GTPASE ACTIVATING COMPLEX FROM ROD PHOTORECEPTORS

这个建议的总体目标是了解感光细胞中G蛋白信号传导持续时间的分子机制。 光感受器G蛋白，transducin，在脊椎动物光转导中起着核心作用，它将信号从激活的受体，光激发的视紫红质，传递到其效应物，cGMP磷酸二酯酶。当光激发的视紫红质催化转导素α亚基上GDP与GTP的交换时，转导素被激活，并继续刺激PDE活性，直到结合的GTP被水解。 由该实验室和其他实验室进行的研究表明，光感受器含有几种蛋白质，其将转导蛋白的缓慢内在GTdR活性加速到足以及时恢复光反应的速率。 这些蛋白质包括转导素的直接靶标、cGMP磷酸二酯酶的γ亚基以及G蛋白信号传导蛋白家族的调节因子（Regulators of G protein Signaling protein family，RGS 9）的第九个成员与5型G蛋白β亚基的长剪接变体（Gp β 5L）之间的复合物。 了解确切的分子机制，该蛋白质合奏的成员有助于以协调的方式激活转导蛋白GT3是本提案的主要目标。 目的1是确定每个蛋白及其功能域在调节GT3转导中的作用和作用机制。 目的2是了解RGS 9-Gbeta 5L复合物与光感受器膜异常紧密结合的性质，并测试这种结合通过与未知跨膜蛋白相互作用介导的可能性。 目的3将利用转基因小鼠技术，以了解哪一个组成部分的GTdR激活合奏作为限速因子在生理上完整的杆光感受器。 解决这个问题不仅有助于理解光响应恢复的分子机制，而且还将使我们能够建立一个总体框架，研究其他细胞内信号通路中信号持续时间的调节。 这项工作也与理解负责确保光反应正常持续时间的生化途径如何在各种遗传性眼病中受到影响有关。

The overall objective of this proposal is to understand the molecular mechanism that governs the duration of G protein signaling in photoreceptor cells. The photoreceptor G protein, transducin, plays a central role in vertebrate phototransduction where it conveys a signal from the activated receptor, photoexcited rhodopsin, to its effector, cGMP phosphodiesterase. Transducin is activated when photoexcited rhodopsin catalyses an exchange of GDP for GTP on the transducin alpha subunit and continues to stimulate PDE activity until the bound GTP is hydrolyzed. Studies conducted by this and other laboratories have shown that photoreceptors contain several proteins which accelerate the slow intrinsic GTPase activity of transducin to a rate sufficient for timely photoresponse recovery. These proteins include the immediate target of transducin, the gamma subunit of cGMP phosphodiesterase and the complex between the ninth member of the Regulators of G protein Signaling protein family (RGS9) and the long splice variant of type 5 G protein beta subunit (Gpbeta5L). Understanding the exact molecular mechanism by which members of this protein ensemble contribute to the activation of transducin GTPase in a coordinated manner is the major goal of this proposal. Aim 1 is to establish a role and mechanism of action for each protein and their functional domains in regulating transducin GTPase. Aim 2 is to understand the nature of an unusually tight association of the RGS9-Gbeta5L complex with photoreceptor membranes and to test possibility that this association is mediated via interactions with an unknown transmembrane protein. Aim 3 will utilize transgenic mouse technology to understand which component of the GTPase activating ensemble acts as the rate-limiting factor in physiologically intact rod photoreceptors. Addressing this problem will not only contribute to the understanding of the molecular mechanism of photoresponse recovery, but also will allow us to establish a general framework for investigating the regulation of signal duration in other intracellular signaling pathways. The work is also relevant to understanding of how biochemical pathways responsible for ensuring normal duration of the photoresponse may be affected in various inherited eye diseases.