REGULATION OF G PROTEIN GTPASE IN PHOTORECEPTORS

光感受器中 G 蛋白 GTP 酶的调节

• 批准号: 6384872
• 负责人: Vladlen Z Slepak
• 金额: $ 29.82万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6384872
• 关键词: G protein; biological signal transduction; guanosinetriphosphatases; protein protein interaction; protein structure function; visual photoreceptor

DESCRIPTION (Verbatim from applicant's abstract): This project will study the molecular mechanisms regulating the kinetics of light response in photoreceptor cells. In vertebrates, rhodopsin triggers the light response by stimulating the binding of GTP to the alpha subunit of the G protein transducin (Gt-alpha), which activates its effector enzyme cGMP phosphodiesterase (PDE). GTP hydrolysis by Gt-alpha terminates this active state, leading to recovery from a light stimulus. The rate of Gt's intrinsic GTPase activity is too slow to explain the rapid termination of photoresponse in vivo. Previous research demonstrated that GTPase activity of Gt could be brought to a sub-second time scale by interaction with the PDE-gamma subunit (PDE-gamma) acting synergistically with the photoreceptor-specific RGS protein RGS9. Importantly, when added separately, neither PDE-gamma nor native RGS9 can act as a GAP (GTPase-activating protein). In contrast, the isolated RGS domain of RGS9 is a GAP, indicating that in situ, RGS9 is inhibited. This application is based on a recent and unexpected discovery that RGS9 is bound to the photoreceptor specific G protein beta subunit, Gbeta5L. Preliminary data with non-photoreceptor isoforms, RGS7 and Gbeta5, suggest that Gbeta5L can attenuate RGS9 activity. The working hypothesis driving this project is that Gbeta5L attenuates RGS9-stimulated GTP hydrolysis until Gt-alpha-GTP interacts with PDE-gamma, resulting in stronger signal amplification by the cascade. Specific aim 1 will investigate the protein complexes involving Gbeta5L and RGS9 in the native extracts of photoreceptors. Using chromatography and immunoprecipitation, these experiments will show whether or not Gbeta5L, Gt-alpha and PDE-gamma can bind to RGS9 simultaneously, and elucidate the role of Gt's GDP/GTP cycle in the formation of these complexes. Aim 2 will study purified proteins in vitro, and particularly, will determine the effect of Gbeta5L on the GAP activity of RGS9. In addition, studies utilizing surface plasmon resonance (SPR) will characterize protein-protein interactions with respect to their kinetics and regulation. Aim 3 will study RGS9 and Gbeta5L by mutational analysis, gaining insight into how these molecules work, and providing molecular tools for the future investigation of their physiological role. This project will result in a better understanding of phototransduction at the molecular level. Many retinopathies occur due to disregulation of signal transduction mechanisms in photoreceptors and, therefore, the knowledge gained by this research will help to develop future therapies.

描述(逐字摘自申请者摘要)：本项目将研究 光感受器光反应动力学的分子调控机制 细胞。在脊椎动物中，视紫红质通过刺激 GTP与G蛋白转导蛋白的α亚基(GT-α)的结合， 激活其效应酶cGMP磷酸二酯酶(PDE)。GTP GT-α的水解会终止这一活跃状态，导致从 光刺激。GT固有的GTPase活性速度太慢，无法 解释体内光反应的快速终止。以前的研究 证明GT的GTP酶活性可降至亚秒级 通过与PDE-伽马亚单位(PDE-伽马亚基)相互作用进行标度 与光感受器特异的RGS蛋白RGS9协同作用。重要的是 单独添加时，PDE-Gamma和本机RGS9都不能作为缺口 (GTP酶激活蛋白)。相比之下，RGS9的分离的RGS结构域是一个 GAP，表明在原位，RGS9被抑制。 此应用程序基于最近意外的发现，即RGS9 结合到光感受器特异的G蛋白β亚基Gbeta5L。 非光感受器亚型RGS7和Gbeta5的初步数据表明 Gbeta5L可抑制RGS9活性。推动这一点的工作假说 项目是Gbeta5L减弱RGS9刺激的GTP水解，直到 GT-α-GTP与PDE-Gamma相互作用，产生更强的信号 通过级联放大。特定目标1将研究该蛋白质 光感受器天然提取物中含有Gbeta5L和RGS9的复合体。 使用层析和免疫沉淀，这些实验将显示 无论Gbeta5L、GT-α和PDE-γ是否能同时与RGS9结合， 并阐明了GT的GDP/GTP循环在形成这些变化中的作用 复合体。AIM 2将在体外研究纯化的蛋白质，特别是将 测定Gbeta5L对RGS9细胞GAP活性的影响。此外, 利用表面等离子体共振(SPR)的研究将表征 蛋白质-蛋白质相互作用的动力学和调节。目标 3将通过突变分析研究RGS9和Gbeta5L，了解如何 这些分子起作用，并为未来提供分子工具 研究它们的生理作用。这个项目将产生一个更好的 在分子水平上对光传导的理解。许多视网膜病变 由于光感受器的信号转导机制失调所致 因此，通过这项研究获得的知识将有助于开发 未来的疗法。