SIGNALING THROUGH RHO GTP/GDP EXCHANGE FACTORS

描述（来自申请人摘要的逐字）：细胞内信号传导 途径依赖于它们的适当和独特的亚细胞位置， 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（例如，从细胞质到血浆 膜或从细胞质到细胞核）响应细胞外刺激。 不正确定位的蛋白质可以阻止特定信号的完成 信号通路或可导致不受调节的信号传导，如不受控制的细胞生长。 了解细胞蛋白质如何在特定时间聚集在一起， 亚细胞位点将导致洞察如何阻止不适当的 在疾病状态如癌症中的信号传导。这项研究经费将用于 在亚细胞定位和相互作用的背景下， p11 5 rhoGEF/PDZrhoGEF和Ga 12/13之间的差异。 异源三聚体（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 效应器一个由a12和a13组成的G蛋白a亚基家族， 激活小GTP酶的rho家族。反过来，Rho激活了 刺激细胞生长和形态变化的途径。最近有两 已经描述了大的多结构域蛋白质p115 rhoGEF和PDZrhoGEF 可以作为12/13和rho之间的直接链接。a12和a13是 通常与质膜紧密结合。另一方面我们 最近的研究表明，在对a13，p11 5 rhoGEF激活的反应中， 从细胞质易位到质膜，并且至少一个rho 家族成员也显示从细胞质重新分布到血浆 膜。 本研究建议的主要目标是阐明 调节p11 5 rhoGEF和PDZrhoGEF的质膜靶向机制， 理解适当定位对适当细胞信号传导的重要性， p11 5 rhoGEF和PDZrhoGEF，并定义关键和特定的相互作用 在a12/13和p11 5 rhoGEF/PDZrhoGEF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 rhoGEF和PDZrhoGEF，并确定它们在 对激活的a12、a13和GPCR的反应。(2)定义等离子体的机制 p115 rhoGEF和PDZrhoGEF的膜定位。(3)调查关系 p115 rhoGEF的亚细胞定位和信号传导能力之间的关系， PDZrhoGEF。(4)研究a12/13和p115 rhoGEF之间的相互作用， PDZrhoGEF。为了实现这些具体目标，本研究资助申请 提出的实验重点是模型哺乳动物细胞培养系统。一 用于p115 rhoGEF和PDZrhoGEF的亚细胞定位的测定的组合， 蛋白质-蛋白质相互作用的测定和rho依赖性信号的测定 将采用转导。

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.