SIGNALING THROUGH RHO GTP/GDP EXCHANGE FACTORS

描述(逐字摘自申请者摘要)：细胞内信号 通路取决于其适当和唯一的亚细胞位置 组成蛋白质。通常，关键的细胞内信号蛋白 从一个亚细胞位置到另一个亚细胞位置(例如，从细胞质到胞浆 细胞膜或从细胞质到细胞核)对细胞外刺激的反应。 错误定位的蛋白质可能会阻止特定信号的完成 途径或可导致不受调控的信号，如不受控制的细胞生长。 了解细胞蛋白质是如何在特定的时间和 亚蜂窝网站将导致对阻止不适当的方法的洞察 在癌症等疾病状态下发出信号。这项研究拨款将解决 在亚细胞定位和相互作用的背景下的这一问题 在p11.5和GA12/13之间。 异三聚体(ABG)G蛋白作为分子开关 从激活的细胞表面受体到适当的细胞内信息 效应器。一个由A12和A13组成的G蛋白a亚基家族可以 激活Rho家族的小GTP酶。反过来，Rho激活信令 刺激细胞生长和形态变化的途径。最近，有两个 已经描述了大的、多结构域的蛋白质，p115rogef和pZrogef 这可能会成为12/13和RHO之间的直接联系。A12和A13是 通常发现与质膜紧密相关。另一方面，我们 最近证明，响应a13的激活，p115ro 从细胞质转位到质膜，至少有一个Rho 家族成员也被证明从细胞质重新分布到血浆中 膜。 这项研究提案的主要目的是阐明潜在的 调控质膜靶向p115和PDZro的作用机制， 通过以下方式理解正确定位对正确细胞信号的重要性 P11 5ROGEM和PDZROGEM，并定义关键和具体的相互作用 在a12/13和p11之间。这些目标将得到解决 通过以下具体目标：(1)定义亚细胞定位 P115，并确定其本地化的变化 对激活的A12、A13和GPCRs的反应。(2)明确了等离子体的作用机制 P115Ro和PDZro Gen的膜定位。(3)调查关系 P115ro-gef亚细胞定位与信号转导能力的关系 PDZROGEM。(4)研究A12/13和p115-Regf之间的相互作用。 PDZROGEM。为了解决这些具体目标，这项研究资助申请 提出了以哺乳动物细胞培养模型系统为重点的实验方案。一个 结合p115ro-Gen和PDZro-Gen的亚细胞定位试验， 蛋白质-蛋白质相互作用的分析和Rho依赖信号的分析 将采用换能器。

DESCRIPTION (Verbatim from the Applicant's Abstract): Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Frequently, key intracellular signaling proteins move from one subcellular location to another (e.g., from cytoplasm to plasma membranes or from cytoplasm to nucleus) in response to extracellular stimuli. Incorrectly localized proteins can prevent completion of a particular signaling pathway or can cause unregulated signaling, such as uncontrolled cell growth. Understanding how cellular proteins come together at specific times and subcellular sites will lead to insight into ways to block inappropriate signaling in disease states such as cancer. This research grant will address this issue in the context of subcellular localization of and interactions between p11 5rhoGEF/PDZrhoGEF and Ga12/13. The heterotrimeric (abg) G proteins act as molecular switches to relay information from activated cell-surface receptors to appropriate intracellular effectors. One family of G protein a subunits, consisting of a12 and a13, can activate the rho family of small GTPases. Rho, in turn, activates signaling pathways that stimulate cell growth and morphological changes. Recently, two large, multi-domain proteins, p115rhoGEF and PDZrhoGEF, have been described that may function as direct links between a 12/13 and rho. a12 and a13 are typically found tightly associated with plasma membranes. On the other hand, we have recently demonstrated that, in response to activation of a13, p11 5rhoGEF translocates from the cytoplasm to plasma membranes, and at least one rho family member has also been shown to redistribute from cytoplasm to plasma membranes. The main objectives of this research proposal are to elucidate the underlying mechanisms of regulated plasma membrane targeting of p11 5rhoGEF and PDZrhoGEF, understand the importance of proper localization for proper cell signaling by p11 5rhoGEF and PDZrhoGEF, and define critical and specific interactions between a12/13 and p11 5rhoGEF/PDZrhoGEF. These objectives will be addressed through the following specific aims: (1) Define subcellular localization of p115rhoGEF and PDZrhoGEF, and determine changes in their localization in response to activated a12, a13 and GPCRs. (2) Define mechanisms of plasma membrane localization of p1l5rhoGEF and PDZrhoGEF. (3) Investigate relationship between subcellular localization and signaling ability for p115rhoGEF and PDZrhoGEF. (4) Investigate interactions between a12/13 and p115rhoGEF and PDZrhoGEF. To address these specific aims, this research grant application proposes experiments focusing on model mammalian cell culture systems. A combination of assays for subcellular localization of p115rhoGEF and PDZrhoGEF, assays for protein-protein interactions, and assays of rho-dependent signal transduction willbe employed.