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，其导致发病率和死亡率。 死亡率在美国和越来越多的世界各地。<$1-肾上腺素能受体（<$1AR）信号传导是 对响应交感神经输入的心脏功能的调节至关重要，但在 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）介导的反式激活可中继心脏保护作用 在慢性升高的儿茶酚胺刺激条件下，维持正常的心脏功能， 通过减少凋亡促进细胞存活。1 AR介导的EGFR的分子机制 反式激活中继心脏保护信号的了解很少，细胞凋亡途径调节 这个过程是未知的。最近的证据表明，1AR和EGFR作为受体复合物缔合， 在G蛋白偶联受体激酶（GRK）依赖的方式，虽然特定的GRK在 1AR-EGFR相关性的调控尚不清楚。此外，差异配体刺激的<$1AR-EGFR 复合物的儿茶酚胺或表皮生长因子显示诱导趋异受体复合物的内化， 下游效应物的运输。虽然EGFR信号传导的蛋白质组学研究已经开始描述蛋白质 网络，或信号体，组装在EGF刺激，EGFR信号体反应， 儿茶酚胺介导的对Δ 1AR-EGFR复合物的刺激尚未被表征。以来 AR-EGFR结合的分子调节和随后的细胞内信号传导可能有助于 在心力衰竭期间有益的心脏结果，这种新的信号传导模式的特征可能导致 心力衰竭的改良治疗方法。因此，本建议的目的是：1）确定 心脏GRKs在调节AR-EGFR的关联，2）确定如何差异刺激的 1AR-EGFR复合物影响受体运输和信号正常和心力衰竭期间，和3） 表征儿茶酚胺刺激<$1AR-EGFR复合物对凋亡信号传导的影响， 正常和心力衰竭期间EGFR信号体的调节。这些目标将通过以下方式进行评估： 通过以下方法阐明了1AR-EGFR结合、运输和下游信号应答的动力学： 利用荧光共振能量转移（FRET）、免疫沉淀、放射性配体结合、RT-PCR 和质谱。正常情况下和心力衰竭期间对AR-EGFR结合的调节将是 使用新生鼠心肌细胞和来自野生型和心脏的全心脏制备物进行评估， 有或没有心肌梗死的GRK基因敲除小鼠。这些研究将提供新的见解， 1AR-EGFR复合物如何发挥心脏保护作用，确定发展的关键信号通路， 心力衰竭治疗的改良分子方法。

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.