The long-term objective of the proposed studies is to understand the molecular properties of the proteins involved in quenching phototransduction. Three proteins are engaged in the process that leads to deactivation of photolyzed rhodopsin: rhodopsin kinase, arrestin and rhodopsin phosphatase. First, rhodopsin kinase catalyzes the phosphorylation of freshly bleached rhodopsin which initiates the deactivation of photolyzed rhodopsin, and then arrestin and rhodopsin phosphatase are involved in the subsequent deactivation steps. In contrast to kinase, only fragmentary information is available about the role arrestin has in phototransduction and about the phosphatase identity. In many cases of retinal degeneration, the primary causes of the disorder appear to be either an abnormality cyclic GMT metabolism or mutations in the rhodopsin gene; alternatively, the degeneration of photoreceptor cells becomes evident when levels of rhodopsin phosphorylation/dephosphorylation are abnormal. To define the molecular events that underlie a complex retinal degeneration in humans, such as retinitis pigmentosa, the various steps of phototransduction therefore must be determined on a very detailed level. The objectives of the planned experiments are (1) to elucidate the crystallographic structure of arrestin. (2) to investigate the status of rhodopsin's chromophore when arrestin is released from the phosphorylated membranes and when rhodopsin is dephosphorylated by rhodopsin phosphatase. (3) to identify competitive inhibitors of the rhodopsin-arrestin interaction. (4) to characterize beta-arrestin, a homolog of arrestin, including its functional and immunological properties. (5) to definitively identify the rhodopsin phosphatase and an inhibitor of its activity.
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