ chelators of varying affinities demonstrates that the Ca2+ influx initially generates a large-amplitude transient that is crucial for negative regulation. Internal Ca2+ stores were revealed by discharging them with thapsigargin. But, in contrast to proposals that IP3-sensitive stores mediate phototransduction, thapsigargin does not mimic or acutely interfere with photoexcitation. Finally, a photoreceptor-specific PKC was identified as essential for normal kinetics of [Ca2+]i recovery (Ranganathan, 1994).

Disruption of phospholipase C-beta (PLC) by the norpA mutations of Drosophila renders flies blind by affecting the light-evoked photoreceptor potential. The norpA-coded PLC modulates the 1,4-dihydropyridine (DHP)-sensitive Ca2+ channels in larval muscles. The DHP-sensitive current is reduced in the norpA mutants. Application of 1 microM phorbol 12-myristate 13-acetate (TPA) and 1 microM phorbol 12,13-didecanoate (PDD), both activators of protein kinase C (PKC), rescues the current in the mutant fibers without significantly affecting the normal current. 4Alpha-phorbol 12,13-didecanoate (4alphaPDD), an inactive analog of PDD, does not affect either the normal or the mutant current. One micromolar bisindolylmaleimide (BIM), an inhibitor of PKC, reduces the current in the normal fibers without affecting the mutant current. 300 microM sn-1,2-dioctanoyl-glycerol (DOG), an analog of diacylglycerol (DAG), increases the current in the mutant fibers. These experiments suggest that the DHP-sensitive Ca2+ channels in Drosophila may be modulated by the PLC-DAG-PKC pathway, and that the same PLC isozyme, which is involved in phototransduction in the adult flies, may also modulate muscle Ca2+ channels in the larval stage of development (Gu, 1997).

Protein Interactions

Interaction of NorpA with G protein alpha49B

The roles of the Drosophila Gq alpha proteins (DGq) were examined in the phototransduction pathway. The DGq proteins immunolocalize to the ocelli and all eight retinular photoreceptor cell rhabdomeres. An affinity-purified anti-DGq alpha immunoglobulin blocks the light-dependent GTP hydrolysis activity associated with Drosophila head membranes in vitro, suggesting that rhodopsin stimulates DGq. Dominantly active DGq1 mutants exhibit a light-independent GTPase activity and abnormal electrophysiological light responses, such as reduced retinal sensitivity and slow response kinetics, as compared with wild-type flies. Dominant DGq2 mutants exhibit a light-independent GTPase activity with normal electrophysiological light responses. Retinas of double mutants of DGq1, but not DGq2, with the light-dependent retinal degeneration mutant rdgB degenerate even in the dark. DGq1 stimulation of rdgB retinal degeneration in the dark is norpA-dependent. These results indicate that DGq1 mediates the stimulation by light-activated rhodopsin of the norpA-encoded phospholipase C in the visual transduction cascade (Y. J. Lee, 1994).

Heterotrimeric G proteins mediate a variety of signaling processes by coupling seven-transmembrane receptors to intracellular effector molecules. The Drosophila phototransduction cascade is a G protein-coupled signaling cascade that utilizes a phospholipase C (PLC beta) effector. PLC beta has been shown to be activated by Gq alpha in reconstituted systems. To determine whether a Gq-like protein couples rhodopsin to PLC, and to study its function, a mutant defective in a photoreceptor-specific Gq protein, DGq, was isolated. Gq is demonstrated to be essential for the activation of the phototransduction cascade in vivo. Transgenic flies were generated expressing DGq under an inducible promoter and it is possible to manipulate the sensitivity of a photoreceptor cell by controlled expression of DGq. Characterization of quantum bumps in mutants expressing less that 1% of the levels of DGq reveals that the rhodopsin-G protein interaction does not determine the gain of the single photon responses. Together, these results provide significant insight into the role of Gq in regulating the output of a photoreceptor cell (Scott, 1995).

Interaction of NorpA with Inad

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 that 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 in three ways: 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).

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 contain the fourth PDZ domain: no indication of eye-PKC/fourth PDZ interaction was found. The constructs tested 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 have been shown to disrupt the eye-PKC binding. None of these amino acid changes are 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).

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