G Proteins and Protein Kinase A Anchoring

描述（由申请人提供）：异源三聚体G蛋白和蛋白激酶A（PKA）是两种重要的信号传导剂，其从多种细胞表面受体转移信号以产生生理反应。响应于各种激素的PKA活化的既定机制涉及刺激性G蛋白Gs，其活化腺苷酸环化酶，导致cAMP的产生。我们发现了一种不需要cAMP的PKA刺激的新机制。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AKAP 110相互作用。我们的数据表明，AKAP 110可能提供了异源三聚体G蛋白和cAMP非依赖性PKA激活之间的联系。了解G13/AKAP 110蛋白质集合体成员对细胞功能的确切分子机制是本提案的主要目标。Galpha 13广泛表达，而AKAP 110的表达仅限于特定的部位，包括小脑。由于Galpha 13和AKAP 110在小脑中的生理作用尚不清楚，因此本研究还旨在了解这些蛋白在神经元细胞系、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AKAP 110可以作为Galpha 13相互作用蛋白，连接Galpha 13与蛋白激酶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Galpha 13和AKAP 110之间的功能相互作用来验证这一假设。目标1.定义AKAP 110与Galpha 13的生化相互作用机制。我们将首先表达AKAP 110和Galpha 13以及含有其各自功能结构域的各种组合的重组蛋白。将使用嵌合G13/Gi 2 α-亚基定位结合AKAP 110所必需的Ga区。将分析在鉴定的Ga区域内具有取代的Ga突变体与AKAP 110相互作用的能力。目标2.分析Galpha 13-AKAP 110信号通路在神经元细胞系和原代培养神经元中的调控。我们将研究原代培养神经元中Galpha 13-AKAP 110相互作用的生理相关性。我们还将分析Galpha 13-AKAP 110及其下游靶点调控的信号通路。目标3.明确Galpha 13和AKAP 110在大脑中的作用。为了进一步了解Galpha 13-AKAP 110在体内的功能，我们将产生具有Galpha 13-AKAP 110通路组成性激活的转基因小鼠。本研究将为异源三聚体G蛋白调控的信号转导和细胞反应提供新的信息。

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.