SIGNALING THROUGH RHO GTP/GDP EXCHANGE FACTORS

通过 RHO GTP/GDP 交换因子发出信号

• 批准号: 6318757
• 负责人: PHILIP B WEDEGAERTNER
• 金额: $ 23.85万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6318757
• 关键词: biological signal transduction; cell growth regulation; cell line; cell membrane; charge coupled device camera; chimeric proteins; cytoplasm; fluorescence microscopy; guanine nucleotide binding protein; guanine nucleotide exchange factors; immunocytochemistry; intracellular transport; molecular site; phosphatidylinositol 3 kinase; phosphorylation; protein localization; protein protein interaction; protein structure function; protein transport; protein tyrosine kinase; site directed mutagenesis; video microscopy

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.

描述（逐字研究申请人的摘要）：细胞内信号传导 途径取决于其适当且独特的亚细胞位置 组成蛋白。通常，关键的细胞内信号蛋白移动 从一个亚细胞位置到另一个位置（例如，从细胞质到血浆 膜或从细胞质到核的膜），以响应细胞外刺激。 错误局部蛋白质可以防止完成特定信号 途径或可能引起不受管制的信号传导，例如不受控制的细胞生长。 了解细胞蛋白在特定时间和 亚细胞站点将深入了解阻止不适当的方法 疾病状态（例如癌症）的信号传导。这项研究赠款将解决 在亚细胞本地化和相互作用的背景下这个问题 在P11 5rhogef/pdzrhogef和Ga12/13之间。 异三聚体（ABG）G蛋白充当分子开关 从活化的细胞表面受体到适当细胞内的信息 效应子。一个由A12和A13组成的G蛋白A亚基的家族可以 激活小GTPases的Rho家族。 RHO反过来激活信号 刺激细胞生长和形态变化的途径。最近，两个 已经描述 这可能充当12/13和Rho之间的直接链接。 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，在激活A13的激活中，P11 5rhogef 从细胞质转移到质膜，至少一个Rho 还显示家庭成员从细胞质重新分配到血浆 膜。 该研究建议的主要目标是阐明基础 P11 5RHOGEF和PDZRHOGEF的质膜靶向的机理， 了解适当定位的重要性对于正确的单元信号传导 P11 5rhogef和pdzrhogef，并定义关键和特定的相互作用 在A12/13和P11 5RHOGEF/PDZRHOGEF之间。这些目标将被解决 通过以下特定目的：（1）定义的亚细胞定位 p115rhogef和pdzrhogef，并确定其本地化的变化 对激活的A12，A13和GPCR的响应。 （2）定义等离子体的机制 P1L5RHOGEF和PDZRHOGEF的膜定位。 （3）研究关系 在P115RHOGEF的亚细胞定位和信号传导能力之间 pdzrhogef。 （4）调查A12/13和P115RHOGEF之间的相互作用以及 pdzrhogef。为了解决这些特定目标，本研究赠款申请 提出了针对哺乳动物模型细胞培养系统的实验。一个 P115rhogef和Pdzrhogef的亚细胞定位测定的组合， 蛋白质蛋白质相互作用的测定和Rho依赖性信号的测定 转导将使用。