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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL080525-08
Application #
8280416
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Wong, Renee P
Project Start
2005-08-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
8
Fiscal Year
2012
Total Cost
$349,718
Indirect Cost
$126,968
Name
Duke University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Jean-Charles, P-Y; Freedman, N J; Shenoy, S K (2016) Chapter Nine - Cellular Roles of Beta-Arrestins as Substrates and Adaptors of Ubiquitination and Deubiquitination. Prog Mol Biol Transl Sci 141:339-69
Feger, Bryan J; Thompson, J Will; Dubois, Laura G et al. (2016) Microgravity induces proteomics changes involved in endoplasmic reticulum stress and mitochondrial protection. Sci Rep 6:34091
Jean-Charles, Pierre-Yves; Zhang, Lisheng; Wu, Jiao-Hui et al. (2016) Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation. J Biol Chem 291:7450-64
Jean-Charles, Pierre-Yves; Rajiv, Vishwaesh; Shenoy, Sudha K (2016) Cover Image, Volume 231, Number 10, October 2016. J Cell Physiol 231:i
Shenoy, Sudha K (2016) Preface. Prog Mol Biol Transl Sci 141:xiii-xiv
Jean-Charles, Pierre-Yves; Rajiv, Vishwaesh; Shenoy, Sudha K (2016) Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction. J Cell Physiol 231:2071-80
Jean-Charles, P-Y; Snyder, J C; Shenoy, S K (2016) Chapter One - Ubiquitination and Deubiquitination of G Protein-Coupled Receptors. Prog Mol Biol Transl Sci 141:1-55
Kommaddi, Reddy Peera; Jean-Charles, Pierre-Yves; Shenoy, Sudha K (2015) Phosphorylation of the deubiquitinase USP20 by protein kinase A regulates post-endocytic trafficking of β2 adrenergic receptors to autophagosomes during physiological stress. J Biol Chem 290:8888-903
Shenoy, Sudha K (2014) Arrestin interaction with E3 ubiquitin ligases and deubiquitinases: functional and therapeutic implications. Handb Exp Pharmacol 219:187-203
Shenoy, Sudha K (2014) Deubiquitinases and their emerging roles in β-arrestin-mediated signaling. Methods Enzymol 535:351-70

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