G PROTEIN COORDINATION BY RGS12 AND RGS14

RGS12 和 RGS14 的 G 蛋白协调

• 批准号: 6698850
• 负责人: David P. Siderovski
• 金额: $ 24.55万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6698850
• 关键词: G protein; SDS polyacrylamide gel electrophoresis; autoradiography; chimeric proteins; enzyme activity; genetic translation; guanosine diphosphate; guanosine triphosphate; guanosinetriphosphatases; immunoprecipitation; protein structure function; receptor coupling; surface plasmon resonance; yeast two hybrid system

DESCRIPTION (Verbatim from the Applicant's Abstract): Hormones and neurotransmitters can activate intracellular signal transduction by binding receptors linked to heterotrimeric guanine nucleotide-binding or "G" proteins. G protein a subunits cycle between active, GTP-bound and inactive, GDP-bound states, and thus signal duration is controlled by their intrinsic GTPase activity. "Regulator of G-protein signaling" (RGS) proteins are considered key desensitizers of G protein-coupled signaling given the ability of their hallmark "RGS-box" domains to accelerate Ga GTPase activity. However, as RGS proteins have only recently been identified, their physiological functions in the overall dynamics of signal onset, integration, and termination are poorly defined. Recent identification of Ras-family GTPase- and Ga-interaction domains ("RFL" and "GoLoco" domains) within RGS 12 and RGS 14 presents the opportunity to define the molecular mechanisms these two RGS proteins use to transact higher-order functions in G protein signaling modulation. This proposal is focused on determining the binding specificities and structural determinants of RGS 12/14 GoLoco and RFL domains, as well as the effects of domain interactions both on the nucleotide cycle of the bound G protein and on RGS-box GTPase-accelerating activity. Studies in Aim 1 test the hypothesis that the GoLoco region binds to GDP-bound Gi-class Ga subunits and acts as a receptor-independent guanine-nucleotide exchange factor. Studies in Aim 2 test the hypothesis that the RFL domains bind GTP-bound Ras-family G proteins and inhibit nucleotide dissociation. Studies in Aim 3 will define the Ga selectivites of RGS12/14 ROS-boxes as well as test the hypothesis that GoLoco/Gax and/or RFL/Ras-family protein interactions modify RGS box function. In all three Aims, binding specificity and affinity will be determined by a combination of yeast two-hybrid analyses, coprecipitation studies, biosensor measurements, and cell co-immunoprecipitation assays; effects of these interactions on nucleotide binding/hydrolysis will be analysed by in vitro nucleotide binding assays, single-turnover and steady state GTP hydrolysis measurements, and cellular readouts of receptor signaling outcomes. As perturbation of G protein-coupled signal transduction can cause human disease, yet forms the basis of many drug actions, defining the mechanisms by which RGS12 and RGS14 assemble and regulate specific heterotrimeric and Ras-family G proteins should ultimately lead to novel drug discovery targets with exquisite specificity.

描述（逐字摘自申请人摘要）：激素和 神经递质可以通过结合激活细胞内信号转导 与异三聚鸟嘌呤核苷酸结合或“G”蛋白连接的受体。 G 蛋白亚基在活性、GTP 结合和非活性、GDP 结合之间循环 状态，因此信号持续时间由其内在的 GTPase 控制 活动。 “G 蛋白信号传导调节器”(RGS) 蛋白被认为是关键 鉴于其能力，G蛋白偶联信号的脱敏剂 标志性“RGS-box”结构域可加速 Ga GTP 酶活性。然而，作为 RGS 蛋白质最近才被鉴定，它们的生理功能 信号开始、整合和终止的整体动态很差 定义的。 Ras 家族 GTPase 和 Ga 相互作用域的最新鉴定 RGS 12 和 RGS 14 内的（“RFL”和“GoLoco”域）提供了机会 定义这两种 RGS 蛋白进行交易的分子机制 G 蛋白信号传导调节中的高阶功能。这个提议是 专注于确定结合特异性和结构决定因素 RGS 12/14 GoLoco 和 RFL 域，以及域相互作用的影响 结合 G 蛋白的核苷酸循环和 RGS-box GTPase 加速活性。目标 1 中的研究检验了以下假设： GoLoco 区域与 GDP 结合的 Gi 类 Ga 亚基结合，并充当 受体独立的鸟嘌呤核苷酸交换因子。目标 2 测试研究 RFL 结构域结合 GTP 结合的 Ras 家族 G 蛋白的假设 抑制核苷酸解离。目标 3 的研究将定义 Ga RGS12/14 ROS-boxes 的选择性以及检验以下假设 GoLoco/Gax 和/或 RFL/Ras 家族蛋白相互作用改变 RGS 盒功能。 在所有三个目标中，结合特异性和亲和力将由 酵母双杂交分析、共沉淀研究、生物传感器的组合 测量和细胞免疫共沉淀测定；这些的影响 核苷酸结合/水解的相互作用将通过体外分析 核苷酸结合测定、单周转和稳态 GTP 水解 测量和受体信号转导结果的细胞读数。作为 G蛋白偶联信号转导的扰动会导致人类疾病， 然而构成了许多药物作用的基础，定义了药物作用的机制 RGS12 和 RGS14 组装和调节特定异源三聚体和 Ras 家族 G 蛋白质最终应该导致新的药物发现目标具有精致的 特异性。