Multiple topological domains mediate subtype-specific internalization of the M-2 muscarinic acetylcholine receptor

Endocytosis of agonist-activated G protein-coupled receptors (GPCRs) is req uired for both resensitization and recycling to the cell surface as well as lysosomal degradation. Thus, this process is crucial for regulation of rec eptor signaling and cellular responsiveness. Although many GPCRs internaliz e into clathrin-coated vesicles in a dynamin-dependent manner, some recepto rs, including the M-2 muscarinic acetylcholine receptor (mAChR), can also e xhibit dynamin-independent internalization. We have identified five amino a cids, located in the sixth and seventh transmembrane domains and the third intracellular loop, that are essential for agonist-induced M-2 mAChR intern alization via a dynamin-independent mechanism in JEG-3 choriocarcinoma cell s. Substitution of these residues into the M-1 mAChR, which does not intern alize in these cells, is sufficient for conversion to the internalization-c ompetent M-2 mAChR phenotype, whereas removal of these residues from the M- 2 mAChR blocks internalization. Cotransfection of a dominant-negative isofo rm of dynamin has no effect on M-2 mAChR internalization. An internalizatio n-incompetent M-2 mutant that lacks a subset of the necessary residues can still internalize via a G protein-coupled receptor kinase-2 and beta-arrest in-dependent pathway. Furthermore, internalization is independent of the si gnal transduction pathway that is activated. These results identify a novel motif that specifies structural requirements for subtype-specific dynamin- independent internalization of a GPCR.