title: 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 beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) 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, Galpha(q) Q209L. In contrast, M(2) mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M(2) 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 M(2) 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.
authors.affiliation: School of Biology and Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332-0363, USA.
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Delaney
authors.forename: Mandi M
authors.initials: MM
authors.lastname: Murph
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Brown
authors.forename: Harish
authors.initials: H
authors.lastname: Radhakrishna
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism* / Q000502:physiology; D004247:DNA / Q000378:metabolism; D034284:Dynamin I; D034281:Dynamins; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D021381:Protein Transport; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D033921:Vesicular Transport Proteins
chemical_list: D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D008164:Luminescent Proteins; D008565:Membrane Proteins; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011990:Receptors, Transferrin; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D049452:Green Fluorescent Proteins; D004247:DNA; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D034281:Dynamins