G PROTEIN COORDINATION BY RGS12 AND RGS14

描述（来自申请人的摘要的逐字）：激素和 神经递质可以通过结合 与异源三聚体鸟嘌呤核苷酸结合或“G”蛋白连接的受体。 G蛋白a亚基在活性的、GTP结合的和非活性的、GDP结合的之间循环 状态，因此信号持续时间由其固有的GTdR控制 活动“G蛋白信号调节器”（RGS）蛋白被认为是关键的 G蛋白偶联信号转导的脱敏剂， 标志性的“RGS-box”结构域以加速Ga GT3活性。然而，作为RGS 蛋白质只是最近才被发现，它们的生理功能， 信号起始、整合和终止的整体动态性较差 定义了Ras家族GTP和Ga相互作用结构域的最新鉴定 RGS 12和RGS 14中的“RFL”和“GoLoco”域提供了机会 为了确定这两种RGS蛋白用于 G蛋白信号调节中的高阶功能。这项建议是 重点是确定结合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 在结合的G蛋白的核苷酸循环和RGS-box上 GTP酶加速活性。目标1中的研究检验了以下假设： GoLoco区结合GDP结合的Gi类Ga亚基，并作为一个 受体非依赖性鸟嘌呤核苷酸交换因子。Aim 2测试 RFL结构域结合GTP结合Ras家族G蛋白的假设， 抑制核苷酸解离。目标3中的研究将定义Ga RGS 12/14 ROS盒的选择性以及检验假设， GoLoco/Gax和/或RFL/Ras家族蛋白相互作用修饰RGS盒功能。 在所有三个目的中，结合特异性和亲和力将通过以下测定： 酵母双杂交分析，共沉淀研究，生物传感器的组合 测量和细胞共免疫沉淀测定;这些的影响 将通过体外试验分析核苷酸结合/水解的相互作用 核苷酸结合试验、单转换和稳态GTP水解 测量和受体信号传导结果的细胞读数。作为 G蛋白偶联信号转导的扰动可引起人类疾病， 但却构成了许多药物作用的基础，定义了药物作用的机制 RGS 12和RGS 14组装并调节特异性异源三聚体和Ras家族G 蛋白质最终将导致新的药物发现目标， 的特异性

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.