SYNERGISTIC REGULATION OF M2 MUSCARINIC ACETYLCHOLINE-RECEPTOR DESENSITIZATION AND SEQUESTRATION BY G-PROTEIN-COUPLED RECEPTOR KINASE-2 ANDBETA-ARRESTIN-1

The m2 muscarinic acetylcholine receptor (m2 mAChR) belongs to the sup erfamily of G protein coupled receptors and is regulated by many proce sses that attenuate signaling following prolonged stimulation by agoni st, We used a heterologous expression system to examine the ability of G protein-coupled receptor kinase-2 (GRK2) and beta-arrestin-1 to reg ulate the phosphorylation state and to promote desensitization and seq uestration of the m2 mAChR. Treatment of JEG-3 cells transiently expre ssing the m2 mAChR with a muscarinic agonist induced an similar to 4- or 8-fold increase in receptor phosphorylation in the absence or prese nce of cotransfected GRK2, respectively, compared with untreated cells transfected with receptor alone. Using the expression of a cAMP-regul ated reporter gene to measure receptor function, we found that transie ntly transfected m2 mAChRs underwent functional desensitization follow ing exposure to agonist, Transfected GRK2 enhanced agonist-induced fun ctional desensitization in a manner that was synergistically enhanced by cotransfection of beta-arrestin-1, which had no effect on m2 mAChR function when coexpressed in the absence of GRK2. Finally, GRK2 and be ta-arrestin-1 synergistically enhanced both the rate and extent of ago nist-induced m2 mAChR sequestration. These results are the first to de monstrate that agonist-induced desensitization and sequestration of th e m2 mAChR in the intact cell can be enhanced by the presence of GRK2, and beta-arrestin-1 and show that these molecules have multiple actio ns on the m2 mAChR.