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越来越多的发病率和死亡率。β 1-肾上腺素能受体（β 1AR）信号传导对交感神经输入引起的心脏功能调节至关重要，但在心力衰竭进展期间对慢性儿茶酚胺刺激的反应变得有害。最近，据报道，β 1 AR介导的表皮生长因子受体（EGFR）的反式激活在慢性升高的儿茶酚胺刺激条件下中继心脏保护，维持正常的心脏功能，并通过减少细胞凋亡促进细胞存活。β 1 AR介导的EGFR反式激活传递心脏保护信号的分子机制知之甚少，受此过程调节的凋亡途径也未知。最近的证据表明，β 1 AR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相关联，尽管特定GRK在调节β 1 AR-EGFR关联中的作用尚不清楚。此外，儿茶酚胺或EGF对β 1 AR-EGFR复合物的不同配体刺激显示诱导趋异受体复合物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蛋白质网络，或信号体，组装在EGF刺激的反应，EGFR信号体的反应，儿茶酚胺介导的刺激的β 1 AR-EGFR复合物还没有得到表征。由于beta1 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心力衰竭期间的有益心脏结局，因此这种新型信号传导模式的表征可能导致心力衰竭的改善治疗方法。因此，本提案的目的是：1）确定心脏GRKs在调节β 1 AR-EGFR结合中的作用，2）确定β 1 AR-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，和3）表征β 1 AR-1的儿茶酚胺刺激的影响，EGFR复合物对凋亡信号传导和EGFR信号体的调节的作用。这些目标将通过使用荧光共振能量转移（FRET）、免疫沉淀、放射性配体结合、RT-PCR和质谱法阐明β 1AR-EGFR结合、运输和下游信号应答的动力学来评估。将使用新生鼠心肌细胞和来自有或无心肌梗死的野生型和心脏特异性GRK敲除小鼠的全心脏制备物评估正常和心力衰竭期间β 1 AR-EGFR结合的调节。这些研究将为beta1 AR-EGFR复合物如何发挥心脏保护作用提供新的见解，确定关键信号通路，以开发改进的心力衰竭治疗分子方法。 公共卫生相关性：心力衰竭仍然是美国发病率和死亡率的主要原因，在国外也越来越多。通过定义导致心力衰竭期间心脏功能保留和存活率增加的分子机制，可以开发新的治疗策略以增强有益的心脏信号传导。

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.