E3 Ligases and Deubiquitinases in GPCR Down Regulation

GPCR 下调中的 E3 连接酶和去泛素酶

• 批准号: 8280416
• 负责人: SUDHA K SHENOY
• 金额: $ 34.97万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8280416
• 关键词: Adaptor Signaling Protein; Address; Adenylate Cyclase; Adrenergic Agents; Adrenergic Antagonists; Adrenergic Receptor; Adrenergic beta-Antagonists; Affect; Affinity; Agonist; Arrestins; Attenuated; Binding; Biological Models; Blood Pressure; Blood Vessels; Cardiac; Cardiac Myocytes; Cardiovascular Physiology; Cardiovascular system; Catecholamines; Cell Line; Cell Surface Receptors; Cell membrane; Cell model; Cell surface; Chronic; Coupled; Coupling; Cyclic AMP; Deubiquitination; Development; Disease; Drug Prescriptions; Epinephrine; Equilibrium; Exercise; Family; Funding; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GRK; Goals; Half-Life; Heart; Heart failure; Homeostasis; Knockout Mice; Knowledge; Left Ventricular Function; Ligase; Link; Lysosomes; Maintenance; Mediating; Molecular; Mus; Muscle Cells; Myocardium; Neonatal; Norepinephrine; Pathway interactions; Patients; Phosphorylation; Physiological; Plasma; Play; Post-Translational Protein Processing; Process; Production; Proteins; RNA Interference; Receptor Down-Regulation; Receptor Signaling; Recycling; Regulation; Relaxation; Role; Signal Transduction; Smooth Muscle Myocytes; Staging; Stress; Testing; Ubiquitination; Ventricular; Work; adrenergic; carvedilol; functional loss; improved; in vivo; novel; novel therapeutics; prevent; public health relevance; receptor; receptor expression; receptor internalization; receptor recycling; response; trafficking; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) constitute the largest cell-surface receptor family and at least 35% of currently prescribed drugs act on these receptor molecules. GPCR signaling is critically involved in many aspects of cardiovascular function. The magnitude and extent of GPCR signaling is determined by several governing factors including the lifetime of the receptor molecule itself. During the first period of funding, we have found that ubiquitination of the cell-surface b2 adrenergic receptor (b2AR) determines its degradation in lysosomes, thus providing an 'off switch' for attenuating cellular responses. We have identified specific enzymatic activities involved in regulating the intracellular trafficking of agonist-activated b2ARs. Thus, the RING-domain containing E3 ubiquitin ligase Mdm2 ubiquitinates the receptor associated adaptor protein b-arrestin2 and is involved in early steps of receptor internalization while the HECT- domain containing E3 ligase Nedd4 ubiquitinates the b2AR leading to receptor degradation in the lysosomes. Recruitment of both ligases to the b2AR is agonist-dependent and occurs sequentially. We have also shown that two related deubiquitinases (DUBS), USP20 and USP33 reverse this ubiquitination and prevent receptor degradation while concomitantly promoting receptor recycling to the plasma membrane. The central hypothesis for the proposed work in this competing continuation application is: "b-adrenergic signaling is intimately linked to trafficking pathways and involves dynamic regulation by distinct E3 ligases and deubiquitinases". By using aortic vascular smooth muscle cells and neonatal ventricular myocytes as cellular model systems, RNAi and knockout mice, we will define the impact of ubiquitination/deubiquitination dynamics on bAR responsiveness in the cardiovascular system. The specific aims are: 1) To determine the effects of lysosomal trafficking in regulating bAR signaling, 2) To elucidate the molecular mechanisms that define the recruitment and/or activation of deubiquitinases during bAR resensitization and 3) To elucidate the mechanistic role of Mdm2 in bAR signaling in the heart. The long-term goal of this project is to understand the molecular mechanisms that integrate G protein-coupled receptor trafficking and signaling, which could play a critical role in balancing physiological responsiveness. PUBLIC HEALTH RELEVANCE: b adrenergic receptors (b1 and b2 ARs) are expressed in the heart and are important for the contractility of heart muscles, especially during stress and exercising. b2ARs also regulate the relaxation of smooth muscle cells that line the wall of blood vessels in our body. The proposed work will elucidate how cell surface expression and function of the bARs are maintained and the knowledge gained will help the development of novel therapeutics, which could be beneficial in the treatment of heart failure and blood pressure disorders.

描述（由申请人提供）：G蛋白偶联受体（GPCR）构成最大的细胞表面受体家族，目前至少35%的处方药物作用于这些受体分子。 GPCR 信号转导与心血管功能的许多方面密切相关。 GPCR 信号传导的强度和范围由几个控制因素决定，包括受体分子本身的寿命。在第一期资助期间，我们发现细胞表面 b2 肾上腺素受体 (b2AR) 的泛素化决定了其在溶酶体中的降解，从而为减弱细胞反应提供了一个“关闭开关”。我们已经确定了参与调节激动剂激活的 b2AR 细胞内运输的特定酶活性。因此，含有 E3 泛素连接酶 Mdm2 的 RING 结构域泛素化受体相关的衔接蛋白 b-arrestin2，并参与受体内化的早期步骤，而含有 E3 连接酶 Nedd4 的 HECT 结构域泛素化 b2AR，导致溶酶体中的受体降解。两种连接酶向 b2AR 的募集是激动剂依赖性的并且依次发生。我们还表明，两种相关的去泛素酶 (DUBS) USP20 和 USP33 可以逆转这种泛素化并防止受体降解，同时促进受体再循环到质膜。这项竞争性延续申请中拟议工作的中心假设是：“b-肾上腺素信号传导与运输途径密切相关，并涉及不同 E3 连接酶和去泛素酶的动态调节”。通过使用主动脉血管平滑肌细胞和新生心室肌细胞作为细胞模型系统、RNAi 和基因敲除小鼠，我们将定义泛素化/去泛素化动力学对心血管系统中 bAR 反应性的影响。具体目标是：1) 确定溶酶体运输对调节 bAR 信号传导的影响，2) 阐明 bAR 重新敏化过程中去泛素酶招募和/或激活的分子机制，3) 阐明 Mdm2 在心脏 bAR 信号传导中的机制作用。该项目的长期目标是了解整合 G 蛋白偶联受体运输和信号传导的分子机制，这可能在平衡生理反应性方面发挥关键作用。 公众健康相关性：b 肾上腺素能受体（b1 和 b2 AR）在心脏中表达，对心肌收缩力很重要，尤其是在压力和运动期间。 b2AR 还调节我们体内血管壁上的平滑肌细胞的松弛。拟议的工作将阐明 bAR 的细胞表面表达和功能是如何维持的，所获得的知识将有助于开发新的疗法，这可能有益于心力衰竭和血压疾病的治疗。