Accumulating evidence indicates that nitric oxide (NO), through S-nitrosylation of Cys residues within multiple, functionally interrelated signaling elements, regulates agonist-induced desensitization and internalization of B- adrenergic receptors (B-ARs). We have demonstrated that NO synthases and the endogenous S-nitrosothiol (SNO), S-nitrosoglutathione, preserve cardiac contractility and prevent down-regulation of B-ARs during maintained agonist stimulation. These effects of NO/SNO on B-AR signaling and trafficking, which can be recapitulated in cellular systems, appear to be mediated, in significant part, by S-nitrosylation of the G protein-coupled receptor (GPCR) kinase, GRK2. Additional components of the B-AR system are also regulated by S-nitrosylation, which suggests a broad functional role for NO/SNO, exerted through targeted S-nitrosylation. In particular, we have recently identified the B-arrestins (Barr1 and Barr2) as targets of B-AR-coupled S-nitrosylation by NO synthases (eNOS and nNOS). S-nitrosylation of Barr2 by eNOS at a single critical site (Cys 410) regulates its protein-protein interactions with clathrin and adapter protein-2 (AP-2) in vitro and in vivo, thereby promoting agonist-mediated B{2}-AR internalization. However, differential regulation of Barr1 and Barr2 by S-nitrosylation and the consequences of these modifications for cardiac function, have not been explored. Our central hypothesis is that S-nitrosylation of the B-arrestins will provide a basis for control by NO of B-AR trafficking and signaling, with important ramifications in healthy and failing hearts. Moreover, we predict that the regulation of Barr1- and Barr2-specific interactomes by S-nitrosylation will provide a principal mechanism through which NO exerts its regulatory influence. We will carry out the following specific aims:
Specific Aim 1. Elucidate the sites of agonist-dependent S-nitrosylation of the B-arrestins by eNOS and nNOS in cells and tissues.
Specific Aim 2. Elucidate the consequences of B-arrestin S-nitrosylation for B-AR internalization and desensitization.
Specific Aim 3. Assess the consequences of B-arrestin S-nitrosylation for p-AR-dependent signaling.
Specific Aim 4. Assess the functional roles of B-arrestin S-nitrosylation in the intact heart. Collectively, these studies should provide fundamental and novel insights into B-AR regulation by NO in both healthy and failing hearts and may open a new area of research.

Agency
National Institute of Health (NIH)
Type
Research Program Projects (P01)
Project #
5P01HL075443-10
Application #
8687715
Study Section
Heart, Lung, and Blood Program Project Review Committee (HLBP)
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Duke University
Department
Type
DUNS #
City
Durham
State
NC
Country
United States
Zip Code
27705
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