PHOSPHORYLATION AND DESENSITIZATION OF HUMAN M2 MUSCARINIC CHOLINERGIC RECEPTORS BY 2 ISOFORMS OF THE BETA-ADRENERGIC-RECEPTOR KINASE

Studies of the human m2 (hm2) muscarinic cholinergic receptors (mAChR) have been performed to provide further insights into the potential re gulation of these receptors by isoforms of the beta-adrenergic recepto r kinase (betaARK). The hm2 mAChR and the isoforms betaARK1 and betaAR K2 were individually expressed in, and purified from, insect Sf9 cells infected with recombinant baculoviruses. The expressed hm2 receptors were tested as substrates for betaARK1 and betaARK2 in vitro using con centrations of receptors and kinases similar to those found in intact cells. The hm2 mAChR were phosphorylated in an agonist-dependent manne r to 4-5 mol of phosphate/mol of receptor by betaARK1 or betaARK2. The reactions were highly dependent on agonist; the antagonist atropine, and heparin, a betaARK inhibitor, both prevented the betaARK-mediated phosphorylation. The rates of phosphorylation catalyzed by both isofor ms were similar, with half-maximal phosphorylation occurring in less t han 5 min. Under the conditions employed the stoichiometries, but not the rates, of phosphorylation catalyzed by both kinases were increased 2-3-fold by either the heterotrimeric G-protein G(o) or the betagamma subunits of transducin. Phosphopeptide mapping experiments indicated that similar sites were phosphorylated by the two betaARK isoforms. In order to test for functional effects of the phosphorylation mediated by the betaARK isoforms, the receptors were reconstituted with purifie d G(o) and were tested for their ability to stimulate guanosine 5'-3-O -(thio)triphosphate (GTPgammaS) binding. The conditions leading to max imal receptor phosphorylation resulted in a 30-50% reduction in the ab ility of the receptors to stimulate GTPgammaS binding to G(o). The res ults demonstrate that the hm2 mAChR are excellent substrates in vitro for both betaARK1 and betaARK2 and that extensive phosphorylation by t hese enzymes occurs in the presence of the betagamma subunits of G pro teins. The betaARK-mediated phosphorylation of the m2 mAChR causes a p erturbation of receptor/G-protein coupling.