SIGNALING THROUGH RHO GTP/GDP EXCHANGE FACTORS

通过 RHO GTP/GDP 交换因子发出信号

• 批准号: 6722887
• 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/6722887
• 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（逐字摘自申请人摘要）：细胞内信号传导 途径取决于其适当且独特的亚细胞位置 组成蛋白质。通常，关键的细胞内信号蛋白会移动 从一个亚细胞位置到另一个亚细胞位置（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。 错误定位的蛋白质可能会阻止特定信号传导的完成 途径或可能导致不受调节的信号传导，例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 亚细胞位点将导致深入了解阻止不当行为的方法 癌症等疾病状态下的信号传导。这项研究补助金将解决 这个问题在亚细胞定位和相互作用的背景下 p11 5rhoGEF/PDZrhoGEF 和 Ga12/13 之间。 异三聚体 (abg) G 蛋白充当分子开关来传递信号 从激活的细胞表面受体到适当的细胞内的信息 效应器。 G 蛋白 a 亚基的一个家族，由 a12 和 a13 组成，可以 激活小 GTP 酶的 rho 家族。 Rho 反过来激活信号传导 刺激细胞生长和形态变化的途径。最近，两 大型多结构域蛋白 p115rhoGEF 和 PDZrhoGEF 已被描述 这可以作为 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，响应 a13、p11 5rhoGEF 的激活 从细胞质易位到质膜，并且至少有一个 rho 家庭成员也被证明可以从细胞质重新分配到血浆 膜。 本研究计划的主要目标是阐明潜在的 p11 5rhoGEF 和 PDZrhoGEF 的调节质膜靶向机制， 了解正确定位对于正确细胞信号传导的重要性 p11 5rhoGEF 和 PDZrhoGEF，并定义关键和特定的相互作用 介于 a12/13 和 p11 5rhoGEF/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 p115rhoGEF 和 PDZrhoGEF，并确定其定位的变化 对激活的 a12、a13 和 GPCR 的反应。 (2) 定义等离子体的机理 p1l5rhoGEF 和 PDZrhoGEF 的膜定位。 (3) 考察关系 p115rhoGEF 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 p115rhoGEF 之间的相互作用 PDZrhoGEF。为了解决这些具体目标，本研究资助申请 提出专注于模型哺乳动物细胞培养系统的实验。一个 p115rhoGEF 和 PDZrhoGEF 亚细胞定位测定的组合， 蛋白质-蛋白质相互作用的测定以及 rho 依赖性信号的测定 将采用转导。