OF WORK: Project 3 ? Regulation of Beta-Adrenergic Receptor Signaling by S-Nitrosylation G protein-coupled receptors (GPCRs) are the most prevalent drug targets in humans and underlie therapy for most cardiovascular disorders. Signal transduction via GPCRs relies critically upon their interactions with the - arrestins ( arr1, arr2), which both desensitize GPCRs and mediate G protein-independent signals that derive from the protein-protein interactions of the arrs with an expanding set of partners including prominently MAP kinase. The demonstration that GPCR signal transduction is mediated by both arrs and G-proteins, and that man-made GPCR ligands can differentially signal through those pathways, has catalyzed an extensive effort to develop drugs that exhibit biased agonism. However, endogenous mechanisms that bias signaling remain largely unknown. Our proposed research is based upon our preliminary discoveries that: 1) S-nitrosylation of arr1 and arr2, mediated by neuronal NOS or induced NOS, inhibits ligand-induced arr recruitment to the 2AR and biases signaling by selectively diminishing arr-mediated transduction; 2) the arrs themselves regulate S-nitrosylation of many cellular substrates; 3) arr S-nitrosylation by n/iNOS is a molecular signature of heart failure. We propose to: 1) characterize the conformational changes induced by arr S-nitrosylation, employing X-ray crystallography and hydrogen-deuterium exchange, in order to elucidate the structural mechanism of inactivation; 2) characterize alterations in the arr interactome induced by arr S-nitrosylation, employing multiplex labeling and mass spectrometry, in order to elucidate the molecular mechanisms of altered signaling consequent upon arr S-nitrosylation; 3) characterize the mediators and effects of arr S- nitrosylation on S-nitrosylation of the constituents of the arr interactome, in order to elucidate the role of the arrs as regulators of cellular S-nitrosylation broadly; 4) characterize the cardiovascular effects of arr S- nitrosylation in vivo, employing mutant mice in which the Cys within arr targeted by S-nitrosylation has been replaced with Ser, in order to elucidate the physiological and pathophysiological roles of arr S-nitrosylation. Our proposed research provides an unprecedented opportunity to explore endogenous mechanisms of signaling bias through GPCRs, and thereby to identify potential novel therapeutic approaches to a broad spectrum of cardiovascular pathophysiologies.
Project 3 ? Regulation of Beta-Adrenergic Receptor Signaling by S-Nitrosylation G protein-coupled receptors (GPCRs) are the most prevalent drug targets in humans. We have discovered that ?-arrestin proteins, critical regulators of GPCRs, are subject to post-translational modification by nitric oxide, which profoundly affects their interactions with GPCRs and other partners. This line of research thus holds great promise for identifying novel drug targets that can influence the function of many GPCRs, with correspondingly broad implications for the treatment of heart disease.
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