New G-Protein Signaling Pathways

新的 G 蛋白信号通路

• 批准号: 6619040
• 负责人: RICHARD M MORTENSEN
• 金额: $ 33.49万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6619040
• 关键词: G protein; apoptosis; beta adrenergic receptor; biological signal transduction; calcium flux; cardiac myocytes; enzyme activity; gene targeting; genetically modified animals; heart contraction; heart function; laboratory mouse; phospholamban; protein isoforms; protein structure function; receptor coupling; receptor expression; tissue /cell culture

DESCRIPTION (provided by applicant): Our overall goal is to determine signaling pathways and function in normal and diseased hearts. We have recently focused on the m2-muscarinic acetylcholine receptor (m2-AchR) activation of the Gi family of heterotrimeric G-proteins which inhibit heart rate, contraction and relaxation. This proposal replaces our previous grant on these pathways. This proposal tests the importance of beta2-adrenergic receptor (beta2AR) coupling Gi family members leading to changes in contraction-relaxation and cell survival both in vitro and in vivo. Critical unknowns are the specific pathways used through these multiple gene products and the physiologic consequences. To determine the specific gene products transducing these signals, we have produced knockouts of each of the Gi family members expressed in heart (Galphai2, Galphai3, and Galphao). Inactivation of the alpha subunits effectively removes the heterotrimer. These knockouts will allow us to determine the mechanism of inhibition of contraction and relaxation, first by determining the specific Gi-proteins, which are mediating the responses, and second determining how each knockout affects other signaling intermediates (Specific Aim 1). The beta2AR-Gi coupling has also been implicated in antiapoptotic pathways (Specific Aim 2). We will extend our analysis to determine the importance of these specific pathways in vivo both with acute and chronic beta-adrenergic stimulation using both pharmacological and genetic probes (Specific Aim 3). These pathways may be critical to the pathogenesis and treatment of cardiac hypertrophy and failure. Equally important, this proposal uses the cardiac cell as a model system and tests a new paradigm for maintaining specificity in G-protein signaling (Specific Aim 4). As discussed in Background and Significance, we have now established that each of the Gi family members have specific functions. In addition, multiple Gi family members activated by a single receptor transduce signals not just by overlapping phenotypes (i.e. family members can substitute for each other) or by a single family member to a single response, but also through required combinations. That much of the specificity is maintained by specific combinations of Gi pathways is a new paradigm. While initially unexpected, we have found a growing number of examples. This new paradigm requires us to broaden and reevaluate our concepts about how G-proteins maintain specificity in signaling and sort multiple inputs and outputs. We will test this paradigm by comparing the Gi family members required for 2 very different receptors.

描述（由申请人提供）：我们的总体目标是确定正常和患病心脏中的信号通路和功能。我们最近集中于抑制心率、收缩和舒张的异源三聚体G蛋白的Gi家族的m2-毒蕈碱乙酰胆碱受体（m2-AchR）激活。这项建议取代了我们以前对这些途径的资助。该提议测试β 2-肾上腺素能受体（β 2AR）偶联Gi家族成员导致体外和体内收缩-舒张和细胞存活变化的重要性。关键的未知因素是通过这些多基因产物使用的特定途径和生理后果。为了确定转导这些信号的特定基因产物，我们已经产生了在心脏中表达的每个Gi家族成员（Galphai 2、Galphai 3和Galphao）的敲除。 α亚基的失活有效地去除了异源三聚体。这些敲除将使我们能够确定抑制收缩和舒张的机制，首先通过确定介导反应的特定Gi蛋白，其次确定每个敲除如何影响其他信号传导中间体（特定目标1）。β 2AR-Gi偶联也涉及抗凋亡途径（特异性目的2）。 我们将扩展我们的分析，以确定这些特定途径在体内的重要性，无论是急性和慢性β-肾上腺素能刺激使用药理学和遗传探针（具体目标3）。这些通路可能对心肌肥厚和心力衰竭的发病机制和治疗至关重要。同样重要的是，该提案使用心脏细胞作为模型系统，并测试了维持G蛋白信号传导特异性的新范例（具体目标4）。正如背景和意义中所讨论的，我们现在已经确定Gi家族的每个成员都有特定的功能。此外，由单个受体激活的多个Gi家族成员不仅通过重叠表型（即家族成员可以相互替代）或通过单个家族成员对单个应答进行信号传导，而且还通过所需的组合进行信号传导。大部分特异性由Gi途径的特定组合维持是一种新的范例。虽然最初出乎意料，但我们发现了越来越多的例子。这种新的范式要求我们拓宽和重新评估我们关于G蛋白如何保持信号特异性和对多个输入和输出进行排序的概念。我们将通过比较两种非常不同的受体所需的Gi家族成员来测试这种范式。