Trimeric G proteins as Novel Targets in Cancer Progression

摘要： 异三聚体G蛋白是控制信号转导的分子开关。G的调节失调 蛋白通路可以导致信号转导的异常，并预示着许多疾病和肿瘤的发生。 虽然传统上已知G蛋白通过G蛋白偶联受体传递信号 (GPCR)，我导师的团队和其他人越来越多的工作已经确定，他们也会转译 另一大类受体的下游信号，生长因子受体酪氨酸激酶 (RTK)，通过知之甚少的机制。最近的研究表明，Gα相互作用 囊泡相关蛋白是一种与RTK和G结合的特殊信号转导蛋白 蛋白质。作为GIV不寻常的模块化组成的直接结果，由多个RTK发起的信号 在GIV平台上聚合以触发三聚体G蛋白的非规范反式激活，GαI.工作 下游的各种生长因子和配体，已被证明其后果 这样的信号是深远的，对一系列不同的生物过程的影响，无论是在健康和 疾病，是巨大的。尽管有了一些见解，但G蛋白在近距离激活的机制 RTKs尚不清楚，该途径如何影响信号转导或细胞表型，以及可能的影响因素 是这种不寻常的RTK-GIV-GI途径的结构基础及其病理生理后果仍然存在 未被开发的。初步结果表明，受体和G蛋白之间的亲和力是必不可少的 对于GαI被多个RTK在三个独特的酪氨酸上的磷酸化，这种磷酸化需要GIV 将G蛋白招募到RTK，这是主要的。这些调查结果将通过 以下三个目的的实验：1)评估G-αI的磷酸化结果(S) 生长因子RTKs在体外和体内的磷酸化检测，蛋白质-蛋白质相互作用的检测 G蛋白的各种调节剂，GI激活的测量，以及评估迁移的表型分析， 表达Gi的WT或Y突变的细胞的侵袭、有丝分裂和存活；2)研究反式激活 通过研究RTK触发的酪氨酸谱在癌症中解除RTK对G蛋白的调控 G-αI在肿瘤细胞转移过程中的磷酸化及一个新的体细胞突变的特征 GαI，其中被RTKs靶向的Tyr(Y)使用类似的生化和细胞生物学被突变为His(H 目标1中概述的分析；和3)阐明磷酸酪氨酸依赖的结构基础 结合蛋白质化学、功能结合分析和 合理设计的突变蛋白质和X射线结晶学。这项提案的总体目标是剖析 多种生长因子RTK反式激活三聚体G蛋白的机制 连接物/平台，GIV从原子水平到肿瘤细胞表型。

ABSTRACT: Heterotrimeric G proteins are molecular switches that control signal transduction. Dysregulation of the G protein pathway can lead to aberrant signal transduction and herald many diseases and oncogenesis. Although G proteins are traditionally known to transduce signals initiated by G protein coupled receptors (GPCRs), a growing body of work by my mentor's group and others have established that they also transduce signaling downstream of yet another large group of receptors, the growth factor receptor tyrosine kinases (RTKs), via mechanisms that are poorly understood. Recent studies have demonstrated that Gα-Interacting Vesicle associated protein (GIV, a.k.a Girdin) is an unusual signal transducer that can bind both RTKs and G proteins. As a direct consequence of an unusual modular makeup of GIV, signals initiated by multiple RTKs converge on the GIV-platform to trigger non-canonical transactivation of trimeric G protein, Gαi. Working downstream of a variety of growth factors and ligands, it has been demonstrated that the consequences of such signaling are far reaching, and that the impact on a diverse set of biological processes, in both health and disease, is enormous. Despite the insights gained, the mechanism of G protein activation in close proximity of RTKs remains unclear, how may this pathway affect signal transduction or cellular phenotype, and what might be the structural basis for this unusual RTK-GIV-Gi pathway and their pathophysiologic consequences remain unexplored. Preliminary results indicate that the proximity between the receptor and the G protein is essential for phosphorylation of Gαi by multiple RTKs at three unique tyrosines, that such phosphorylation requires GIV to recruit G proteins to the RTKs, and that one of the major. These findings will be studied in-depth through the experiments in the following 3 aims – 1) assess the consequence(s) of phosphorylation of Gαi by multiple growth factor RTKs using in vitro and in vivo phosphorylation assays, protein-protein interaction assays with various modulators of G proteins, measures of Gi activation, and phenotypic assays to evaluate migration, invasion, mitosis, and survival in cells expressing WT or Y mutants of Gi; 2) investigate how transactivation of G proteins by RTKs is deregulated in cancers by studying the profile of RTK-triggered tyrosine phosphorylation of Gαi in tumor cells during metastasis and by characterization of a novel somatic mutation in Gαi where the tyr (Y) that is targeted by RTKs is mutated to his (H) using similar biochemical and cell biological assays as outlined in Aim 1; and 3) elucidate the structural basis for phosphotyrosine-dependent transactivation of G proteins using a combination of protein chemistry, functional binding assays with rationally designed mutant proteins, and x-ray crystallography. The overall goal of this proposal is to dissect the mechanisms by which multiple growth factor RTKs transactivate trimeric G proteins via the novel linker/platform, GIV from an atomic level to tumor cell phenotype.