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===========================================

超过提供的空间。异三聚体G蛋白和蛋白激酶A（PKA）是两个重要的信号发射器，它们从各种细胞表面受体转移信号以产生物理反应。 PKA激活对各种恐怖的既定机制涉及刺激G蛋白GS，它激活腺苷酸环化酶，从而导致CAMP的产生。我们发现了一种新型的PKA刺激机制，不需要扎营。使用酵母两杂交筛选，我们发现GI3蛋白的X亚基与PKA锚定蛋白家族的成员AKAP110相互作用。我们的数据表明，AKAP110可以提供异三聚体G蛋白与cAMP独立于PKA的激活之间的联系。了解G13/AKAPLL0蛋白质集合成员有助于细胞功能的确切分子机制是该建议的主要目标。 GCO3普遍表达，而AKAP 110表达仅限于特定位置，包括小脑。由于G_13和AKAPLL0在小脑中的身体作用尚不清楚，因此该建议还旨在了解这些蛋白质在神经元细胞系，原代培养神经元和转基因小鼠模型中受调节的分子机制和细胞功能。我们假设AKAPLL0可以用作GO_13相互作用的蛋白质，将GCTL3与蛋白激酶A的调节亚基联系起来，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检验这一假设。 AIM 1。定义AKAP110与G0D3的生化相互作用的机制。我们将首先表达AKAPII0和GCTL3以及包含其各个功能域组合的重组蛋白。与AKAP1 L0结合所需的GTZ区域将使用嵌合G13/GI2 _-subunits定位。将分析已确定的G0_Rigions内部取代的GC_突变体，以与AKAPI10相互作用的能力进行分析。 AIM 2。分析神经元细胞系和原发性培养神经元中GTXL3-AKAPLL0信号通路的调节。我们将研究原发性培养神经元中GTXL3-AKAP110相互作用的物理相关性。我们还将分析由GCTL3-AKAPLL0调节的信号通路及其下游目标。 AIM 3定义了G_13和AKAP110在大脑中的作用。为了进一步了解体内的GTXL3-AKAP1 L0功能，我们将通过构成性GCTL3-AKAP110途径生成转基因小鼠。这项研究将提供有关异三聚体G蛋白调节的信号传导和细胞反应的新信息。性能站点====段end =====