G Proteins and Protein Kinase A Anchoring

超出所提供的空间。异三体G蛋白和蛋白激酶A （PKA）是两种重要的信号递质，它们传递来自多种细胞表面受体的信号以产生生理反应。PKA对各种激素的激活机制涉及刺激G蛋白，Gs激活腺苷酸环化酶导致cAMP的产生。我们发现了一种新的不需要cAMP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锚定蛋白家族的成员AKAP110相互作用。我们的数据表明，AKAP110可能在异源三聚体G蛋白和不依赖camp的PKA激活之间提供了联系。了解G13/AKAPll0蛋白集合成员参与细胞功能的确切分子机制是本提案的主要目标。GcO3普遍表达，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，本研究也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的分子机制和细胞功能调控。我们假设AKAPll0可以作为连接Gctl3与蛋白激酶A调节亚基的go_13相互作用蛋白，从而调节细胞功能。我们将在体外和体内实验中通过确定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以及包含其各自功能域的各种组合的重组蛋白。结合akap110所需的Gtz区域将使用嵌合G13/Gi2 _-亚基定位。在鉴定出的g0区域内进行替换的Gc_突变体将分析其与AKAPI10相互作用的能力。目标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作用。我们将研究Gtxl3-AKAP110相互作用在原代培养神经元中的生理相关性。我们还将分析Gctl3-AKAPll0及其下游靶点调控的信号通路。目的3明确G_13和AKAP110在脑中的作用。为了进一步了解gtxl3 - akap110在体内的功能，我们将产生Gctl3-AKAP110通路组成性激活的转基因小鼠。本研究将为异源三聚体G蛋白调控的信号和细胞反应提供新的信息。网站性能 ======================================== 节结束 ===========================================

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