E3 Ligases and Deubiquitinases in GPCR Down Regulation

描述（由申请人提供）：G蛋白偶联受体（gpcr）是最大的细胞表面受体家族，目前至少35%的处方药作用于这些受体分子。GPCR信号在心血管功能的许多方面起着至关重要的作用。GPCR信号的大小和范围是由几个控制因素决定的，包括受体分子本身的寿命。在资助的第一个阶段，我们发现细胞表面b2肾上腺素能受体（b2AR）的泛素化决定了它在溶酶体中的降解，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b2ARs的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。这两种连接酶对b2AR的募集是激动剂依赖性的，并且是顺序发生的。我们还发现，两种相关的去泛素酶（DUBS） USP20和USP33可以逆转这种泛素化，防止受体降解，同时促进受体再循环到质膜。在这一竞争性继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肌细胞作为细胞模型系统、RNAi和敲除小鼠，我们将确定泛素化/去泛素化动力学对心血管系统中bAR反应性的影响。具体目的是：1)确定溶酶体转运在调节bAR信号传导中的作用；2)阐明在bAR重敏过程中去泛素酶的募集和/或激活的分子机制；3)阐明Mdm2在心脏中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的分子机制，这可能在平衡生理反应中发挥关键作用。

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.