New G-Protein Signaling Pathways

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的信号通路和功能。我们最近关注的是异三聚体g蛋白的Gi家族的m2-毒蕈碱乙酰胆碱受体（m2-AchR）的激活，其抑制心率、收缩和舒张。这项提案取代了我们之前对这些途径的拨款。本研究在体外和体内验证了β -肾上腺素能受体（β - 2ar）偶联Gi家族成员在收缩-舒张和细胞存活变化中的重要性。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，我们敲除了在心脏中表达的Gi家族成员（Galphai2、Galphai3和galphai）。α亚基的失活有效地去除异源三聚体。这些基因敲除将使我们能够确定收缩和松弛的抑制机制，首先通过确定介导反应的特定gi蛋白，然后确定每种基因敲除如何影响其他信号中间体（specific Aim 1）。beta2AR-Gi偶联也与抗凋亡途径有关（Specific Aim 2）。我们将扩展我们的分析，以确定这些特定途径在体内急性和慢性β -肾上腺素能刺激的重要性，同时使用药理学和遗传学探针（specific Aim 3）。这些途径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，本研究使用心脏细胞作为模型系统，并测试了维持g蛋白信号特异性的新范例（Specific Aim 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，由单一受体激活的多个Gi家族成员不仅通过重叠表型（即家族成员可以相互替代）或单一家族成员对单一反应转导信号，而且还通过所需的组合转导信号。许多特异性是通过胃肠道通路的特定组合来维持的，这是一个新的范例。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扩大和重新评估我们关于g蛋白如何在信号传导中保持特异性和分类多重输入和输出的概念。我们将通过比较两种非常不同的受体所需的Gi家族成员来验证这一范式。

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.