G PROTEIN COORDINATION BY RGS12 AND RGS14

描述(摘自申请者摘要)：荷尔蒙和 神经递质通过结合激活细胞内信号转导 与异三聚体鸟嘌呤核苷酸结合或“G”蛋白相连的受体。 G蛋白A亚基在活跃的GTP结合和非活跃的GDP结合之间循环 状态，因此信号持续时间由其固有的GTP酶控制 活动。“G蛋白信号调节蛋白”(RGS)蛋白被认为是关键 G蛋白偶联信号的脱敏剂 标记“RGS-box”结构域以加速GA GTP酶活性。然而，作为RGS 蛋白质是最近才被发现的，它们的生理功能 信号的开始、积分和终止的整体动态很差 已定义。Ras家族GTPase和Ga相互作用结构域的最新鉴定 RGS 12和RGS 14内的(“RFL”和“GoLoco”域)提供了这样的机会 为了确定这两种RGS蛋白用于交易的分子机制 高阶蛋白在G蛋白信号调节中的作用。这项建议是 重点确定了结合的特异性和结构决定因素 RGS 12/14 GoLoco和RFL结构域，以及结构域相互作用的影响 结合G蛋白和RGS-box的核苷酸循环 GTP酶加速活性。Aim 1中的研究验证了这样的假设 GoLoco区域与GDP结合的Gi类GA亚基，并作为 受体非依赖性鸟嘌呤核苷酸交换因子。关于AIM 2测试的研究 RFL结构域与GTP结合的RAS家族G蛋白和 抑制核苷酸解离。目标3中的研究将定义气体 RGS12/14 ROS盒的选择性以及检验假设 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，结合的特异性和亲和力将由 酵母双杂交分析、共沉淀研究、生物传感器的结合 测量和细胞免疫共沉淀分析；这些的影响 核苷酸结合/水解的相互作用将通过体外分析 核苷酸结合分析、单周转和稳态GTP水解法 测量，以及受体信号结果的细胞读数。AS G蛋白偶联信号转导的干扰会导致人类疾病， 但却构成了许多药物作用的基础，定义了 RGS12和RGS14组装和调节特定的异三聚体和RAS家族G 蛋白质应该最终导致新的药物发现靶点的精致 专一性。

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.