Interaction of RGS Protein with G beta subunit G beta 5

RGS 蛋白与 G beta 亚基 G beta 5 的相互作用

• 批准号: 7214804
• 负责人: Vladlen Z Slepak
• 金额: $ 28.54万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214804
• 关键词: Adenylate Cyclase; Affect; Animals; Attenuated; Binding; Biochemical; Biological Assay; Brain; Catalytic Domain; Cell model; Cells; Class; Complex; Cyclic AMP; Data; Disruption; Drug Delivery Systems; Eating Disorders; Enzymes; Exhibits; Family; Fluorescence Resonance Energy Transfer; Future; G-Protein-Coupled Receptors; GTP-Binding Protein Regulators; GTP-Binding Proteins; GTPase-Activating Proteins; Gases; Genes; Genetic; Goals; Guanine Nucleotide Dissociation Inhibitors; Guanosine Triphosphate; Human; Hydrolysis; In Vitro; Investigation; Ion Channel; Kinetics; Knockout Mice; Lead; Measures; Mediating; Molecular; Mus; Neurons; Numbers; Pathway Analysis; Pathway interactions; Pattern; Phenotype; Phosphatidylinositols; Primary Cell Cultures; Production; RGS Domain; RGS Proteins; Receptor Mediated Signal Transduction; Receptor Signaling; Regulation; Research; Research Personnel; Role; Role playing therapy; Second Messenger Systems; Series; Signal Pathway; Signal Transduction; Specificity; System; Targeted Research; Testing; Time; Tissues; Yeasts; attenuation; cell type; dimer; in vitro Assay; in vivo; insight; member; multidisciplinary; novel; programs; protein protein interaction; receptor; reconstitution; research study; response; second messenger; size; stem; success; therapeutic target; yeast two hybrid system

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.

描述（由申请人提供）：G蛋白偶联受体（GPCRS）控制关键的第二使者，例如CA，磷酸肌醇（IP）和CAMP。与激活的GPCR相互作用后，G蛋白结合GTP并将其分离为GAGTP和紧密相关的GBY二聚体，从而调节其靶效应酶或离子通道的活性。尽管建立了这些基本步骤，但对这些途径在特异性方面的调节机制却不了解不同的G蛋白回路之间的动力学和串扰。该研究程序源于PI的早期发现，表明神经元细胞包含一种新型的G蛋白异二聚体 - GB5和RGS蛋白的复合物。 RGS蛋白是G蛋白信号传导的大型调节剂，可作为GA亚基的GTPase激活蛋白（GAP）。只有一个RGS蛋白的亚家族蛋白，该蛋白（含有GY样（GGL）结构域）与GB5相互作用，并且该项目集中在GB5-RGS7复合物上，该复合物在大脑中广泛表达。在该实验室和其他实验室中的研究表明，GB5和RGS7始终在天然组织中相关。 GB5-RGS7复合物的每个亚基在没有另一个的情况下会迅速降解，因此在GPS敲除小鼠中，RGS7不存在。在重构的系统中，GB5-RGS7可以减弱GI-和GQ介导的信号，但基本的分子机制知之甚少，尚不清楚GI和GQ途径是否在体内调节。此外，该提案的意外初步数据将GS确定为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RGS7的RGS域，而是其独特的DEP域。该建议将使用生化，细胞和遗传策略进一步揭示GB5-RGS7在信号转导中的作用。具体目标1将研究使用转染模型细胞中GQ耦合的GPCR重构GB5-RGS7抑制GQ信号传导的机制，并测量Ca2+释放和IP生产的动力学。特定的目标2将通过一系列蛋白质 - 蛋白质相互作用分析以及调节营地产量的途径分析GB5-RGS7与气体之间的相互作用。特定的目标3将使用缺乏GB5-RGS复合物的小鼠比较从野生型GB5 - / - 小鼠获得的原代培养神经元的信号转导模式。总的来说，该多学科计划将实现以下目标：（i）确定本机细胞中GB5-RGS7调节的G蛋白途径，并研究该调节是如何在分子水平上发生的； （ii）检验了GB5-RGS7可以以受体选择方式作用的假设。脑GPCR是主要的和未来的治疗靶标，这项研究将导致对调节这些受体信号传导的机制的新见解。