Reinhard Paulsen

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.

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.

Light-regulated subcellular translocation of Drosophila TRPL channels induces long-term adaptation and modifies the light-induced current

Drosophila phototransduction results in the opening of two classes of cation channels, composed of the channel subunits transient receptor potential (TRP), TRP-like (TRPL), and TRPgamma. Here, we report that one of these subunits, TRPL, is translocated back and forth between the signaling membrane and an intracellular compartment by a light-regulated mechanism. A high level of rhabdomeral TRPL, characteristic of dark-raised flies, is functionally manifested in the properties of the light-induced current. These flies are more sensitive than flies with no or reduced TRPL level to dim background lights, and they respond to a wider range of light intensities, which fit them to function better in darkness or dim background illumination. Thus, TRPL translocation represents a novel mechanism to fine tune visual responses.

Phosphorylation of the InaD Gene Product, a Photoreceptor Membrane Protein Required for Recovery of Visual Excitation

In an approach directed to isolate and characterize key proteins of the transduction cascade in photoreceptors using the phosphoinositide signaling pathway, we have isolated the Calliphora homolog of the Drosophila InaD gene product, which in Drosophila InaD mutants causes slow deactivation of the light response. By screening a retinal cDNA library with antibodies directed against photoreceptor membrane proteins, we have isolated a cDNA coding for an amino acid sequence of 665 residues (M = 73,349). 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 revealed 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. As 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 revealed that the InaD protein is a phosphoprotein. In vitro phosphorylation is, at least to some extent, Ca-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 Ca channel, and the norpA-encoded phospholipase C, the primary target enzyme of the transduction pathway. It is proposed that the rise of the intracellular Ca 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.

The TRP Ca2+ channel assembled in a signaling complex by the PDZ domain protein INAD is phosphorylated through the interaction with protein kinase C (ePKC)

Photoreceptors which use a phospholipase C‐mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cβ (PLCβ), the light‐activated Ca2+ channel TRP, and an eye‐specific protein kinase C (ePKC) are clustered by the PDZ domain protein INAD. Here we investigated the function of ePKC 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 (MW=78 036) and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti‐ePKC antibodies led to the co‐precipitation of PLCβ, TRP, INAD and ePKC but not of rhodopsin. Phorbolester‐ and Ca2+‐dependent protein phosphorylation revealed 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 which regulates Ca2+ influx through the TRP channel.

Molecular cloning of Drosophila Rh6 rhodopsin: the visual pigment of a subset of R8 photoreceptor cells 1

By screening retinal cDNA libraries for photoreceptor‐specifically expressed genes we have isolated and sequenced a cDNA clone encoding the rhodopsin (Rh6) of a subset of R8 photoreceptor cells of the Drosophila compound eye. Compared to the other visual pigments of Drosophila, this rhodopsin is equally homologous to Rh1 and Rh2 (51% amino acid identity) but shows only 32% and 33% amino acid identity with Rh3 and Rh4, respectively. The open reading frame codes for a protein of 369 amino acids (MW=41 691). The primary structure of Rh6 displays sites typical for rhodopsin molecules in general, for example, a chromophore binding site in transmembrane domain VII, sequence motifs in the intracellular loops 2 and 3 required for the binding of a heterotrimeric G‐protein, and a glycosylation site near the N‐terminus which seems to be important for protein transport and maturation. Since R8 cells are founder cells in the developing compound eye, the isolation of a rhodopsin gene expressed in these cells may aid the understanding of terminal differentiation of photoreceptor cells.

The Visual G Protein of Fly Photoreceptors Interacts with the PDZ Domain Assembled INAD Signaling Complex via Direct Binding of Activated Gαq to Phospholipase Cβ

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 Ca2+ channel transient receptor potential, an eye-specific protein kinase C, and thenorpA-encoded phospholipase Cβ, 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 Gαq- guanosine 5′-O-(thiotriphosphate) andAlF 4 − -activated Gαq, but not Gβγ, form a stable complex with the INAD signaling complex. This interaction requires the presence ofnorpA-encoded phospholipase Cβ, indicating that phospholipase Cβ is the target of activated Gαq. Our data establish that the INAD signaling complex is a light-activated target of the phototransduction pathway, with Gαq forming a molecular on-off switch that shuttles the visual signal from activated rhodopsin to INAD-linked phospholipase Cβ.

Site-directed mutagenesis of highly conserved amino acids in the first cytoplasmic loop of Drosophila Rh1 opsin blocks rhodopsin synthesis in the nascent state.

The cytoplasmic surface of Drosophila melanogaster Rh1 rhodopsin (ninaE) harbours amino acids which are highly conserved among G‐protein‐coupled receptors. Site‐directed mutations which cause Leu81Gln or Asn86Ile amino acid substitutions in the first cytoplasmic loop of the Rh1 opsin protein, are shown to block rhodopsin synthesis in the nascent, glycosylated state from which the mutant opsin is degraded rapidly. In mutants Leu81Gln and Asn86Ile, only 20–30% and <2% respectively, of functional rhodopsins are synthesized and transported to the photoreceptive membrane. Thus, conserved amino acids in opsins cytoplasmic surface are a critical factor in the interaction of opsin with proteins of the rhodopsin processing machinery. Photoreceptor cells expressing mutant rhodopsins undergo age‐dependent degeneration in a recessive manner.

A Novel Gγ Isolated from Drosophila Constitutes a Visual G Protein γ Subunit of the Fly Compound Eye

Visual transduction in the compound eye of flies is a well established model system for the study of G protein-coupled transduction pathways. To characterize key components of the phototransduction cascade we performed substractive hybridization screening. We cloned the cDNA coding for the visual Gγ (Gγe) subunit from Drosophila which had so far eluded identification at the molecular level. Northern blot analysis revealed the presence of a major, 1.4-kilobase(kb) Gγe transcript and two minor transcripts of 1.8 and 6 kb in size. The major 1.4-kb mRNA is expressed preferentially in the eye. The spatial expression pattern determined for Gγe as well as co-immunoprecipitation experiments demonstrated that Gγe dimerizes with Gβe to form the heterodimeric Gβγ subunit which functions in visual transduction in the Drosophila compound eye. Gγe shares common characteristics with the visual Gγ subunits of human rod and cone photoreceptors although different classes of Gα subunits are employed in vertebrate and invertebrate phototransduction. By the molecular cloning and characterization of the visual γ subunit ofDrosophila one of the few missing links in the well studiedDrosophila phototransduction cascade has been characterized to complete our knowledge about the Drosophila visual transduction pathway.

Rhodopsin patterning in central photoreceptor cells of the blowfly Calliphora vicina: cloning and characterization of Calliphora rhodopsins Rh3, Rh5 and Rh6

SUMMARY The ommatidia that constitute the compound eyes of flies contain eight photoreceptor cells, which are divided into two classes: the peripheral photoreceptors, R1–6, and the central photoreceptors, R7 and R8. In the fruit fly, Drosophila, R1–6 express the same rhodopsin (Rh1), whilst the R7 and R8 of a given ommatidium express either Rh3 and Rh5, or Rh4 and Rh6, respectively. We have studied whether this expression pattern of rhodopsins is conserved in the blowfly Calliphora vicina. We have cloned three novel Calliphora rhodopsins, which are homologues of Drosophila Rh3, Rh5 and Rh6, with an amino acid sequence identity of 80.7%, 60.9% and 86.1%, respectively. Immunocytochemical studies with antibodies specific for Rh3, Rh5 and Rh6 revealed that Rh3 is expressed in a subset of R7 cells, while Rh5 and Rh6 are expressed in a non-overlapping subset of R8 cells. Rh3 and Rh5 are present in most cases in the same ommatidia, which account for approximately 27% of all ommatidia, and Rh6 is found in the complementary 73%. The similarity of the rhodopsin expression pattern of Calliphora with that of Drosophila suggests that the developmental mechanism regulating the terminal differentiation of R7 and R8 cells are highly conserved between these fly species.

The PDZ scaffold protein INAD abolishes apparent store-dependent regulation of the light-activated cation channel TRP

In fly photoreceptor cells, light initiates a G protein‐coupled phospholipase Cβ‐dependent signaling cascade that results in the depolarization of the cell membrane, which is mediated by the cation channels TRP and TRPL. Together with phospholipase Cβ and an eye‐specific protein kinase C, TRP is tethered to the scaffolding protein INAD, which forms a multimolecular signaling complex. Divergent data from expressed TRP and studies from photoreceptor cells have brought up a controversy whether or not a capacitative calcium entry (CCE) mechanism is involved in the Drosophila phototransduction pathway. Our initial characterization of TRP from photoreceptors of Calliphora vicina supported the hypothesis of a CCE mechanism, as heterologously expressed TRP was stimulated after application of thapsigargin. The situation changed when the PDZ domain protein INAD was coexpressed with TRP. In cells coexpressing TRP and INAD, no calcium entry was detectable on application of store depletion protocols. Suppression of CCE by INAD was not observed when the described interaction was disrupted by mutations in TRP and INAD. Our data show that apparent activation of TRP by CCE is abolished by INAD. Within the complex, the proteins necessary for phototransduction mutually influence their activities. The results support the hypothesis of a store‐independent activation of TRP.