Abstract 240: Phosphorylation of β2-Adrenoceptor by cAMP-Dependent Protein Kinase Is Neither Necessary nor Sufficient to Mediate the Receptor Coupling to Gi Proteins
β2-Adrenoceptor (β2AR) couples dually to Gs and Gi proteins, resulting in functionally opposing effects on cardiomyocyte contractility. Recent studies have suggested that phosphorylation of β2AR by protein kinase A (PKA) is both necessary and sufficient for the receptor coupling to Gi proteins. Our previous study has demonstrated that while most β2-agonists activate both Gs and Gi, fenoterol, a full agonist of β2AR, preferentially activates Gs. In the present study, we seek to investigate the receptor-G protein coupling characteristics of different stereoisomers of fenoterol in a physiologically relevant context, adult rat cardiac myocytes. We demonstrate that fenoterol-induced β2AR-Gi coupling is dependent on the chirality of the agonist rather than the phosphorylation of the receptor by PKA. First, R,R′-fenoterol mediated β2AR contractile response is not sensitized by disrupting Gi signaling with pertussis toxin (PTX), whereas S,R′-fenoterol induced β2AR contractile response exhibits a robust PTX sensitivity. Similarly, in human embryonic kidney (HEK) 293 cells, stimulation of endogenous β2AR with S,R′-fenoterol, but not R,R′-fenoterol, increases phosphorylation of extracellular signal-regulated kinases (ERK1/2) in a PTX-sensitive manner, indicating that R,R′-fenoterol and S,R′-fenoterol preferentially activate Gs and Gi, respectively. Indeed, using photoaffinity G protein labeling technique, we have shown that R,R′-fenoterol and S,R′-fenoterol exhibit opposite G protein coupling selectivity with the former for Gs and the latter for Gi (Gi2 and Gi3). To determine whether there is a causal relationship between β2AR phosphorylation by PKA and the receptor coupling to Gi proteins, we have assayed PKA-mediated phosphorylation of β2AR at Ser262 with a site-specific antibody, and found that R,R′-fenoterol is much more potent than S,R-fenoterol in increasing phosphorylation of β2AR-Ser262 in rat cardiomyocytes overexpressing the human β2AR. Thus, enhanced phosphorylation of β2AR by PKA is neither necessary nor sufficient to mediate the receptor-Gi coupling.