Multiple subtypes of phospholipase C are encoded by the norpA gene of Drosophila melanogaster.

Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA.

The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is essential for phototransduction. Besides being found abundantly in retina, norpA gene products are expressed in a variety of tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to phototransduction. We have identified a second subtype of norpA protein that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon that is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in adult retina, while subtype II transcripts are most abundant in adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, we conclude that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways.

PMID: 7540168 [PubMed - indexed for MEDLINE]

Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants.

Department of Biological Science, Purdue University, West Lafayette, Indiana 47907, USA.

Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.

PMID: 8617767 [PubMed - indexed for MEDLINE]

NorpA and itpr mutants reveal roles for phospholipase C and inositol (1,4,5)- trisphosphate receptor in Drosophila melanogaster renal function.

Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK.

Mutants of norpA, encoding phospholipase C beta (PLC beta), and itpr, encoding inositol (1,4,5)-trisphosphate receptor (IP(3)R), both attenuate response to diuretic peptides of Drosophila melanogaster renal (Malpighian) tubules. Intact tubules from norpA mutants severely reduced diuresis stimulated by the principal cell- and stellate cell-specific neuropeptides, CAP(2b) and Drosophila leucokinin (Drosokinin), respectively, suggesting a role for PLC beta in both these cell types. Measurement of IP(3) production in wild-type tubules and in Drosokinin-receptor-transfected S2 cells stimulated with CAP(2b) and Drosokinin, respectively, confirmed that both neuropeptides elevate IP(3) levels. In itpr hypomorphs, basal IP(3) levels are lower, although CAP(2b)-stimulated IP(3) levels are not significantly reduced compared with wild type. However, CAP(2b)-stimulated fluid transport is significantly reduced in itpr alleles. Rescue of the itpr(90B.0) allele with wild-type itpr restores CAP(2b)-stimulated fluid transport levels to wild type. Drosokinin-stimulated fluid transport is also reduced in homozygous and heteroallelic itpr mutants. Measurements of cytosolic calcium levels in intact tubules of wild-type and itpr mutants using targeted expression of the calcium reporter, aequorin, show that mutations in itpr attenuated both CAP(2b)- and Drosokinin-stimulated calcium responses. The reductions in calcium signals are associated with corresponding reductions in fluid transport rates. Thus, we describe a role for norpA and itpr in renal epithelia and show that both CAP(2b) and Drosokinin are PLC beta-dependent, IP(3)-mobilising neuropeptides in DROSOPHILA: IP(3)R contributes to the calcium signalling cascades initiated by these peptides in both principal and stellate cells.

PMID: 12547945 [PubMed - indexed for MEDLINE]

Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development.

Institut de Neurobiologie Alfred Fessard, CNRS UPR 2216 (NGI), 91198 Gif-sur-Yvette, France.

The visual system is one of the input pathways for light into the circadian clock of the Drosophila brain. In particular, extra-retinal visual structures have been proposed to play a role in both larval and adult circadian photoreception. We have analyzed the interactions between extra-retinal structures of the visual system and the clock neurons during brain development. We first show that the larval optic nerve, or Bolwig nerve, already contacts clock cells (the lateral neurons) in the embryonic brain. Analysis of visual system-defective genotypes showed that the absence of the afferent Bolwig nerve resulted in a severe reduction of the lateral neurons dendritic arborization, and that the inhibition of nerve activity induced alterations of the dendritic morphology. During wild-type development, the loss of a functional Bolwig nerve in the early pupa was also accompanied by remodeling of the arborization of the lateral neurons. Approximately 1.5 days later, visual fibers that came from the Hofbauer-Buchner eyelet, a putative photoreceptive organ for the adult circadian clock, were seen contacting the lateral neurons. Both types of extra-retinal photoreceptors expressed rhodopsins RH5 and RH6, as well as the norpA-encoded phospholipase C. These data strongly suggest a role for RH5 and RH6, as well as NORPA, signaling in both larval and adult extra-retinal circadian photoreception. The Hofbauer-Buchner eyelet therefore does not appear to account for the previously described norpA-independent light input to the adult clock. This supports the existence of yet uncharacterized photoreceptive structures in Drosophila.

PMID: 11880353 [PubMed - indexed for MEDLINE]

The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.

Zoological Institute I, University of Karlsruhe, Germany.

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, we find that Trp is 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 analyzed here is 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.

PMID: 9003779 [PubMed - indexed for MEDLINE]

PMCID: PMC452529

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

Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-6600, USA. shiehb@ctrvax.vanderbilt.edu

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.

PMID: 9356510 [PubMed - indexed for MEDLINE]

PMCID: PMC25084

Two distantly positioned PDZ domains mediate multivalent INAD-phospholipase C interactions essential for G protein-coupled signaling.

Departments of Physiology and Neuroscience, The Johns Hopkins University School of Medicine, 725 N.Wolfe Street, WBSB 216, Baltimore, MD 21205, USA.

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 mislocalization of signaling molecules of phototransduction which include the essential effector, phospholipase C-beta (PLC-beta), which is also known as NORPA. The molecular and biochemical details of this functional link are unknown. We report that 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). The NORPA proteins lacking binding sites, which display normal basal PLC activity, 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.

PMID: 9545241 [PubMed - indexed for MEDLINE]

PMCID: PMC1170572

Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster.

Department of Biological Sciences, State University of New York, Buffalo 14260, USA.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific phospholipase C (PLC), which enzymes play a pivotal role in one of the largest classes of signaling pathways known. A chimeric norpA minigene was constructed by placing the norpA cDNA behind an R1-6 photoreceptor cell-specific rhodopsin promoter. This minigene was transferred into norpAP24 mutant by P-element-mediated germline transformation to determine whether it could rescue the phototransduction defect concomitant with restoring PLC activity. Western blots of head homogenates stained with norpA antiserum show that norpA protein is restored in heads of transformed mutants. Moreover, transformants exhibit a large amount of measurable PLC activity in heads, whereas heads of norpAP24 mutant exhibit very little to none. Immunohistochemical staining of tissue sections using norpA antiserum confirm that expression of norpA protein in transformants localizes in the retina, more specifically in rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. Furthermore, electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in R1-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells. This is the strongest evidence thus far supporting the hypothesis that the norpA gene encodes phospholipase C that is utilized in phototransduction.

PMID: 7768926 [PubMed - indexed for MEDLINE]

The Drosophila dgq gene encodes a G alpha protein that mediates phototransduction.

Department of Biological Sciences, University of Notre Dame, Indiana 46556.

We examined the roles of the Drosophila Gq alpha proteins (DGq) in the phototransduction pathway. The DGq proteins immunolocalized to the ocelli and all eight retinular photoreceptor cell rhabdomeres. An affinity-purified anti-DGq alpha immunoglobulin blocked the light-dependent GTP hydrolysis activity associated with Drosophila head membranes in vitro, suggesting that rhodopsin stimulated DGq. Dominantly active DGq1 mutants exhibited a light-independent GTPase activity and abnormal electrophysiological light responses, such as reduced retinal sensitivity and slow response kinetics compared with wild-type flies. Dominant DGq2 mutants exhibited 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 degenerated even in the dark. DGq1 stimulation of rdgB retinal degeneration in the dark was 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.

PMID: 7946351 [PubMed - indexed for MEDLINE]

G protein control of Drosophila photoreceptor phospholipase C.

Howard Hughes Medical Institute, University of Washington, Seattle 98195, USA.

Light stimulates phosphatidylinositol bisphosphate phospholipase C (PLC) activity in Drosophila photoreceptors. We have investigated the mechanism of this reaction 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.

PMID: 7759511 [PubMed - indexed for MEDLINE]

The roles of PDZ-containing proteins in PLC-beta-mediated signaling.

Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang 790-784, South Korea. pgs@postech.ac.kr

Mammalian phospholipase C-beta isozymes are activated by a heterotrimeric GTP-binding protein linked to various cell surface receptors. Recent reports suggest that PDZ domain proteins play a significant role of PDZ-containing proteins in the regulation of mammalian PLC-beta isozymes. PDZ-containing proteins mediate the clustering of receptors and signaling molecules and thereby regulate agonist-induced signal transduction in polarized cells such as neuronal and epithelial cells. NORPA, a Drosophila PLC-beta, is known to be a component of a signaling complex that includes TRP and rhodopsin through interaction with INAD, a PDZ-containing protein. Mammalian PLC-beta1 and -beta2 isoforms interact with a PDZ-containing protein NHERF which is coupled to Trp4, a Ca(2+) channel. In addition, PLC-beta3 specifically interacts with E3KARP, another protein closely related to NHERF, through its C-terminal PDZ-binding motif. E3KARP up-regulates the PLC-beta3 activation coupled to muscarinic receptor. In this review, the role of signaling complexes mediated by PDZ-containing proteins in the regulation of PLC-beta isoforms will be discussed. Copyright 2001 Academic Press.

PMID: 11594744 [PubMed - indexed for MEDLINE]

Limited role of developmental programmed cell death pathways in Drosophila norpA retinal degeneration.

Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556-0369, USA.

We examined the role of programmed cell death (PCD) pathways in retinal degeneration caused by a mutation in the norpA gene. norpA degeneration shows morphological hallmarks of programmed cell death, specifically cytoplasmic condensation and engulfment of the dying photoreceptor cells by neighboring retinal pigment cells. However, genetic mosaic analysis of adult photoreceptors lacking rpr, hid, and grim show that these PCD inducers are not required for norpA degeneration. We showed previously that ectopic expression of either rpr or hid triggers rapid PCD in adult photoreceptors, and this is completely suppressed by the coexpression of the baculoviral P35 caspase inhibitor. In contrast, expression of P35 does not suppress norpA retinal degeneration, although a small delay in the rate of degeneration is observed in low light-low temperature conditions. P35 does not alter the morphological characteristics of norpA cell death. Overexpression of the Drosophila inhibitor of apoptosis Diap1 or a dominant-negative form of the Dronc caspase, even when coexpressed with P35, does not dramatically alter the time course of norpA degeneration. These results establish that the pathways responsible for PCD in development do not play a major role in adult retinal degeneration caused by norpA.

PMID: 14724249 [PubMed - indexed for MEDLINE]

Comment in:

Neuron. 2008 Jan 10;57(1):1-2.

The SOCS box protein STOPS is required for phototransduction through its effects on phospholipase C.

Department of Biological Chemistry, Center for Sensory Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Phosphoinositide-specific phospholipase C (PLC) isozymes play roles in a diversity of processes including Drosophila phototransduction. In fly photoreceptor cells, the PLCbeta encoded by norpA is critical for activation of TRP channels. Here, we describe a PLCbeta regulator, STOPS, which encodes a SOCS box protein. Mutation of stops resulted in a reduced concentration of NORPA and a defect in stopping signaling following cessation of the light stimulus. NORPA has been proposed to have dual roles as a PLC- and GTPase-activating protein (GAP). We found that the slow termination resulting from expressing low levels of wild-type NORPA was suppressed by addition of normal amounts of an altered NORPA, which had wild-type GAP activity, but no PLC activity. STOPS is the first protein identified that specifically regulates PLCbeta protein concentration. Moreover, this work demonstrates that a PLCbeta derivative that does not promote TRP channel activation, still contributes to signaling in vivo.

PMID: 18184564 [PubMed - indexed for MEDLINE]

PMCID: PMC2253723

Splicing of the period gene 3'-terminal intron is regulated by light, circadian clock factors, and phospholipase C.

Graduate Program in Biochemistry, Rutgers University Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.

The daily timing of circadian ( congruent with 24-h) controlled activity in many animals exhibits seasonal adjustments, responding to changes in photoperiod (day length) and temperature. In Drosophila melanogaster, splicing of an intron in the 3' untranslated region of the period (per) mRNA is enhanced at cold temperatures, leading to more rapid daily increases in per transcript levels and earlier "evening" activity. Here we show that daily fluctuations in the splicing of this intron (herein referred to as dmpi8) are regulated by the clock in a manner that depends on the photoperiod (day length) and temperature. Shortening the photoperiod enhances dmpi8 splicing and advances its cycle, whereas the amplitude of the clock-regulated daytime decline in splicing increases as temperatures rise. This suggests that at elevated temperatures the clock has a more pronounced role in maintaining low splicing during the day, a mechanism that likely minimizes the deleterious effects of daytime heat on the flies by favoring nocturnal activity during warm days. Light also has acute inhibitory effects, rapidly decreasing the proportion of dmpi8-spliced per transcript, a response that does not require a functional clock. Our results identify a novel nonphotic role for phospholipase C (no-receptor-potential-A [norpA]) in the temperature regulation of dmpi8 splicing.

PMID: 15060157 [PubMed - indexed for MEDLINE]

PMCID: PMC381688

Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.

PMID: 1662208 [PubMed - indexed for MEDLINE]

The rpa (receptor potential absent) visual mutant of the blowfly (Calliphora erythrocephala) is deficient in phospholipase C in the eye.

Department of Biological Sciences, State University of New York, Buffalo 14260.

The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the norpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, norpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila.

PMID: 7965386 [PubMed - indexed for MEDLINE]

The visual G protein of fly photoreceptors interacts with the PDZ domain assembled INAD signaling complex via direct binding of activated Galpha(q) to phospholipase cbeta.

Department of Cell, Institute of Zoology, University of Karlsruhe, D-76128 Karlsruhe, Germany.

Visual transduction in the compound eye of flies is a well-established model system for the study of G protein-coupled transduction pathways. Pivotal components of this signaling pathway, including the principal light-activated Ca(2+) channel transient receptor potential, an eye-specific protein kinase C, and the norpA-encoded phospholipase Cbeta, are assembled into a supramolecular signaling complex by the modular PDZ domain protein INAD. We have used immunoprecipitation assays to study the interaction of the heterotrimeric visual G protein with this INAD signaling complex. Light-activated Galpha(q)- guanosine 5'-O-(thiotriphosphate) and AlF(4)(-)-activated Galpha(q), but not Gbetagamma, form a stable complex with the INAD signaling complex. This interaction requires the presence of norpA-encoded phospholipase Cbeta, indicating that phospholipase Cbeta is the target of activated Galpha(q). Our data establish that the INAD signaling complex is a light-activated target of the phototransduction pathway, with Galpha(q) forming a molecular on-off switch that shuttles the visual signal from activated rhodopsin to INAD-linked phospholipase Cbeta.

PMID: 10644758 [PubMed - indexed for MEDLINE]

Specific molecular alterations in the norpA-encoded phospholipase C of Drosophila and their effects on electrophysiological responses in vivo.

Graduate School of Biotechnology, KyungHee University, Yongin City, Kyungki-Do, Korea.

A large number of mutants in the norpA gene, which encodes the phospholipase C (PLC) involved in Drosophila phototransduction, is available for the investigation of the effects of specific amino acid substitutions in PLC on biochemical and electrophysiological properties of these mutants. Of the 47 norpA mutants screened for PLC protein content, all but one (H43) displayed drastically decreased amounts of the protein suggesting that almost any mutational alteration has a deleterious effect on the integrity of the protein. Three new amino acids were identified in the catalytic domains X and Y that are important for PLC catalytic activity and the generation of photoreceptor responses (ERG). One of them was found substituted in H43, which showed a low specific PLC activity, a pronounced decrease in ERG sensitivity, and a wild-type-like response termination time. The response termination times obtained from three mutants was found to be approximately inversely proportional to the amount of PLC. In addition, we show that (i) the specific PLC activity is a key factor determining the photoreceptor sensitivity; (ii) the catalytic activity and response termination are separable functions of PLC; and (iii) a mutation in the putative G alpha-interacting C2 domain causes a preferentially strong defect in latency.

PMID: 15140198 [PubMed - indexed for MEDLINE]

Functional relevance of the disulfide-linked complex of the N-terminal PDZ domain of InaD with NorpA.

Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

In Drosophila, phototransduction is mediated by G(q)-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 require the multi-domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospho lipase C-beta isozyme. We have determined the crystal structure of the N-terminal PDZ domain of InaD bound to a peptide corresponding to the C-terminus of NorpA to 1.8 A 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.

PMID: 11500369 [PubMed - indexed for MEDLINE]

PMCID: PMC125561

Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C.

Department of Genetics, University of Leicester, Leicester LE1 7RH, United Kingdom.

Drosophila melanogaster locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3' intron in the period mRNA transcript. Here we demonstrate that the control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, we observed that a mutation in the no-receptor-potential-A(P41) (norpA(P41)) gene, which encodes phospholipase-C, generated an extremely high level of 3' splicing. This cannot be explained simply by the mutation's effect on the visual pathway and suggests that norpA(P41) is directly involved in thermosensitivity.

PMID: 14766972 [PubMed - indexed for MEDLINE]

PMCID: PMC357032