The phototransduction messenger, cGMP, mediates rod and cone response to light. The synthesis of cGMP by retinal guanylyl (guanylate) cyclase (RetGC), controlled by calcium through guanylyl cyclase activating proteins (GCAPs), is one of the most critical steps in photoresponse recovery. The defects in its regulation cause multiple forms of congenital human blindness. While the general importance and basic principles of the RetGC regulation have been established, some of the key mechanistic aspects remain poorly understood, especially how protein-protein interactions in GCAP and RetGC result in the cyclase activation and inhibition or triggering retinal diseases. Seeking answers to the questions addressed in this proposal conforms to the NEI mission to """"""""conduct research and disseminate information with respect to blinding eye diseases, mechanisms of visual function and preservation of sight"""""""". This proposal is based on characterization of new RetGC mutations causing Leber congenital amaurosis (LCA) and novel findings about the RetGC regulation: 1) that GCAP1 desensitized by disease-causing mutations preferentially targets RetGC1 isozyme in vivo;2) that GCAP1 acts as the 'first-response'Ca2+ sensor activating RetGC1 early in photoresponse;3) that N-fatty acylation in GCAP1 affects its function as a calcium sensor for RetGC1 via an intramolecular 'tug'action;4) that two regions in GCAP1 molecule emerge as a likely cyclase-binding interface;5) that a photoreceptor protein, RD3, acts as a potent inhibitor of RetGC activity, but fails to inhibit the cyclase when affected by LCA-related mutations. We propose a broad integrated approach to verify new hypotheses and delineate mechanisms in RetGC/GCAP regulatory pathways using a combination of protein biochemistry, molecular biology, and molecular genetics.
Aim 1 will address the molecular structure of GCAP1 with the emphasis on establishing the key to the conformational transition of GCAP1 into its RetGC activator state, elucidating the protein architecture for the disease-causing constitutive active GCAP1 mutants, and testing a hypothesis of """"""""Ca2+-myristoyl tug"""""""" - across-the-molecule action of the N-fatty acyl group that controls Ca2+ sensitivity of GCAP1.
Aim 2 will investigate how the molecular mechanisms of RetGC catalytic activity and regulation become altered by newly characterized mutations causing LCA1 blindness.
Aim 3 will seek understanding of biological role of RD3 as a novel, linked to LCA and cone-rod degeneration, negative regulator of the RetGC/GCAP pathway. By completing these specific aims, we expect to overcome some critical barriers in understanding of RetGC regulation and its role in normal photoreceptor physiology and in retinal diseases.
Mutations altering cGMP synthesis in photoreceptors cause severe congenital blinding diseases including cone dystrophy, cone-rod dystrophy (CORD), and Leber's congenital amaurosis (LCA). In this application we seek better understanding of the molecular mechanisms underlying regulation of cGMP synthesis in rods and cones and expect that this new knowledge will help better design strategies for treatment of such diseases.
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