X-linked retinitis pigmentosa (XLRP) is a relatively severe retinal degeneration with six distinct mapped loci; nonetheless, mutations in the RPGR gene are detected in 70-80% of XLRP and 25% of simplex RP males, making it the most common single cause of inherited retinal degeneration (15-20% of RP). All human mutations are detected in the RPGRORF15 isoforms that primarily localize to ciliary axoneme, basal body and centrosome. RPGR interacts with several ciliary and microtubule transport proteins. The reported flpgr-knockout mouse is not a complete null, and only limited analysis has been performed for two canine RPGR mutants. Precise biochemical functions of RPGR are not yet understood, and mechanisms underlying the disease pathogenesis have not been elucidated. ? ? The connecting cilium of the photoreceptor serves as a conduit for bidirectional transport of macromolecular complexes using the microtubule network. Continuous turnover of photoreceptor outer segments demands efficient functioning of the ciliary transport process. We hypothesize that RPGR facilitates the assembly of transport protein complexes by interacting with distinct ciliary axoneme - basal body - centrosome (CABC) proteins, and that RPGR's localization in the photoreceptor cilia is necessary for efficient inter-segmental transport. Hence, the primary goals of this project are to define the RPGR interaction network as it pertains to ciliary transport in the retina and determine in vivo relevance of selected interactions.
Aim 1 will identify direct interactions of RPGRORF15 with candidate CABC proteins, characterize the interacting domains, and examine the effects of selected disease-causing RPGR mutations.
In Aim 2, we will evaluate in vivo effects of RPGR mutations on its interactions and assembly of protein complexes, by comprehensive analysis of three different Rpgr mouse mutants and two canine models.
Aim 3 will examine mouse mutants of selected RPGR-interacting proteins for RPGR localization and assembly/stability of RPGR-complexes.
In Aim 4, we will purify RPGR-complexes from bovine retinal ciliary fraction and determine constituent proteins by mass spectrometry-based methods. A particular focus will be on uncovering GTP-binding proteins. ? ? The proposed studies (years 19-23) are expected to provide significant mechanistic insights into RPGR function in the retina and clues to pathogenic mechanisms. Elucidation of binary interactions and complexes involved in microtubule transport network should have broader implications for syndromic ciliopathies that include retinal degeneration phenotype. Our investigations should also allow better design of therapies for XLRP and other human diseases involving ciliary dysfunction. ? ? ?
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