The visual pigment rhodopsin is both a G protein-coupled receptor (GPCR) and a critical structural component of the outer segment of photoreceptor cells. Mutations in rhodopsin are the most common cause of autosomal dominant retinitis pigmentosa (RP), a blinding disease that afflicts more than 1.5 million individuals world- wide. Rhodopsin can activate the visual signaling cascade as a monomer, but it self-associates to form dimers and higher order multimers that have been proposed to be important for phototransduction. We discovered that certain rhodopsin mutants that cause RP through unknown mechanisms appear to be function normally as visual pigments but unlike wild type rhodopsin, they fail to dimerize when reconstituted into lipid vesicles. We hypothesize that the lack of dimerization in these mutants prevents normal photoreceptor function, leading eventually to loss of photoreceptor cells, retinal degeneration and RP pathology. The overall goal of this application is to elucidate the biological and pathobiological aspects of photoreceptor functions that rely on rhodopsin dimerization. The RP mutants we propose to analyze provide novel tools and an exciting new opportunity to study the molecular basis of rhodopsin dimerization and the specific role of dimerization in phototransduction and maintaining disc architecture. In our two specific aims, we propose to conduct a comprehensive analysis of rhodopsin dimerization from its molecular basis (and why it fails in RP mutants) to its biological role in living photoreceptors. In the first aim we will use multiscale computational approaches, combined with experimental probing and verification, to test specific hypotheses about the driving forces for rhodopsin dimerization, why dimerization is perturbed in the RP-associated rhodopsin mutants, whether heterodimerization between mutant and wildtype protein occurs and whether we can identify compensatory mutations that restore dimerization. In iterative fashion, predictions from analyses in silico will be tested in direct biochemical assays and the results will serve to refine models and computational investigations. In the second aim we will comprehensively characterize two mouse strains that homozygously express non-dimerizing rhodopsin mutants, one each corresponding to point mutations in TM1 (F45L) and TM5 (F220C) to reflect the different dimer interfaces that are directly implicated. Our goal is to conduct a ?360 degree? analysis of the heterozygous and homozygous knock-in mouse phenotypes by examining photoreceptor morphology, analyzing intracellular targeting of outer segment- resident proteins, biochemical characterization of phototransduction, and electrophysiological assessment of light responses produced by the mutant rods.
Mutations in rhodopsin are the most common cause of autosomal dominant retinitis pigmentosa (RP), a blinding disease that afflicts more than 1.5 million individuals world-wide. We discovered that some RP- associated rhodopsin mutants fail to self-associate and we will test the hypothesis that this deficit prevents normal photoreceptor function, leading eventually to loss of photoreceptor cells, retinal degeneration and RP pathology. The overall goal of this application is to elucidate the mechanism of rhodopsin self-association and to understand the biological and pathobiological aspects of photoreceptor functions that rely on it.
|Pandey, Kalpana; Ploier, Birgit; Goren, Michael A et al. (2017) An engineered opsin monomer scrambles phospholipids. Sci Rep 7:16741|