DEACTIVATION OF RHODOPSIN

光转导的分子细节已经被密集地 过去十年的研究和过程的许多特征 潜在的激活已经被很好地描述了。相比之下， 失活和光激活的潜在机制的细节 都没有被很好地理解。 这项提案中概述的研究的总体目标是 阐明光敏色素失活的分子机制 确定视紫红质磷酸化在这一过程中的确切作用 视紫红质分子的处理及结合部位的鉴定 视紫红质激活剂和arrestin。实验方法 将联合使用视紫红质的定点突变 使用体外重建检测系统。 这项提议有三个具体目标。首先，要确定 氨基酸残基，其磷酸化是必需的 停用。第二，测定氨基酸残基 影响视紫红质与arrestin和视紫红质的结合 以确定这些残基是否也改变了转导蛋白的结合。 第三，确定构成活性视紫红质突变体[视黄素 在体外激活转导蛋白的分子，在没有 发色团和无光]由相同的 机制如野生型视紫红质。 这项资助中描述的实验具有重要意义，因为它们 旨在了解构成其基础的分子细节 感光色素失活。此外，似乎有一些形式的 视网膜退行性变可能是由于视网膜持续激活 视觉传导级联。因此，对分子的洞察 视紫红质失活的细节肯定会导致 对导致结构性活动和 视网膜变性。

The molecular details of phototransduction have been intensively studied over the last ten years and many features of the processes underlying activation have been well characterized. In contrast, the details of the mechanism underlying inactivation and light-activation are not as well understood . The broad aim of the research outlined in this proposal is to elucidate the molecular mechanism of photopigment deactivation by determining the precise role of rhodopsin phosphorylation in this process and identifying the binding sites on the rhodopsin molecule for both rhodopsin kinase and arrestin. The experimental approach will be to use site-directed mutagenesis of rhodopsin in conjunction with an in vitro reconstitution assay system. There are three specific aims of this proposal. First, to identify the amino acid residues whose phosphorylations are essential for deactivation. Second, to determine the amino acid residues that affect the binding of arrestin and rhodopsin kinase to rhodopsin and to determine if these residues also alter the binding of transducin. Third, to determine if constitutively active rhodopsin mutants [opsin molecules that in vitro activate transducin in the absence of chromophore and the absence of light] are inactivated by the same mechanisms as wild type rhodopsin. The experiments described in this grant are significant for they are aimed at understanding the molecular details that underlie photopigment deactivation. In addition, it appears that some forms of retinal degeneration maybe caused by persistent activation of the visual transduction cascade. Therefore, insights into the molecular details of rhodopsin deactivation will certainly lead to an understanding of pathologies that result in constitutive activity and retinal degeneration.