New G-Protein Signaling Pathways

描述(由申请者提供)：我们的总体目标是确定正常和疾病心脏中的信号通路和功能。我们最近关注的是Gi家族异三聚体G蛋白的M2-M受体(M2-ACHR)激活，它抑制心率、收缩和松弛。这项提议取代了我们之前对这些路径的拨款。这项建议测试了β2-肾上腺素能受体(Beta2AR)偶联GI家族成员的重要性，导致了体外和体内收缩-松弛和细胞存活的变化。临界未知数是通过这些多个基因产物及其生理后果所使用的特定途径。为了确定转导这些信号的特定基因产物，我们对心脏表达的每个GI家族成员(Galphai2、Galphai3和Galpho)进行了敲除。α亚基的失活有效地去除了杂三聚体。这些敲除将使我们能够确定抑制收缩和松弛的机制，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中间体(特定目标1)。β2AR-GI偶联也与抗细胞凋亡途径有关(特定目标2)。我们将扩展我们的分析，以确定这些特定的途径在体内的重要性，无论是在急性和慢性β-肾上腺素能刺激下，使用药理学和遗传学探针(特定目标3)。这些通路可能在心肌肥厚和衰竭的发病机制和治疗中起关键作用。同样重要的是，这项建议使用心肌细胞作为模型系统，并测试了一种保持G蛋白信号特异性的新范式(特定目标4)。正如在背景和意义中所讨论的，我们现在已经确定，每个GI家庭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的表型(即家族成员可以相互替代)或由单个家族成员向单一反应传递信号，而且还通过所需的组合传递信号。GI途径的特定组合保持了大部分的特异性，这是一种新的范式。虽然最初是意想不到的，但我们发现越来越多的例子。这一新的范式要求我们拓宽和重新评估我们的概念，即G蛋白如何在信号传递中保持特异性，并对多个输入和输出进行排序。我们将通过比较两个非常不同的受体所需的GI家族成员来测试这一范式。

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.