Two homologous phosphorylation domains differentially contribute to desensitization and internalization of the m2 muscarinic acetylcholine receptor.

Short term exposure of m2 muscarinic acetylcholine receptors (m2 mAChRs) to agonist causes a rapid phosphorylation of the activated receptors, followed by a profound loss in the ability of the m2 mAChR to activate its signaling pathways. We have used site-directed mutagenesis to identify two clusters of Ser/Thr residues in the third intracellular loop of the m2 mAChR that can serve as redundant targets for agonist-dependent phosphorylation. Mutation of both clusters of Ser/Thr residues to alanines abolished agonist-dependent phosphorylation, while wild-type levels of m2 mAChR phosphorylation were observed in mutant receptors with only one or the other cluster mutated. However, the functional effects of phosphorylation of these two "redundant" clusters were not equivalent. No receptor desensitization was observed in an m2 mAChR with residues Thr307-Ser311 mutated to alanine residues. In contrast, mutation of the other Ser/Thr cluster, residues Ser286-Ser290, to alanines produced a receptor that continued to desensitize. Internalization of the m2 mAChR was promoted by phosphorylation of either cluster, suggesting that distinct mechanisms with unique structural requirements act downstream of m2 mAChR phosphorylation to mediate receptor desensitization and receptor internalization.