G Proteins and Protein Kinase A Anchoring

G 蛋白和蛋白激酶 A 锚定

• 批准号: 6843820
• 负责人: TATYANA A VOYNO-YASENETSKAYA
• 金额: $ 33.98万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6843820
• 关键词: Proteins; Protein; Kinase; Anchoring

EXCEED THE SPACE PROVIDED. Heterotrimerie G proteins and protein kinase A (PKA) are two important signal transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation in response to various hormones involves stimulatory G protein, Gs, which activates adenylyl cyclase resulting in production of cAMP. We have discovered a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the ¿x subunit of GI3 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Our data suggested that AKAP110 may provide a link between heterotrimeric G proteins and cAMP-independent activation of PKA. Understanding the exact molecular mechanism by which members of the G13/AKAPll0 protein ensemble contribute to cellular functions is the major goal of this proposal. GcO3 is expressed ubiquitously, whereas AKAP 110 expression is restricted to particular locations, including cerebellum. As physiological role of G_13 and AKAPll0 in cerebellum is unknown, this proposal is also aimed to understand molecular mechanisms and cellular functions regulated by these proteins in neuronal cell lines, primary cultured neurons, and transgenic mice models. We hypothesize that AKAPll0 can serve as Go_13-interacting protein of that connects Gctl3 with regulatory subunit of protein kinase A and thus regulates cell functions. We will test this hypothesis by determining functional interactions between Gctl3 and AKAPll0 in in vitro and in vivo experiments. Aim 1. Define mechanisms of biochemical interaction of AKAP110 with G0d3. We will first express AKAPII0 and Gctl3 as well as recombinant proteins containing various combinations of their individual functional domains. The Gtz regions necessary for binding to AKAP1 l0 will be localized using chimeric G13/Gi2 _-subunits. Gc_ mutants with substitutions within identified G0_regions will be analyzed for their ability to interact with AKAPI10. Aim 2. Analyze the regulation of Gtxl3-AKAPll0 signaling pathway in neuronal cell lines and primary cultured neurons. We will investigate the physiological relevance of the Gtxl3-AKAP110 interaction in primary cultured neurons. We also will analyze signaling pathways regulated by Gctl3-AKAPll0 and their downstream targets. Aim 3 Define the role of G_13 and AKAP110 in brain. To further understand the Gtxl3-AKAP1 l0 function in vivo, we will generate transgenic mice with constitutive activation of Gctl3-AKAP110 pathway. This study will provide new information about signaling and cellular responses regulated by heterotrimeric G proteins. PERFORMANCE SITE ========================================Section End===========================================

超出提供的空间。异三聚体G蛋白和蛋白激酶A(PKA)是两种重要的信号传递体，它们从多种细胞表面受体传递信号以产生生理反应。各种激素激活PKA的机制包括刺激性G蛋白Gs，它激活腺苷环化酶，导致cAMP的产生。我们发现了一种不需要cAMP的新的PKA刺激机制。通过酵母双杂交筛选，我们发现GI3蛋白的？x亚基与PKA锚定蛋白家族的一个成员AKAP110相互作用。我们的数据提示，AKAP110可能在异三聚体G蛋白和cAMP非依赖性激活PKA之间提供了联系。了解G13/AKAPll0蛋白家族成员对细胞功能做出贡献的确切分子机制是这项提议的主要目标。GcO_3广泛表达，而AKAP 110的表达仅限于特定部位，包括小脑。由于G_13和AKAPll0在小脑中的生理作用尚不清楚，本研究还旨在了解这些蛋白在神经细胞系、原代培养神经元和转基因小鼠模型中调节的分子机制和细胞功能。我们推测，AKAPll0可以作为GO_13的相互作用蛋白，连接Gctl3和蛋白激酶A的调节亚基，从而调节细胞功能。我们将通过在体外和体内实验中确定Gctl3和AKAPll0之间的功能相互作用来验证这一假设。目的1.明确AKAP110与G0d3的生化相互作用机制。我们将首先表达AKAPII0和Gctl3以及包含它们各自功能结构域的各种组合的重组蛋白。与AKAP1 l0结合所需的Gtz区域将使用嵌合的G13/GI2_-亚基进行定位。在已确定的G0_区内有替换的GC_突变体将被分析其与AKAPI10相互作用的能力。目的2.分析Gtxl3-AKAPll0信号通路在神经细胞系和原代培养神经元中的调控。我们将研究Gtx13-AKAP110相互作用在原代培养神经元中的生理学相关性。我们还将分析Gctl3-AKAPll0调控的信号通路及其下游靶点。目的3明确G_13和AKAP110在脑内的作用。为了进一步了解Gtx13-AKAP1 10在体内的功能，我们将建立具有Gctl3-AKAP110途径组成性激活的转基因小鼠。这项研究将提供有关异源三聚体G蛋白调控的信号和细胞反应的新信息。表演网站========================================Section End===========================================