Protein Kinase A-mediated Phosphorylation of the (cid:1) 2 -Adrenergic Receptor Regulates Its Coupling to G s and G i DEMONSTRATION IN A RECONSTITUTED SYSTEM*

While classically viewed as a prototypic G s and adeny- lyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of (cid:1) 2 -adrener- gic receptor ( (cid:1) 2 -AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G i /G o proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the (cid:1) 2 -AR regulates its coupling specificity with respect to G s and G i . Using a Chinese hamster ovary cell system, we show that mutant (cid:1) 2 -ARs with Ala substituted for Ser at con- sensus PKA sites stimulate robust cyclic AMP accumulation (G s ) but are unable to activate ERK (G i ). In con- trast, Ser 3 Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser 3 Ala mutant receptors is not altered by pertussis toxin, whereas adenylyl cyclase centri- determination of cAMP of were determined using a H-labeled cAMP assay system according to the manufacturer’s The final picomoles of cAMP/mg of