Molecular Mechanisms of Cardiac beta1AR-EGFR Association and Signaling

描述(申请人提供)：心力衰竭仍然是最普遍和最昂贵的综合征之一，在美国和世界各地导致发病率和死亡率越来越高。β1肾上腺素能受体(Beta1AR)信号在交感神经传入时对心功能的调节至关重要，但在心力衰竭的发展过程中，由于慢性儿茶酚胺刺激而变得有害。最近有报道称，在儿茶酚胺长期升高的刺激条件下，β1AR介导的表皮生长因子受体(EGFR)的反式激活可以传递心脏保护作用，维持正常的心功能，并通过减少细胞凋亡来促进细胞存活。β1受体介导的表皮生长因子受体反式激活传递心肌保护信号的分子机制(S)尚不清楚，受此过程调控的细胞凋亡途径也不清楚。最近的研究表明，β1AR和EGFR以G蛋白偶联受体激酶(GRK)依赖的受体复合体的形式结合在一起，尽管特定的GRK在调节β1AR和EGFR结合中的作用尚不清楚。此外，儿茶酚胺或EGF对Beta1AR-EGFR复合体的不同配体刺激可诱导不同受体复合体的内化和下游效应分子的运输。虽然对EGFR信号的蛋白质组学研究已经开始描述EGF刺激下组装的蛋白质网络或信号小体，但EGFR信号小体对儿茶酚胺介导的Beta1AR-EGFR复合体刺激的反应还没有被表征。由于β1AR-EGFR结合的分子调控和随后的细胞内信号可能有助于心力衰竭期间有益的心脏结局，表征这一新的信号范例可能导致改进心力衰竭的治疗方法。因此，这项建议的目的是：1)明确心脏GRKs在调节β1 AR-EGFR结合中的作用；2)确定不同刺激β1 AR-EGFR复合体如何影响正常和心力衰竭期间受体的运输和信号转导；3)表征儿茶酚胺刺激Beta1AR-EGFR复合体对正常和心力衰竭期间细胞凋亡信号和EGFR信号体调节的影响。这些目的将通过使用荧光共振能量转移(FRET)、免疫沉淀、放射性配基结合、RT-PCR和质谱学来阐明β1AR-EGFR结合、运输和下游信号反应的动力学来评估。将使用新生小鼠心肌细胞和来自野生型和心脏特异性GRK基因敲除小鼠的全心脏标本来评估正常和心力衰竭期间对Beta1AR-EGFR结合的调节。这些研究将为Beta1AR-EGFR复合体如何发挥心脏保护作用提供新的见解，为开发一种改进的心力衰竭治疗的分子方法确定关键的信号通路。 与公共卫生相关：在美国和越来越多的国外，心力衰竭仍然是发病率和死亡率的主要原因。通过确定在心力衰竭期间保护心脏功能和提高存活率的分子机制，可以开发新的治疗策略来增强有益的心脏信号。

DESCRIPTION (provided by applicant): 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. Beta1-adrenergic receptor (beta1AR) 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, beta1AR- 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 beta1AR-mediated EGFR transactivation relays cardioprotective signaling are poorly understood and the apoptotic pathways regulated by this process are unknown. Recent evidence showed that beta1AR 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 beta1AR-EGFR association is unknown. Further, differential ligand stimulation of the beta1AR-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 beta1AR-EGFR complex has not been characterized. Since the molecular regulation of beta1AR-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 beta1AR-EGFR association, 2) determine how differential stimulation of the beta1AR-EGFR complex impacts receptor trafficking and signaling normally and during heart failure, and 3) characterize the impact of catecholamine stimulation of the beta1AR-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 beta1AR-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 beta1AR-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 beta1AR-EGFR complex exerts cardioprotection, identifying key signaling pathways for development of an improved molecular approach to heart failure therapy. PUBLIC HEALTH RELEVANCE: Heart failure remains a leading cause of morbidity and mortality in the United States and increasingly abroad. By defining the molecular mechanisms that lead to preservation of cardiac function and increased survival during heart failure, new therapeutic strategies can be developed to augment beneficial cardiac signaling.