REGULATION OF G-PROTEIN COUPLING BY PHOSDUCIN

描述：细胞表面受体和受体之间的偶联 细胞内效应物由一系列G蛋白介导 由Gα和Gβ/伽马亚基组成的复合体。G的激活 蛋白质涉及受体催化的核苷酸交换反应，该反应 产生游离G-α/GTP和G-β/伽马，从而激活 效应酶。磷酸脱氢酶是一种磷化蛋白，它形成一种特定的 与G-β/γ结合，抑制G-蛋白偶联的级联反应。 这项拟议的研究的目标是阐明分子 光导蛋白与G-β/γ的相互作用。三种不同的方法 都会被带走。(1)光还原蛋白/G-β/γ的溶液动力学 配合物将借助荧光光谱学进行研究。一个 将使用荧光探针标记G-β/伽马以监测 络合物的形成和解离。各种G-的亲和力 光还原蛋白的β/γ亚基及其络合物的稳定性 关于磷酸脱氢酶的磷酸化，将进行评估。(2) 光导蛋白/G-β/伽马复合体的分子结构将是 由X射线结晶学测定。光合作用的单晶 和来自视网膜光感受器的转导蛋白G-β/γ亚单位复合体 已经获得了细胞。将致力于解决 复合体的三维结构。我们将尝试 获得与其他G-β/γ络合物类似的硫代蛋白晶体 亚单位。(3)酵母发达的分子遗传学 酿酒酵母将被用来研究G- 活体内的β/伽马。光导蛋白基因将被导入酵母中以 监测突变对酵母交配反应的影响，这是 受G-β/伽马刺激。光导蛋白与T-β的相互作用 将在酵母中使用双杂交系统进行遗传分析。 将努力在酵母中鉴定硫代蛋白的同源物 为了研究自然信号中G-β/伽马活性的调节 适用于基因分析的系统。综合所获得的结果 从生化表征、X-射线衍射和分子检测等方面 基因研究，将有助于确定 光导蛋白、G-β和G-γ与光导蛋白/G-γ的调节 贝塔/伽马复合体。了解系统的结构限制 光导蛋白和G-β/γ之间的相互作用可能导致新的 治疗骨质疏松症的药物设计方法探讨 涉及G蛋白偶联作用的疾病。

DESCRIPTION: The coupling between cell surface receptors and intracellular effectors is mediated by a family of G proteins composed of a complex of G alpha and G beta/gamma subunits. The activation of G proteins involves a receptor-catalyzed nucleotide exchange reaction that generates free G-alpha/GTP and G-beta/gamma leading to activation of the effector enzymes. Phosducin is a phosphoprotein that forms a specific complex with G-beta/gamma and inhibits the G-protein coupled cascade. The goal of the proposed research is to elucidate the molecular interaction of phosducin and G-beta/gamma. Three different approaches will be taken. (1) The solution dynamics of the phosducin/G-beta/gamma complex will be studied with the aid of fluorescence spectroscopy. A fluorescent probe will be used to label G-beta/gamma to monitor the formation and dissociation of the complex. The affinity of various G- beta/gamma subunits for phosducin and the stability of the complex with respect to the phosphorylation of phosducin will be evaluated. (2) The molecular structure of the phosducin/G-beta/gamma complex will be determined by X-ray crystallography. Single crystals of the phosducin and transducin G- beta/gamma subunit complex from retinal photoreceptor cells have been obtained. A major effort will be devoted to solving the three-dimensional structure of the complex. Attempts will be made to obtain similar crystals of phosducin complexed with other G-beta/gamma subunits. (3) The well-developed molecular genetics of the yeast Saccharomyces cerevisiae will be exploited to study regulation of G- beta/gamma in vivo. The phosducin gene will be introduced into yeast to monitor the effects of mutations on the yeast mating response, which is stimulated by G-beta/gamma. The interaction between phosducin and T-beta will be analyzed genetically using the two-hybrid system in yeast. Efforts will be made to identify a homologue of phosducin in yeast in order to study modulation of G-beta/gamma activity in a native signaling system amenable to genetic analysis. Integrating the results obtained from biochemical characterization, X-ray diffraction and the molecular genetic study, will facilitate determination of the interaction among phosducin, G-beta and G-gamma and the regulation of the phosducin/G- beta/gamma complex. Understanding the structural constraints of the interaction between phosducin and G-beta/gamma may lead to novel approaches in the design of drugs for the treatment of diseases involving G-protein coupling action.