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

超出所提供的空间。 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因子，它们从多种细胞表面受体传递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GI 3蛋白的<$x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。理解G13/AKAP 110蛋白系综成员对细胞功能的确切分子机制是本提案的主要目标。GcO 3是普遍表达的，而AKAP 110的表达仅限于特定的位置，包括小脑。由于G_13和AKAP 110在小脑中的生理作用尚不清楚，因此本研究也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细胞功能的分子机制。我们推测AKAP 110可以作为Gct 13的相互作用蛋白，连接Gct 13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Gct 13和AKAP 110之间的功能相互作用来测试这一假设。目标1.定义AKAP 110与G 0 d3的生化相互作用机制。我们将首先表达AKAPII 0和Gctl 3以及含有它们各自功能结构域的各种组合的重组蛋白。结合AKAP 110所必需的Gtz区将使用嵌合G13/G12_亚基定位。将分析在鉴定的G 0_区内具有取代的Gc_突变体与AKAPI 10相互作用的能力。目标2.分析Gtxl 3-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Gtxl 3-AKAP 110相互作用在原代培养的神经元中的生理相关性。我们还将分析Gctl 3-AKAP 110及其下游靶点调控的信号通路。目的3明确G_13和AKAP 110在脑内的作用。为了进一步理解Gtxl 3-AKAP 110在体内的功能，我们将产生具有Gtxl 3-AKAP 110途径的组成型激活的转基因小鼠。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。性能现场=