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A variety of rod opsin mutations result in autosomal dominant retinitis pigmentosa and congenital night blindness in humans. One subset of these mutations encodes constitutively active forms of the rod opsin protein. Some of these dominant rod opsin mutant proteins, which desensitize transgenic Xenopus rods, provide an animal model for congenital night blindness. In a genetic screen to identify retinal degeneration mutants in Drosophila, we identified a dominant mutation in the ninaE gene (NinaEee100) that encodes the rhodopsin that is expressed in photoreceptors R1-R6. Deep pseudopupil analysis and histology showed that the degeneration was attributable to a light-independent apoptosis. Whole-cell recordings revealed that the NinaEpp100 mutant photoreceptor cells were strongly desensitized, which partially masked their constitutive activity. This desensitization primarily resulted from both the persistent binding of arrestin (ARR2) to the NINAEpp100 mutant opsin and the constitutive activity of the phototransduction cascade. Whereas mutations in several Drosophila genes other than ninaE were shown to induce photoreceptor cell apoptosis by stabilizing a rhodopsinarrestin complex, NinaEpp100 represented the first rhodopsin mutation that stabilized this protein complex. Additionally, the NinaEpp100 mutation led to elevated levels of Gqα in the cytosol, which mediated a novel retinal degeneration pathway. Eliminating both Gqα and arrestin completely rescued the NinaE pp100-dependent photoreceptor cell death, which indicated that the degeneration is entirely dependent on both Gqα and arrestin. Such a combination of multiple pathological pathways resulting from a single mutation may underlie several dominant retinal diseases in humans. [ABSTRACT FROM AUTHOR]

Bacterial GTPase-activating proteins (GAPs) subvert their host''s eukaryotic Rho GTPases to their own advantage. Studies of bacterial GAPs extend our understanding of the action of eukaryotic GAPs, provide new tools for studies of cytoskeletal dynamics and offer new targets for anti-bacterial drugs. [Copyright &y& Elsevier]

Comparisons of different protein structures are commonly carried out by superimposing the coordinates of the protein backbones or selected parts of the proteins. When the objective is analysis of similarities and differences in the enzyme''s active site, there is an inherent problem in using the same domains for the superimposition. In this work we use a comparative approach termed here “Substrate Directed SuperImposition” (SDSI). It entails the superimposition of multiple protein–substrate structures using exclusively the coordinates of the comparable substrates. SDSI has the advantage of unbiased comparison of the active-site environment from the substrate''s point of view. Our analysis extends previous usage of similar approaches to comparison of enzyme catalytic machineries.We applied SDSI to various G-protein structures for dissecting the mechanism of the GTPase reaction that controls the signaling activity of this important family. SDSI indicates that dissimilar G-proteins stabilize the transition state of the GTPase reaction similarly and supports the commonality of the critical step in this reaction, the reorientation of the critical arginine and glutamine. Additionally, we ascribe the catalytic inefficiency of the small G-protein Ras to the great flexibility of its active site and downplay the possible catalytic roles of the Lys16 residue in Ras GTPase. SDSI demonstrated that in contrast to all other Gly12 Ras mutants, which are oncogenic, the Gly12→Pro mutant does not interfere with the catalytic orientation of the critical glutamine. This suggests why this mutant has a higher rate of GTP hydrolysis and is non-transforming. Remarkably, SDSI also revealed similarities in the divergent catalytic machineries of G-proteins and UMP/CMP kinase. Taken together, our results promote the use of SDSI to compare the catalytic machineries of both similar and different classes of enzymes. [Copyright &y& Elsevier]

Heterotrimeric G-proteins relay signals between membrane-bound receptors and downstream effectors. Little is known, however, about the regulation of Gα subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that tight causes massive and reversible translocation of the visual Gqα to the cytosot, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqα affect these processes. Epistatic analysis showed that Gqα is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqβ is essential for targeting of Gqα to the membrane and suggested that Gqβ is also needed for efficient activation of Gqα by rhodopsin. Our results support the 'two-signal model' hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity-dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors. [ABSTRACT FROM AUTHOR]

Rhodopsin kinase activity of Musca domestica was characterized in a reconstitution assay, using urea-treated eye membranes as substrate and a purified fraction of eye cytosol as the enzyme. Analysis of kinase activity in fly eye, brain and abdomen extracts by reconstitution assays revealed that fly rhodopsin kinase is an eye-specific enzyme. It preferentially phosphorylates the light-activated form of rhodopsin (metarhodopsin) and has little activity with other protein substrates. Rhodopsin kinase binds to metarhodopsin and is released from rhodopsin-containing membranes. Metarhodopsin is a poor substrate for kinases from tissues other than the eye, making it a unique substrate for rhodopsin kinase. Rhodopsin kinase is inhibited by heparin, but not by the protein inhibitor of cAMP-dependent protein kinase. Its Km for ATP is 9 μM. Since fly rhodopsin is coupled to phospholipase C, studies of the interaction of rhodopsin with rhodopsin kinase can be useful in analysis of the reactions that lead to termination of the inositol-phospholipid-signaling pathway. [ABSTRACT FROM AUTHOR]

The total sulfhydryl content of sarcoplasmic reticulum determined in the presence of sodium dodecylsulfate is 14 sulfhydryl equivalents per 105 g of membrane protein, but in the absence of sodium dodecylsutfate only 10 sulfhydryl groups are titrateable with 5,5'-dithiobis (2-nitrobenzoic acid). The rate of ATPase inactivation by N-ethylmaleimide in the presence of ATP yields an ATP-ATPase dissociation constant of 45 µM. When the sulfhydryl group essential for ATPase activity was reacted with N-ethyl-[1-14C]maleimide after all the sulfhydryl groups unrelated to ATPase activity had been reacted previously with unlabeled reagent, only one protein band in electrophorograms was strongly labeled. The presence of ATP during trypsin proteolysis preserves ATPase activity and the band that can be specifically labeled with N-ethylmaleimide is demonstrated in gel electrophorograms. When trypsin proteolysis was carried out in the absence of ATP this band can no longer be seen. The N-ethyl-[1-14C]maleimide labeledband is tentatively identified with the ATPase. [ABSTRACT FROM AUTHOR]