E3 Ligases and Deubiquitinases in GPCR Down Regulation

GPCR 下调中的 E3 连接酶和去泛素酶

基本信息

批准号：
7987494
负责人：
SUDHA K SHENOY
金额：
$ 35.33万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/7987494
关键词：
Adaptor Signaling Protein Address Adenylate Cyclase Adrenergic Agents 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 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 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 internalization receptor recycling response trafficking ubiquitin ligase ubiquitin-protein ligase

项目摘要

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）构成了最大的细胞表面受体家族，至少35％的当前处方药对这些受体分子作用。 GPCR信号与心血管功能的许多方面至关重要。 GPCR信号传导的大小和程度由几个管理因子（包括受体分子本身的寿命）确定。在第一阶段的资金阶段，我们发现细胞表面B2肾上腺素能受体（B2AR）的泛素化确定其在溶酶体中的降解，因此为减弱细胞反应提供了“关闭开关”。我们已经确定了调节激动剂激活的B2AR的细胞内贩运涉及的特定酶活性。因此，含有E3泛素连接酶MDM2的环域泛素泛素可泛素相关的辅助蛋白B- arrestin2，并参与受体内在化的早期步骤，而含有E3 rigase NEDD4的高域将B2AR泛素泛滥导致受体Degradation in Lysosomes in lysosossomessys in lysososemess。将两个连接酶募集到B2AR都是激动剂依赖性的，并且依次发生。我们还表明，两个相关的去泛素酶（DUB），USP20和USP33反转了这种泛素化，并防止受体降解，同时同时促进受体回收到质膜。在此竞争连续应用中提出的工作的中心假设是：“ B-肾上腺素能信号与运输途径密切相关，并涉及不同的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儿心室心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棒反应性的影响。具体目的是：1）确定溶酶体运输在调节棒信号传导中的影响，2）阐明定义棒状素化酶的募集和/或激活的分子机制，并在棒上激活脱普素化酶，并阐明MDM2在MDM2中的机械作用酒吧在心脏中发出信号。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中起关键作用。公共卫生相关性：B肾上腺素能受体（B1和B2 AR）在心脏中表达，对于心脏肌肉的收缩力很重要，尤其是在压力和锻炼过程中。 B2AR还调节平滑肌细胞的放松，该平滑肌细胞在我们体内的血管壁上排列。提出的工作将阐明如何维持棒的细胞表面表达和功能，并获得知识将有助于开发新的治疗疗法，这可能对治疗心力衰竭和血压障碍的治疗有益。