Interaction of RGS Protein with G beta subunit G beta 5

描述（由申请人提供）：G蛋白偶联受体（gpcr）控制关键的第二信使，如Ca，磷酸肌苷（IP）和cAMP。在与活化的GPCR相互作用后，G蛋白结合GTP并解离成Ga-GTP和紧密相关的GBy二聚体，它们调节其靶效应酶或离子通道的活性。虽然这些基本步骤已经建立，但这些途径的特异性、动力学和不同G蛋白回路之间的串扰调控机制尚不清楚。这个研究项目源于Pi的早期发现，即神经细胞含有一种新型的G蛋白异二聚体——GB5和RGS蛋白的复合物。RGS蛋白是一大家族的G蛋白信号调节因子，作为Ga亚基的GTPase激活蛋白（gap）。RGS蛋白中只有一个亚家族，即含有Gy-like （GGL）结构域的亚家族与GB5相互作用，本项目主要研究在大脑中广泛表达的GB5- rgs7复合物。本实验室和其他实验室的研究表明，GB5和RGS7在原生组织中总是相关的。GB5-RGS7复合物的每一个亚基都在缺乏另一个亚基的情况下迅速降解，因此在GPS敲除小鼠中，RGS7缺失。在重组系统中，GB5-RGS7可以减弱Gi-和Gq介导的信号，但其潜在的分子机制尚不清楚，也不清楚Gi和Gq途径是否在体内都受到调节。此外，本提案的意外初步数据确定Gs是GB5-RGS7复合物的潜在结合伙伴。令人惊讶的是，与Gs的相互作用涉及的不是RGS7的RGS结构域，而是其独特的DEP结构域。本研究将利用生物化学、细胞和遗传策略进一步阐明GB5-RGS7在信号转导中的作用。特异性目的1将在转染的模型细胞中使用Gq偶联gpcr重组GB5-RGS7抑制Gq信号传导的机制，并测量Ca2+释放和IP产生的动力学。Specific Aim 2将通过一系列蛋白质相互作用实验和cAMP产生调控途径分析来探索GB5-RGS7与Gas之间的相互作用。特异性Aim 3将使用缺乏GB5- RGS复合物的小鼠，比较从GB5-/-小鼠获得的原代培养神经元与野生型的信号转导模式。总之，这个多学科项目将实现以下目标：(i)确定天然细胞中哪些G蛋白通路受GB5-RGS7调控，并研究这种调控在分子水平上是如何发生的；（ii）验证GB5-RGS7以受体选择性方式起作用的假设。脑gpcr是当前和未来的主要治疗靶点，这项研究将为调节这些受体的信号传导机制提供新的见解。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) control key second messengers such as Ca , phosphoinositides (IP) and cAMP. Upon the interaction with activated GPCR, the G protein binds GTP and dissociates into the Ga-GTP and the tightly associated GBy dimer, which modulate the activity their target effector enzymes or ion channels. While these basic steps are established, mechanisms of regulation of these pathways with respect to specificity, kinetics and crosstalk between distinct G protein circuits, are not understood. This research program stems from the Pi's earlier discovery showing that neuronal cells contain a novel kind of G protein heterodimer - the complex of GB5 and RGS proteins. RGS proteins are a large family of regulators of G protein signaling that act as GTPase activating proteins (GAPs) for Ga subunits. Only one subfamily of RGS proteins, those containing a Gy-like (GGL) domain, interacts with GB5, and this project is focused on GB5-RGS7 complex, which is widely expressed in the brain. Studies in this and other labs showed that GB5 and RGS7 are always associated in native tissues. Each subunit of the GB5-RGS7 complex rapidly degrades in the absence of the other, and so in GPS knock-out mice, RGS7 is absent. In reconstituted systems, GB5-RGS7 can attenuate Gi-, and Gq-mediated signals but underlying molecular mechanisms are poorly understood and it is not known if both Gi and Gq pathways are regulated in vivo. In addition, unexpected preliminary data of this proposal identified Gs as a potential binding partner of the GB5-RGS7 complex. Surprisingly, the interaction with Gs involved not the RGS domain of RGS7 but its unique DEP domain. This proposal will use biochemical, cellular and genetic strategies to further unravel the role of GB5-RGS7 in signal transduction. Specific Aim 1 will investigate the mechanism of inhibition of Gq signaling by GB5-RGS7 using reconstitution with Gq-coupled GPCRs in transfected model cells, and measuring kinetics of Ca2+ release and IP production. Specific Aim 2 will explore the interaction between GB5-RGS7 and Gas by a series of protein-protein interaction assays and analysis of pathways regulating cAMP production. Specific Aim 3 will use mice lacking GB5- RGS complex to compare signal transduction patterns of primary cultured neurons obtained from GB5-/- mice with wild-type. In all, this multidisciplinary program will achieve the following goals: (i) establish which G protein pathways are regulated by GB5-RGS7 in native cells, and study how this regulation occurs at the molecular level; (ii) test the hypothesis that GB5-RGS7 can act in receptor-selective manner. Brain GPCRs are a major current and future therapeutic target, and this research will lead to new insights into the mechanisms that regulate signaling from these receptors.