Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis

Abstract

Upon agonist stimulation, many G protein-coupled receptors such as β2-adrenergic receptors are internalized via β-arrestin- and clathrin-dependent mechanisms, whereas others, like M2 muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M2 mAChR endocytosis, we investigated the post-endocytic trafficking of M2 mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M2 mAChR internalization. Here, we report that M2 mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Gαq Q209L. In contrast, M2 mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M2 mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M2 mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.