G Proteins and Protein Kinase A Anchoring

描述(申请人提供)：异三聚体G蛋白和蛋白激酶A(PKA)是两种重要的信号传递器，它们从各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G13蛋白的α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/AKAP110蛋白家族成员对细胞功能做出贡献的确切分子机制是本提案的主要目标。Galpha13广泛表达，而AKAP110的表达仅限于特定部位，包括小脑。由于Galpha13和AKAP110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，AKAP110可以作为Galpha13相互作用的蛋白，连接Galpha13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Galpha13和AKAP110之间的功能相互作用来验证这一假设。目的1.明确AKAP110与Galpha13的生化相互作用机制。我们将首先表达AKAP110和Galpha13以及包含它们各自功能结构域的各种组合的重组蛋白。与AKAP110结合所需的Galpha区域将使用嵌合的G13/Gi2Alpha亚基进行定位。在已确定的Galpha区域内具有替换的Galpha突变体将被分析其与AKAP110相互作用的能力。目的2.分析Galpha13-AKAP110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Galpha13-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Galpha13-AKAP110调控的信号通路及其下游靶点。目的3.明确Galpha13和AKAP110在脑中的作用。为了进一步了解Galpha13-AKAP110在体内的功能，我们将建立具有Galpha13-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。

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.