G Proteins and Protein Kinase A Anchoring

G 蛋白和蛋白激酶 A 锚定

• 批准号: 6574732
• 负责人: TATYANA A VOYNO-YASENETSKAYA
• 金额: $ 31.03万
• 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/6574732
• 关键词: A kinase anchoring protein; G protein; biological signal transduction; cAMP response element binding protein; chimeric proteins; enzyme activity; enzyme mechanism; functional /structural genomics; genetically modified animals; immunoprecipitation; laboratory mouse; neuroregulation; protein kinase A; protein protein interaction; proteomics; recombinant proteins; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): HeterotrimericG 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 alpha subunit of G13 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/AKAP110 protein ensemble contribute to cellular functions is the major goal of this proposal. Galpha13 is expressed ubiquitously, whereas AKAP 110 expression is restricted to particular locations, including cerebellum. As the physiological role of Galpha13 and AKAP110 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 AKAP110 can serve as Galpha13-interacting protein that connects Galpha13 with regulatory subunit of protein kinase A and thus regulates cell functions. We will test this hypothesis by determining functional interactions between Galpha13 and AKAP110 in in vitro and in vivo experiments. Aim 1. Define mechanisms of biochemical interaction of AKAP110 with Galpha13. We will first express AKAP110 and Galpha13 as well as recombinant proteins containing various combinations of their individual functional domains. The Galpha regions necessary for binding to AKAP110 will be localized using chimeric G13/Gi2alpha-subunits. Galpha mutants with substitutions within identified Galpha regions will be analyzed for their ability to interact with AKAP110. Aim 2. Analyze the regulation of Galpha13-AKAP110 signaling pathway in neuronal cell lines and primary cultured neurons. We will investigate the physiological relevance of the Galpha13-AKAP110 interaction in primary cultured neurons. We also will analyze signaling pathways regulated by Galpha13-AKAP110 and their downstream targets. Aim 3. Define the role of Galpha13 and AKAP110 in brain. To further understand the Galpha13-AKAP110 function in vivo, we will generate transgenic mice with constitutive activation of Galpha13-AKAP110 pathway. This study will provide new information about signaling and cellular responses regulated by heterotrimeric G proteins.

描述（由申请人提供）：异三聚合物蛋白和蛋白激酶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生理反应。 PKA激活对各种激素的既定机制涉及刺激G蛋白GS，它激活腺苷酸环化酶，导致CAMP产生。我们发现了一种新型的PKA刺激机制，不需要扎营。使用酵母双杂交筛选，我们发现G13蛋白的α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。理解G13/AKAP110蛋白质合奏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。 Galpha13普遍表达，而AKAP 110表达仅限于特定位置，包括小脑。由于Galpha13和Akap110在小脑中的生理作用尚不清楚，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调节的分子机制和细胞功能。我们假设AKAP110可以用作GALPHA13相互作用的蛋白，该蛋白将Galpha13与蛋白激酶A的调节亚基连接起来，从而调节细胞功能。我们将通过在体外和体内实验中确定Galpha13和AKAP110之间的功能相互作用来检验这一假设。 AIM 1。定义AKAP110与Galpha13的生化相互作用的机制。我们将首先表达AKAP110和GALPHA13，以及包含其各个功能域组合的重组蛋白。与AKAP110结合所需的Galpha区域将使用嵌合G13/GI2Alpha-subunits进行定位。将分析已确定的Galpha区域内取代的Galpha突变体，以与AKAP110相互作用的能力分析。 AIM 2。分析神经元细胞系和原发性培养神经元中Galpha13-Akap110信号通路的调节。我们将研究原发性培养神经元中Galpha13-Akap110相互作用的生理相关性。我们还将分析由Galpha13-Akap110及其下游目标调节的信号通路。 AIM 3。定义Galpha13和AKAP110在大脑中的作用。为了进一步了解体内的galpha13-Akap110功能，我们将通过构成性激活Galpha13-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。