The long-term objective of the proposed studies is to understand the biochemical steps in phototransduction, leading from rhodopsin activation, through its inactivation, and to its regeneration. The importance of inactivation and regeneration of photolyzed rhodopsin has become apparent recently, as malfunctions of these biochemical events during phototransduction lead to degeneration of photoreceptors and impairment of vision. Arrestin is an important component of inactivation of phototransduction and a potent auto-antigen. Elucidation of the structure and function of arrestin will greatly enhance our understanding of the pathogenesis of disease involving dysfunction of this protein. The applicant proposes to examine the structural properties of arrestin and P44 (a splice variant of arrestin) and how they participate in the quanching of phototransduction and in the visual cycle. The applicant seeks to understand: (1) the functional and structural domains of arrestin, continuing his crystallographic approaches; (2) the functional properties of truncated arrestin found in Oguchi Disease, and the molecular basis of arrestin heterogeneity; (3) the functional differences between arrestin and p44 using electrophysiological and biochemical methods; (4) which regions in the primary sequence of arrestin and p44 interact with photolyzed rhodopsin, phosphorylated at physiologically relevant sites (Ser338, Ser343, and Ser334); and finally (5) inactivation of photolyzed rhodopsin and steps in the visual cycle of mutant mice in which the rhodopsin kinase or arrestin genes were disrupted by targeted mutagenesis.
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