The purpose of the proposed study is to define new processes that regulate phototransduction and adaptation in visual receptors. The long-term goal is to determine the molecular mechanisms that regulate synthesis of cyclic GMP, the second messenger of phototransduction, by retinal membrane guanylyl cyclases. The proposal is based on the findings that photoreceptor-specific calcium binding proteins, GCAPs, are essential for stimulation of cGMP synthesis in response to the change in the intracellular free Ca2+ concentrations during recovery and light adaptation of photoreceptors. Recent evidence demonstrate that the ability of GCAPs to stimulate the cyclase is determined by their Ca2+ binding domains and three other fragments of their primary structure. Therefore, the first aim of this study is to identify by using directed mutagenesis the specific amino acid residues in GCAPs that: constitute a switch between the GCAP activator and inhibitor states and determine the ability of GCAPs to activate the cyclase. GCAPs act via the cyclase intracellular domain, and there are recent evidence that dimerization of GCAPs control the interaction between the cyclase subunits. Therefore, the second aim of this proposal is to determine the structural changes in the cyclase upon its activation by GCAP and to reveal the structure of the cyclase/GCAP complexes by using protein engineering and high resolution chromatography. Mutations that affect regulation of retGC by GCAPs have been recently linked to human retinal degeneration.
The third aim of this proposal is to characterize biochemical and biological effects of a GCAP mutation associated with a human autosomal dominant cone degeneration by using biochemical assays for its activity in vitro and by studying the morphological and the functional changes in transgenic retinas. The experiments proposed here are relevant to understanding of the intricate feedback mechanisms that regulate photoreceptor activity and, when perturbed, cause inherited retinal diseases.
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