Intracellular localization of the M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization is mediated by a C-terminal tryptophan-based motif.
The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W(442) or I(445) with alanine residues abolished constitutive internalization. This WxxI motif resembles YxxΦ, which is the canonical binding motif for the μ2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 μ2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-μ2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophan-based motif, which mediates constitutive internalization.
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Mecicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan Organization for Life Science Advancement Programs, University of Fukui, Eiheiji, Fukui 910-1193, Japan uwada@u-fukui.ac.jp.
Junsuke
J
Uwada
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Mecicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan.
Hatsumi
H
Yoshiki
Department of Pharmacology, School of Medicine, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan.
Takayoshi
T
Masuoka
Department of Pharmacology, School of Medicine, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan.
Matomo
M
Nishio
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Mecicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan Organization for Life Science Advancement Programs, University of Fukui, Eiheiji, Fukui 910-1193, Japan Child Development Research Center, Graduate School of Medicine, University of Fukui, Eiheiji, Fukui 910-1193, Japan.
Ikunobu
I
Muramatsu
D000818:Animals; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D043643:Receptor, Muscarinic M1 / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D014162:Transfection; D014364:Tryptophan / Q000378:metabolism*
D002966:Clathrin; D043643:Receptor, Muscarinic M1; D043562:Receptors, G-Protein-Coupled; D014364:Tryptophan
AP-2; Clathrin; Internalization; Muscarinic acetylcholine receptor