DEACTIVATION OF RHODOPSIN

光转导的分子细节已经被深入研究， 在过去的十年里，研究了许多过程的特点， 潜在的激活已经被很好地表征。 而反观 失活和光激活机制的详细信息 还没有得到很好的理解。 本提案中概述的研究的广泛目标是 阐明了色素失活的分子机理， 确定视紫红质磷酸化在这一过程中的确切作用， 处理和鉴定视紫红质分子上的结合位点 视紫红质激酶和抑制蛋白。 实验方法 将使用视紫红质的定点突变， 用体外重构测定系统。 这项建议有三个具体目标。 首先确定 其磷酸化是必需的氨基酸残基 去活化 第二，确定氨基酸残基， 影响抑制蛋白和视紫红质激酶与视紫红质的结合， 以确定这些残基是否也改变转导素的结合。 第三，确定组成型活性视紫红质突变体[视蛋白 在体外激活转导素的分子， 发色团和缺乏光]被相同的失活 野生型视紫红质的机制。 在这个补助金中描述的实验是重要的，因为他们是 目的是了解 色素失活。 此外，似乎有些形式的 视网膜变性可能是由持续激活的 视觉传导级联 因此，深入了解分子 视紫红质失活的细节肯定会导致 理解导致组成性活动的病理学， 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.