REGULATION OF G PROTEIN GTPASE IN PHOTORECEPTORS

描述（申请人的摘要逐字）：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蛋白转导素α亚基（GT-α）的结合， 其激活其效应酶cGMP磷酸二酯酶（PDE）。GTP 通过GT-α的水解终止了这种活性状态，导致从 光刺激GT的内在GT酶活性的速率太慢， 解释了体内光反应的快速终止。以前的研究 证明Gt的GT3活性可以被带到亚秒的时间 通过与PDE-γ亚基（PDE-γ）作用的相互作用进行缩放 与光受体特异性RGS蛋白RGS 9协同作用。重要的是， 当单独添加时，PDE-γ和天然RGS 9都不能充当GAP （GTP酶活化蛋白）。相比之下，RGS 9的分离的RGS结构域是 GAP，表明在原位，RGS 9被抑制。 该应用程序是基于最近的一个意外发现，RGS 9是 与感光细胞特异性G蛋白β亚基G β 5L结合。 非光感受器亚型RGS 7和Gbeta 5的初步数据表明， Gbeta 5L可减弱RGS 9活性。驱动这一现象的工作假设 Gbeta 5L减弱RGS 9刺激的GTP水解， Gt-alpha-GTP与PDE-gamma相互作用，产生更强的信号 通过级联放大。具体目标1将研究蛋白质 涉及光感受器的天然提取物中的G β 5L和RGS 9的复合物。 使用层析和免疫沉淀，这些实验将表明 G β 5L、GT-α和PDE-γ是否可以同时结合RGS 9， 并阐明GT的GDP/GTP循环在这些形成中的作用 配合物目的2将在体外研究纯化的蛋白质，特别是将 确定Gbeta 5L对RGS 9差距活性的影响。此外，本发明还提供了一种方法， 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。目的 3将通过突变分析研究RGS 9和Gbeta 5L，深入了解如何 这些分子起作用，为未来提供分子工具， 研究其生理作用。该项目将带来更好的 在分子水平上理解光传导。许多视网膜病变 由于光感受器中的信号转导机制失调而发生 因此，这项研究所获得的知识将有助于发展 未来的治疗

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.