title: Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor.
abstract: Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence (374)KKKPPPS(380) servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching (374)KKKPPPS(380) to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, (361)VARKIVKMTKQPA(373), which is normally masked in the presence of the downstream sequence (374)KKKPPPS(380). Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent.
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Min
authors.initials: M
authors.lastname: Wan
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Wenhua
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Yangli
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Chanjuan
authors.initials: C
authors.lastname: Xu
authors.affiliation: 1] Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China [2] National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Liu
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Rongying
authors.initials: R
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology*; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000378:metabolism*; D043588:Receptor, Muscarinic M4 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D002966:Clathrin; D043585:Receptor, Muscarinic M2; D043588:Receptor, Muscarinic M4; D002217:Carbachol