UV-light-dependent binding of a visual arrestin 1 isoform to photoreceptor membranes in a neuropteran (Ascalaphus) compound eye.

Arrestins are regulators of the active state of G-protein-coupled receptors. Towards elucidating the function of different arrestin subfamilies in sensory cells, we have isolated a novel arrestin 1, Am Arr1, from the UV photoreceptors of the neuropteran Ascalaphus macaronius. Am Arr1 forms a phylogenetic clade with antennal and visual Arr1 isoforms of invertebrates. Am Arr1 undergoes a light-dependent binding cycle to photoreceptor membranes, as reported earlier only for members of the arrestin 2 subfamily. This suggests a common control mechanism for the active state of invertebrate rhodopsins and G-protein-coupled receptors of antennal sensory cells. Furthermore, it implies that a strict correlation of distinct arrestin isoforms to distinct functions is not a general principle for invertebrate sensory cells.

Patterning of the R7 and R8 photoreceptor cells of Drosophila: evidence for induced and default cell-fate specification.

Opsin gene expression in the R7 and R8 photoreceptor cells of the Drosophila compound eye is highly coordinated. We have found that the R8 cell specific Rh5 and Rh6 opsins are expressed in non-overlapping sets of R8 cells, in a precise pairwise fashion with Rh3 and Rh4 in the R7 cells of individual ommatidia. Removal of the R7 cells in sevenless, boss or sina mutants, disrupts Rh5 expression and dramatically increases the number of Rh6-expressing R8 cells. This suggests that the expression of Rh5 may be induced by an Rh3-expressing R7 cell, whereas Rh6 expression is most likely a default state of the R8 cell. We found that the paired expression of opsin genes in the R7 and R8 cells occurs in a sevenless and boss independent manner. Furthermore, we found that the generation of both Rh3- and Rh4-expressing R7 cells can occur in the absence of an R8 cell. These results suggest that the specification of opsin expression in the R7 cells may occur autonomously, whereas the R7 photoreceptor cell may be responsible for regulating a binary developmental switch between induced and default cell-fates in the R8 cell.

Rhodopsin mutations as the cause of retinal degeneration. Classification of degeneration phenotypes in the model system Drosophila melanogaster.

Insight into the molecular basis of inherited photoreceptor cell degeneration has been rapidly evolving during the last decade. The Drosophila Rh1 rhodopsin gene was the first gene shown to cause retinal degeneration when mutated. Many more degeneration-causing mutations in genes encoding rhodopsin and other photoreceptor proteins have been isolated since then in both, Drosophila and humans. To date some 70 mutations of the Drosophila Rh1 gene have been isolated, most of them have been characterized at the molecular level, and more than 60% of them cause retinal degeneration. This review lists the known Rh1 mutations that cause retinal degeneration up to April 1998, gives an overview on the ultrastructural and biochemical correlates of photoreceptor cell degeneration, and suggests a system for the classification of degeneration-causing Rh1 mutations.

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

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 = 41691). 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.

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 opsin's 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.

An arrestin homolog of blowfly photoreceptors stimulates visual-pigment phosphorylation by activating a membrane-associated protein kinase.

An arrestin homolog (Arr2, 49-kDa protein) of blowfly (Calliphora erythrocephala) retinae undergoes light-dependent reversible binding to the photoreceptor membrane. In order to characterize this arrestin homolog and to study its function in a well-defined experimental system, we developed a purification scheme which used microvillar photoreceptor membranes as an affinity binding matrix. Additional purification steps included ammonium sulfate precipitation, gel filtration and binding to heparin-agarose. The molecular mass of purified Arr2, as judged by SDS/PAGE, is in the range 45-49 kDa. The isoelectric point, as judged by gel isoelectric focussing, is 8.7. Arr2 is specific to the retina, where it is subject to phosphorylation at multiple sites. Binding of purified Arr2 to isolated photoreceptor membranes efficiently activates the light-induced phosphorylation of visual pigment. Since the assay system used is deficient in rhodopsin phosphatase activity, the arrestin-stimulated phosphate incorporation into rhodopsin results solely from the activation of a protein kinase. Phosphorylation experiments with highly purified membrane preparations indicate that rhodopsin kinase is tightly associated with the rhabdomeric membrane or the microvillar cytoskeleton. Rhodopsin kinase is released from the membrane or inactivated upon treatment with urea. It is concluded that this arrestin is a regulator protein that controls visual-pigment phosphorylation by affecting the interaction of metarhodopsin and rhodopsin (metarhodopsin) kinase.

Light-modulated ADP-ribosylation, protein phosphorylation and protein binding in isolated fly photoreceptor membranes.

Rhodopsin (P, lambda max 480 nm) of blowfly photoreceptors R1-6 is converted by light into a thermally stable metarhodopsin (M, lambda max 565 nm). In isolated blowfly rhabdoms photoconversion of P to M affects bacterial toxin-catalyzed ADP-ribosylation of a 41-kDa protein, activates phosphorylation of opsin and induces the binding of a 48-kDa phosphoprotein to the rhabdomeric membrane. ADP-ribosylation of the 41-kDa protein is catalyzed by cholera toxin and is inhibited by P----M conversion. The 41-kDa protein might represent the alpha-subunit of the G-protein, proposed to be part of the phototransduction mechanism [Blumenfeld, A. et al. (1985) Proc. Natl Acad. Sci. USA 82, 7116-7120]. P----M conversion leads to phosphorylation of opsin at multiple binding sites: up to 4 mol phosphate are bound/mol M formed. Dephosphorylation of the phosphate binding sites is induced by photoconversion of M to P. High levels of calcium (2 mM) inhibit phosphorylation of M and increase dephosphorylation of P. Protein patterns obtained by sodium dodecyl sulfate gel electrophoresis of irradiated retina membranes show an increased incorporation of label from [gamma-32P]ATP also into protein bands of 48 kDa, 68 kDa and 200 kDa. Binding studies reveal that in the case of the 48-kDa protein this effect is primarily due to a light-induced binding of the protein to the photoreceptor membrane. The binding of the 48-kDa phosphoprotein is reversible: after M----P conversion the protein becomes extractable by isotonic buffers. These data suggest that in rhabdomeric photoreceptors of invertebrates light-activation of rhodopsin is coupled to an enzyme cascade in a similar way as in the ciliary photoreceptors of vertebrates, although there may be differences, e.g. in the type of G-protein which mediates between the activated state of metarhodopsin and a signal-amplifying enzyme reaction.

Activation of rhodopsin phosphorylation is triggered by the lumirhodopsin-metarhodopsin I transition.

The absorption of light by the chromophore of rhodopsin initiates a series of interconversions between spectrally distinct intermediates. The possibility has been raised that one of these transitions is accompanied by a change in the state of rhodopsin, and that it is this change which instigates visual excitation via a cascade of enzyme catalysed reactions. It has been suggested that the initial step of this cascade, which leads to the activation of cyclic GMP phosphodiesterase (PDE), involves the interaction of a GTP-binding regulatory (G) protein with rhodopsin. The ability of rhodopsin to activate PDE may be inhibited by the phosphorylation of sites exposed on the opsin surface as a result of light-induced conformational changes. To obtain more information about the relationship between the postchemical and biochemical reactions of rhodopsin we have investigated which transition leads to the activation of rhodopsin as a substrate for rhodopsin kinase, and report here that it is the transition from lumirhodopsin to metarhodopsin I.