2 HOMOLOGOUS PHOSPHORYLATION DOMAINS DIFFERENTIALLY CONTRIBUTE TO DESENSITIZATION AND INTERNALIZATION OF THE M2 MUSCARINIC ACETYLCHOLINE-RECEPTOR

Short term exposure of m2 muscarinic acetylcholine receptors (m2 mAChR s) to agonist causes a rapid phosphorylation of the activated receptor s, followed by a profound loss in the ability of the m2 mAChR to activ ate its signaling pathways, We have used site-directed mutagenesis to identify two clusters of Ser/Thr residues in the third intracellular l oop of the m2 mAChR that can serve as redundant targets for agonist-de pendent phosphorylation, Mutation of both clusters of Ser/Thr residues to alanines abolished agonist-dependent phosphorylation, while wild-t ype levels of m2 mAChR phosphorylation were observed in mutant recepto rs with only one or the other cluster mutated, However, the functional effects of phosphorylation of these two ''redundant'' clusters were n ot equivalent, No receptor desensitization was observed in an m2 mAChR with residues Thr(307)-Ser(311) mutated to alanine residues, In contr ast, mutation of the other Ser/Thr cluster. residues Ser(286)-Ser(290) , to alanines produced a receptor that continued to desensitize, Inter nalization of the m2 mAChR was promoted by phosphorylation of either c luster, suggesting that distinct mechanisms with unique structural req uirements act downstream of m2 mAChR phosphorylation to mediate recept or desensitization and receptor internalization.