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Molecular Mechanisms of Cardiac beta1AR-EGFR Association and Signaling

心脏 β1AR-EGFR 关联和信号转导的分子机制

基本信息

批准号：
8794455
负责人：
Douglas Tilley
金额：
$ 37.68万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-15 至 2016-03-10
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8794455
关键词：
ADRBK1 gene Address Adrenergic Receptor Apoptosis Apoptotic Binding Cardiac Cardiac Myocytes Catecholamines Cell Culture System Cell Culture Techniques Cell Survival Chronic Complex Data Development EGF gene Energy Transfer Epidermal Growth Factor Receptor G protein coupled receptor kinase GRK5 gene Gene Expression Heart Heart failure Immunoprecipitation Kinetics Knockout Mice Lead Ligands Mass Spectrum Analysis Mediating Modeling Molecular Morbidity - disease rate Mus Myocardial Infarction Neonatal Outcome Pathway interactions Pattern Phosphorylation Preparation Process Protein Isoforms Proteins Proteomics Receptor Activation Receptor Protein-Tyrosine Kinases Receptor Signaling Regulation Reporting Reverse Transcriptase Polymerase Chain Reaction Role Signal Pathway Signal Transduction Syndrome Testing Therapeutic Transactivation United States heart preservation improved in vivo in vivo Model insight mortality novel novel therapeutic intervention novel therapeutics radioligand receptor receptor internalization response therapy development trafficking

项目摘要

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.

项目概要心力衰竭仍然是最常见和最昂贵的综合征之一，导致发病率和死亡率美国和世界各地的死亡率不断上升。 ¿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调节至关重要，但在以下方面变得有害心力衰竭进展期间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长期升高的儿茶酚胺刺激条件下，维持正常的心脏功能和通过减少细胞凋亡促进细胞存活。 1AR介导的EGFR的分子机制反式激活中继心脏保护信号传导知之甚少，细胞凋亡途径受到调节这个过程是未知的。最近的证据表明 ¿1AR 和 EGFR 作为受体复合物结合以 G 蛋白偶联受体激酶 (GRK) 依赖性方式，尽管特定 GRK 的作用 ¿1AR-EGFR 关联的调节尚不清楚。此外，1AR-EGFR 的差异配体刺激儿茶酚胺或 EGF 复合物被证明可以诱导不同的受体复合物内化，并且下游效应器的贩运。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，响应 EGF 刺激而组装，EGFR 信号体响应儿茶酚胺介导的 ¿1AR-EGFR 复合物的刺激尚未得到表征。自从 ¿1AR-EGFR 关联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有益的心脏结果，这种新颖的信号传导范式的特征可能会导致一种改进的心力衰竭治疗方法。因此，本提案的目的是： 1) 定义心脏 GRK 在 ¿1AR-EGFR 关联的调节中，2) 确定如何差异刺激 ¿1AR-EGFR 复合物影响正常情况下和心力衰竭期间的受体运输和信号传导，以及 3) 描述儿茶酚胺刺激 ¿1AR-EGFR 复合物对细胞凋亡信号传导的影响正常情况下和心力衰竭期间 EGFR 信号体的调节。这些目标将由通过阐明 ¿1AR-EGFR 关联、运输和下游信号反应的动力学使用荧光共振能量转移 (FRET)、免疫沉淀、放射性配体结合、RT-PCR 和质谱分析。正常情况下和心力衰竭期间 ¿1AR-EGFR 关联的调节将是使用新生小鼠心肌细胞和来自野生型和心脏的全心脏制剂进行评估患有或不患有心肌梗塞的特定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一种改进的心力衰竭治疗分子方法。