Structure-function studies of visual arrestin

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DESCRIPTION (provided by applicant): Visual arrestins are key players in photoreceptor signaling, governing the rate of signal shutoff and photo response recovery. Closely related non-visual arrestins orchestrate signaling and trafficking of hundreds of different G protein-coupled receptors expressed in virtually every eukaryotic cell. Here we propose to elucidate molecular mechanisms of arrestin function in photoreceptors, focusing on arrestin1 (a.k.a. "rod" arrestin), which is expressed at high level in both rods and cones. Using a combination of biochemical and biophysical methods we propose to determine the conformation of rhodopsin-bound arrestin and the shape of the arrestin-rhodopsin complex. This will allow us to understand how arrestin complexes with hyper- phosphorylated rhodopsin contribute to photoreceptor death in cases of retinitis pigmentosa associated with constitutive formation of these "aberrant" complexes. We propose to test whether arrestin preferentially interacts with monomeric or dimeric rhodopsin, thereby defining the stoichiometry of the biologically relevant arrestin-rhodopsin complex. Based on our studies of the mechanism of arrestin self-association, we propose to elucidate the biological role of this process in photoreceptor cells in mice by replacing wild type arrestin with self-association-impaired mutants that retain all other arrestin functions. Based on the success of our initial proof-of-principle experiments, where we showed that arrestin mutants with high affinity for light-activated unphosphorylated rhodopsin improve the survival and facilitate photo response recovery in rhodopsin phosphorylation-deficient rods, we propose to design new "enhanced" arrestins with better ability to compensate for the defects of rhodopsin phosphorylation in mouse models of congenital visual disorders. We believe that this "compensational" approach will have high therapeutic value in all inherited disorders caused by gain-of-function receptor mutants, where traditional gene replacement approaches, that cannot "silence" excessive signaling by a mutant receptor, are ineffective. PUBLIC HEALTH RELEVANCE: Visual arrestins are key players in photoreceptor signaling, governing the rate of signal shut-off and photo response recovery. Here we propose to elucidate molecular mechanisms of arrestin function, focusing on the arrestin interaction with rhodopsin, the conformation of rhodopsin-bound arrestin and the stoichiometry and shape of the arrestin-rhodopsin complex, as well as on the mechanism and biological role of arrestin self- association in photoreceptor cells. We propose to use this structural information to construct "custom- designed" arrestin proteins with high affinity for light-activated unphosphorylated rhodopsin and test the ability of these "enhanced" mutants to compensate for the defects of rhodopsin phosphorylation in mouse models of congenital visual disorders.