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、磷酸肌醇（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和紧密缔合的GBy二聚体，其调节其靶效应酶或离子通道的活性。虽然建立了这些基本步骤，但这些途径在特异性、动力学和不同G蛋白回路之间的串扰方面的调节机制尚不清楚。这项研究计划源于Pi的早期发现，即神经元细胞含有一种新型的G蛋白异源二聚体-GB 5和RGS蛋白的复合物。RGS蛋白是G蛋白信号传导的调节剂的大家族，其充当Ga亚基的GT3活化蛋白（GAP）。只有一个RGS蛋白亚家族，那些含有Gy样（GGL）结构域，与GB 5相互作用，本项目的重点是GB 5-RGS 7复合物，它在大脑中广泛表达。这个实验室和其他实验室的研究表明，GB 5和RGS 7总是在天然组织中相关。GB 5-RGS 7复合物的每个亚基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，RGS 7不存在。在重构系统中，GB 5-RGS 7可以减弱Gi和Gq介导的信号，但对潜在的分子机制了解甚少，并且不知道Gi和Gq途径是否在体内受到调节。此外，该提议的意外初步数据将Gs鉴定为GB 5-RGS 7复合物的潜在结合配偶体。令人惊讶的是，与Gs的相互作用不涉及RGS 7的RGS结构域，但其独特的DEP结构域。该提案将使用生物化学，细胞和遗传策略来进一步阐明GB 5-RGS 7在信号转导中的作用。具体目标1将使用在转染的模型细胞中用Gq偶联的GPCR重建，并测量Ca 2+释放和IP产生的动力学，研究GB 5-RGS 7抑制Gq信号传导的机制。具体目标2将通过一系列蛋白质-蛋白质相互作用测定和调节cAMP产生的途径分析来探索GB 5-RGS 7和Gas之间的相互作用。具体目标3将使用缺乏GB 5- RGS复合物的小鼠来比较从GB 5-/-小鼠与野生型小鼠获得的原代培养神经元的信号转导模式。总之，该多学科计划将实现以下目标：（i）确定天然细胞中哪些G蛋白通路受GB 5-RGS 7调节，并研究这种调节如何在分子水平上发生;（ii）测试GB 5-RGS 7可以以受体选择性方式发挥作用的假设。脑GPCR是当前和未来的主要治疗靶点，这项研究将导致对调节这些受体信号传导机制的新见解。

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.