title: Contrasting effects of allosteric and orthosteric agonists on m1 muscarinic acetylcholine receptor internalization and down-regulation.
abstract: A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M(1) mACh receptor internalization and down-regulation, as well as functional coupling in a Chinese hamster ovary (CHO) cell line. In contrast to full and partial orthosteric agonists, which cause significant receptor internalization and down-regulation, prolonged exposure to AC-42 did not significantly alter either cell-surface or total cellular M(1) mACh receptor expression. 77-LH-28-1, an AC-42 homolog, did cause some receptor internalization, but not down-regulation. The presence of atropine completely prevented the orthosteric agonist-induced adaptive changes in receptor populations; however, in contrast, the copresence of atropine and AC-42 significantly increased both cell-surface receptor and total M(1) mACh receptor expression. Maximal phosphoinositide hydrolysis responses to the partial agonist arecoline were similar in CHO-M(1) cells pretreated for 24 h with either AC-42 or vehicle; in contrast, these responses were markedly reduced when cells were pretreated with oxotremorine-M or pilocarpine. These data indicate that, whereas AC-42 binding to the M(1) mACh receptor can initiate signal transduction, the AC-42-liganded receptor is resistant to the usual mechanisms regulating receptor internalization and down-regulation. In addition, our data suggest unusual interactions between allosteric agonists and orthosteric antagonists to regulate cell-surface and total cellular receptor expression.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Room 4/04, Henry Wellcome Building, Lancaster Road, Leicester, LE1 9HN, United Kingdom.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Thomas
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Langmead
authors.forename: Martyn D
authors.initials: MD
authors.lastname: Wood
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D008024:Ligands; D018721:Muscarinic Agonists / Q000494:pharmacology*; D010880:Piperidines / Q000494:pharmacology*; D011485:Protein Binding; D015363:Quinolones / Q000494:pharmacology*; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000096:biosynthesis*; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000096:biosynthesis; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C456520:4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)-piperidine hydrogen chloride; C531940:77-LH-28-1; D008024:Ligands; D018721:Muscarinic Agonists; D010880:Piperidines; D015363:Quinolones; D043643:Receptor, Muscarinic M1; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3