title: Synergistic regulation of m2 muscarinic acetylcholine receptor desensitization and sequestration by G protein-coupled receptor kinase-2 and beta-arrestin-1.
abstract: The m2 muscarinic acetylcholine receptor (m2 mAChR) belongs to the superfamily of G protein-coupled receptors and is regulated by many processes that attenuate signaling following prolonged stimulation by agonist. We used a heterologous expression system to examine the ability of G protein-coupled receptor kinase-2 (GRK2) and beta-arrestin-1 to regulate the phosphorylation state and to promote desensitization and sequestration of the m2 mAChR. Treatment of JEG-3 cells transiently expressing the m2 mAChR with a muscarinic agonist induced an approximately 4- or 8-fold increase in receptor phosphorylation in the absence or presence of cotransfected GRK2, respectively, compared with untreated cells transfected with receptor alone. Using the expression of a cAMP-regulated reporter gene to measure receptor function, we found that transiently transfected m2 mAChRs underwent functional desensitization following exposure to agonist. Transfected GRK2 enhanced agonist-induced functional 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 beta-arrestin-1 synergistically enhanced both the rate and extent of agonist-induced m2 mAChR sequestration. These results are the first to demonstrate that agonist-induced desensitization and sequestration of the 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 actions on the m2 mAChR.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Schlador
authors.forename: N M
authors.initials: NM
authors.lastname: Nathanson
mesh_terms: D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D018721:Muscarinic Agonists / Q000494:pharmacology; D010766:Phosphorylation; D017510:Protein Folding; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D051552:beta-Adrenergic Receptor Kinases; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; D019390:Arrestins; D017362:Cyclic AMP Response Element-Binding Protein; D018076:DNA, Complementary; D018721:Muscarinic Agonists; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D005576:Colforsin; D002217:Carbachol; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases