Abstract 10956: Phosphorylation of AdipoR1 Following Myocardial Ischemia Modifies Its Association from Gs-Protein to β-Arrestin
Adiponectin (APN) exerts its cardiac regulatory and protective actions largely through APN receptor 1 (AdipoR1), a 7-transmembrane domain protein structurally and topologically distinct from GPCRs. The current study investigated whether AdipoR1 signaling involves the G-protein system, and whether modifications occur post-MI. In isolated cardiomyocytes (CM), APN increased intracellular cAMP in a dose- and time-dependent fashion, followed by PKA activation. These effects were blocked by siRNA-mediated AdipoR1 knockdown. APN significantly inhibited TNFα-induced iNOS/NADPH oxidase expression and NO/superoxide/peroxynitrite overproduction. These effects were blocked by pharmacological or genetic PKA inhibition. To obtain direct evidence that G-protein is involved in AdipoR1 signaling in CM, the interaction between AdipoR1 and various G-proteins was determined by co-immunoprecipitation. During resting conditions, AdipoR1 selectively interacts with Gs-protein. This interaction progressively decreases after MI, reaching levels only 40% of control 3-days post-MI. While AdipoR1/β-arrestin complex was not detected in the normal heart, it was detected 3 days after MI. Moreover, serine/threonine phosphorylation of AdipoR1 is detected 24 hours post-MI, and progressively increases thereafter. Bio-informatics analysis predicted the presence of multiple phosphorylation sites in the N-terminal intracellular domain of AdipoR1, with GRK2, GRK3, and GRK5 as potential kinases. No significant increase in GRK3 and GRK5 expression was observed until 3 days post-MI, a time point when significant AdipoR1 phosphorylation and AdipoR1/β-arrestin interaction had already developed. In contrast, GRK2 is significantly elevated 3 hours after MI, and remains significantly elevated thereafter. Finally, a significant positive relationship between GRK2 expression and AdipoR1 phosphorylation levels was observed in cardiac tissue obtained 3 days post-MI. Collectively, we have demonstrated that the G-protein system is involved in AdipoR1-mediated APN signaling in CM. Moreover, our results suggest that post-MI phosphorylation of AdipoR1 by GRK2 inhibits cardioprotective signaling of APN, potentially contributing to post-MI cardiac injury.
- © 2012 by American Heart Association, Inc.