Molecular Mechanisms of Cardiac beta1AR-EGFR Association and Signaling

描述（由申请人提供）：心力衰竭仍然是最普遍和最昂贵的综合征之一，导致美国和世界各地的发病率和死亡率越来越高。β -肾上腺素能受体（β - 1ar）信号对交感神经输入对心功能的调节至关重要，但在心力衰竭的进展过程中，慢性儿茶酚胺刺激对心功能的调节是有害的。最近，有报道称β 1ar介导的表皮生长因子受体（EGFR）的转激活在儿茶酚胺长期升高的刺激条件下传递心脏保护，维持正常的心功能，并通过减少细胞凋亡促进细胞存活。beta1ar介导的EGFR转激活传递心脏保护信号的分子机制尚不清楚，该过程调节的凋亡途径尚不清楚。最近的证据表明，β - 1ar和EGFR以G蛋白偶联受体激酶（GRK）依赖的方式作为受体复合物结合，尽管特异性GRK在调节β - 1ar -EGFR关联中的作用尚不清楚。此外，儿茶酚胺或EGF对β - 1ar - egfr复合物的不同配体刺激被证明可以诱导不同受体复合物的内化和下游效应物的运输。虽然EGFR信号的蛋白质组学研究已经开始描述响应EGF刺激而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β - 1ar -EGFR复合物刺激的反应尚未被表征。由于beta1AR-EGFR关联的分子调控和随后的细胞内信号传导可能有助于心力衰竭期间有益的心脏结果，因此表征这种新的信号传导模式可能会导致心力衰竭治疗方法的改进。因此，本提案的目的是：1)确定心脏GRKs在调节β - 1ar -EGFR关联中的作用；2)确定β - 1ar -EGFR复合物的差异刺激如何影响正常和心力衰竭期间的受体运输和信号传导；3)表征儿茶酚胺刺激β - 1ar -EGFR复合物对正常和心力衰竭期间凋亡信号传导和EGFR信号体调节的影响。这些目标将通过荧光共振能量转移（FRET）、免疫沉淀、放射寡核苷酸结合、RT-PCR和质谱分析来阐明β - 1ar - egfr关联、运输和下游信号反应的动力学来评估。在正常和心力衰竭期间，将使用新生小鼠心肌细胞和野生型和心脏特异性GRK敲除小鼠的全心制剂来评估β - 1ar - egfr关联的调节，不论有无心肌梗死。这些研究将为β - 1ar - egfr复合物如何发挥心脏保护作用提供新的见解，为开发改进的心力衰竭分子治疗方法确定关键的信号通路。

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.