GTPASE ACTIVATING COMPLEX FROM ROD PHOTORECEPTORS

这个建议的总体目标是了解控制光感受器细胞中G蛋白信号持续时间的分子机制。光感受器G蛋白转导蛋白在脊椎动物的光转导中起着核心作用，它将信号从激活的受体光激发视紫红质传递到其效应器cGMP磷酸二酯酶。当光激发的视紫红质催化转导蛋白α亚基上的GDP交换为GTP时，转导蛋白被激活，并继续刺激PDE活性，直到结合的GTP被水解。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，这些蛋白质可以加速转导蛋白缓慢的内在GTP酶活性，使其达到足以及时恢复光反应的速度。这些蛋白质包括转导蛋白的直接靶标、cGMP磷酸二酯酶的伽马亚基以及G蛋白信号蛋白家族第九调节成员(RGS9)和5G蛋白β亚基的长剪接变异体(Gpbeta5L)之间的复合体。了解这一蛋白质集合成员以协调方式促进转导蛋白GTP酶激活的确切分子机制是本提案的主要目标。目的1是确定每种蛋白及其功能域在调节转导蛋白GTP酶中的作用和机制。目的2是了解RGS9-Gbeta5L复合体与光感受器膜异常紧密结合的性质，并测试这种结合是通过与未知跨膜蛋白相互作用而介导的可能性。目的3将利用转基因小鼠技术来了解GTP酶激活系综中的哪一组分在生理上完整的杆状感光细胞中起到限速因子的作用。解决这一问题不仅有助于理解光响应恢复的分子机制，而且将使我们能够建立一个通用的框架，用于研究其他细胞内信号通路中信号持续时间的调节。这项工作还与理解负责确保正常光反应持续时间的生化途径在各种遗传性眼病中可能受到的影响有关。

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.