title: Residues specifically involved in down-regulation but not internalization of the m1 muscarinic acetylcholine receptor.
abstract: Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [35S]GTP gamma S binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.
authors.affiliation: Department of Biopharmaceutical Sciences, University of California at San Francisco 94143-0446, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Shockley
authors.forename: N T
authors.initials: NT
authors.lastname: Burford
authors.forename: W
authors.initials: W
authors.lastname: Sadée
authors.forename: J
authors.initials: J
authors.lastname: Lameh
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000201:enzymology; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D015536:Down-Regulation / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D018613:Microscopy, Confocal; D018721:Muscarinic Agonists / Q000494:pharmacology; D009154:Mutation / Q000502:physiology; D011956:Receptors, Cell Surface / Q000819:agonists; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013347:Subcellular Fractions / Q000737:chemistry; D013462:Sulfur Radioisotopes; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D007295:Inositol Phosphates; D018721:Muscarinic Agonists; D011956:Receptors, Cell Surface; D011976:Receptors, Muscarinic; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002217:Carbachol; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases