INTERNALIZATION OF THE M2 MUSCARINIC ACETYLCHOLINE-RECEPTOR - ARRESTIN-INDEPENDENT AND ARRESTIN-DEPENDENT PATHWAYS

Recent studies have identified agonist-dependent phosphorylation as a critical event in the rapid uncoupling of the m2 muscarinic cholinergi c receptors (mAChR) rom G-proteins and sequestration of the receptors away hom the cell surface, However, mutant m2 mAChRs were identified t hat were phosphorylated but unable to desensitize in adenylyl cyclase assays, while they internalized like wild type (WT) mAChRs, Ne have te sted whether these properties might stem from differences in the abili ties of the WT and mutant mAChR to bind arrestins, proteins implicated in both receptor-/G-protein uncoupling and internalization, We have d e determined that arresting binding requires phosphorylation at a clus ter of Ser/Thr residues ia amino acids 307-311 in the m2 mAChR, A stro ng correlation was found between the ability of WT and mutant receptor s to bind arrestins in vitro or in vivo and to desensitize in adenylyl cyclase assays, However, the phosphorylation dependent internalizatio n of the m2 mAChR in HEK-tsA201 cells did not require arrestins and di d not proceed via clathrin-mediated endocytosis, While the m2 mAChR wa s able to enter a clathrin-and arrestin-dependent pathway when arresti n 2 or arrestin 3 was significantly overexpressed, the preferred pathw ay al internalization of WT and certain mutant m2 mAChR in HEK-tsA201 cells did not involve participation of arrestins. The results suggest that the phosphorylation-mediated regulation of the m2 mAChR may invol ve arrestin-dependent and -independent events.