The death of retinal photoreceptors as a result of genetic mutations or environmental abuse leads to irreversible loss of vision. X-linked retinitis pigmentosa (XLRP) is perhaps the most devastating of such diseases because of relative severity of phenotype and an early age of onset. Although several genetic loci have been mapped, a majority of XLRP can be accounted by RP3 and RP2 subtypes; the corresponding genes for the two loci, RPGR and RP2, respectively, have recently been isolated. RPGR and RP2 mutations have so far been identified in <50 percent of North American XLRP families. The RPGR protein shows homology to RCC1 (a guanine nucleotide exchange factor for Ran-GTPase), demonstrates a complex isoform profile because of alternative splicing, and is suggested to be involved in intracellular transport. The RP2 protein has similarity to cofactor C, which is a GTPase-activating protein involved in tubulin processing. The functions of RPGR and RP2 proteins in the retina are unknown. The mechanisms of photoreceptor degeneration caused by mutations in RPGR or RP2 are not understood. No treatment or cure is currently available for XLRP. This competing renewal application requests support to continue our decade-long investigations on the pathogenesis of XLRP. We hypothesize that RPGR and RP2 participate in intracellular transport and/or signaling pathways and that their functions are critical in the photoreceptors or retinal pigment epithelium (RPE).
The specific aims of the project are: (1) To determine the expression and subcellular localization of RPGR and RP2 proteins in the developing and mature retina by using a battery of well-characterized polyclonal and monoclonal antibodies. (2) To identify proteins that interact and form complexes with RPGR and RP2 in the retina and develop biochemical assays to assess their function. We are using the yeast two-hybrid strategy to initially identify interacting proteins. The specificity of interaction will be further evaluated by GST-pull down and co-immunoprecipitation assays. Immuno-affinity chromatography will be used to purify RPGR- and RP2-containing complexes from bovine retina. Studies will be initiated to examine the biochemical activity of RPGR and RP2 and their interacting proteins. (3) To create conditional null mutations of mouse Rpgr and Rp2in retinal photoreceptors and RPE and investigate the pathogenesis of disease. Cre-lox technology will be utilized to achieve this goal. The proposed studies seek to unravel the biological functions of RPGR and RP2 in the retina with a goal of understanding how mutations in these two cause retinal degeneration. These investigations promise to provide significant insights into the pathogenesis of XLRP and may lead to novel targets for gene-based therapy.
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