Molecular Mechanisms of Cardiac beta1AR-EGFR Association and Signaling

项目摘要 心力衰竭仍然是最普遍和最昂贵的综合征之一，导致发病率和 美国的死亡率和世界各地的死亡率都在上升。肾上腺素能受体(1AR)信号转导 对交感神经输入反应的心功能调节至关重要，但在 心力衰竭进展过程中对慢性儿茶酚胺刺激的反应。最近，1AR- 据报道，表皮生长因子受体(EGFR)介导的反式激活可传递心脏保护作用 在儿茶酚胺刺激慢性升高的情况下，维持正常的心功能和 通过减少细胞凋亡促进细胞存活。受体介导表皮生长因子受体的分子机制(S) 对心肌保护信号转导途径知之甚少，对细胞凋亡途径的调控也很少。 通过这一过程是未知的。最近的证据表明，1AR和EGFR以受体复合体的形式联系在一起 在G蛋白偶联受体激酶(GRK)依赖的方式中，尽管特定的GRK在 1AR-EGFR关联的调节尚不清楚。此外，差异配体对1AR-EGFR的刺激作用 儿茶酚胺或EGF的络合物可诱导发散受体复合体内化和 下游效应器的贩运。虽然对EGFR信号的蛋白质组学研究已经开始描述蛋白质 网络，或信号体，为响应EGF刺激而组装，EGFR信号体对 儿茶酚胺对1AR-EGFR复合体的刺激作用尚未明确。自.以来 1AR-EGFR结合的分子调控和随后的细胞内信号转导可能有助于 在心力衰竭期间有益的心脏结果，这种新的信号范式的特征可能导致 一种治疗心力衰竭的改进方法。因此，本提案的目的是：1)界定 心脏GRKs在AR-EGFR关联的调节中，2)决定如何区分刺激 ？1AR-EGFR复合体影响正常和心力衰竭期间受体的转运和信号转导，以及3) 表征儿茶酚胺刺激1AR-EGFR复合体对细胞凋亡信号和 正常和心力衰竭时EGFR信号小体的调节。这些目标将通过以下方式进行评估 阐明1AR-EGFR结合、转运和下游信号反应的动力学 荧光共振能量转移(FRET)、免疫沉淀、放射性配基结合、RT-PCR的应用 和质谱学。正常和心力衰竭期间1AR-EGFR结合的调节将是 用新生小鼠心肌细胞和来自野生型和心脏的全心脏标本进行评估 有或没有心肌梗死的特异性GRK基因敲除小鼠。这些研究将提供新的洞察力 1AR-EGFR复合体如何发挥心脏保护作用，识别促进血管生成的关键信号通路 心力衰竭治疗的改进分子方法。

Project Summary Heart failure remains one of the most highly prevalent and costly syndromes that leads to morbidity and mortality in the United States and increasingly around the world. ¿1-adrenergic receptor (¿1AR) signaling is critical to the regulation of cardiac function in response to sympathetic input but becomes deleterious in response to chronic catecholamine stimulation during the progression of heart failure. Recently, ¿1AR- mediated transactivation of epidermal growth factor receptor (EGFR) was reported to relay cardioprotection under conditions of chronically elevated catecholamine stimulation, maintaining normal cardiac function and promoting cell survival via reduced apoptosis. The molecular mechanism(s) by which ¿1AR-mediated EGFR transactivation relays cardioprotective signaling are poorly understood and the apoptotic pathways regulated by this process are unknown. Recent evidence showed that ¿1AR and EGFR associate as a receptor complex in a G protein-coupled receptor kinase (GRK)-dependent manner, though the role of specific GRKs in the regulation of ¿1AR-EGFR association is unknown. Further, differential ligand stimulation of the ¿1AR-EGFR complex by catecholamine or EGF was shown to induce divergent receptor complex internalization and trafficking of downstream effectors. While proteomic studies of EGFR signaling have begun to describe protein networks, or signalosomes, assembled in response to EGF stimulation, the EGFR signalosome response to catecholamine-mediated stimulation of the ¿1AR-EGFR complex has not been characterized. Since the molecular regulation of ¿1AR-EGFR association and subsequent intracellular signaling may contribute to beneficial cardiac outcomes during heart failure, characterization of this novel signaling paradigm may lead to an improved therapeutic approach to heart failure. Thus, the aims of this proposal are to: 1) define the role of cardiac GRKs in the regulation of ¿1AR-EGFR association, 2) determine how differential stimulation of the ¿1AR-EGFR complex impacts receptor trafficking and signaling normally and during heart failure, and 3) characterize the impact of catecholamine stimulation of the ¿1AR-EGFR complex on apoptotic signaling and regulation of the EGFR signalosome normally and during heart failure. These aims will be assessed by elucidating the kinetics of ¿1AR-EGFR association, trafficking and downstream signaling responses through the use of fluorescent resonance energy transfer (FRET), immunoprecipitation, radioligand binding, RT-PCR and mass spectroscopy. Regulation of ¿1AR-EGFR association normally and during heart failure will be assessed using neonatal murine cardiomyocytes and whole heart preparations from wild-type and cardiac- specific GRK knockout mice with or without myocardial infarction. These studies will provide novel insight into how the ¿1AR-EGFR complex exerts cardioprotection, identifying key signaling pathways for development of an improved molecular approach to heart failure therapy.