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Contrasting Effects of Allosteric and Orthosteric Agonists on M1 Muscarinic Acetylcholine Receptor Internalization and Down-regulation

By Rachel L. Thomas, Christopher J. Langmead, Martyn D. Wood and R.A. John Challiss


This paper was published as Journal of Pharmacology and Experimental Therapeutics, 2009, 331 (3), pp. 1086-1095. It is available from Doi: 10.1124/jpet.109.160242Metadata only entryA 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 M1 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 M1 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 M1 mACh receptor expression. Maximal phosphoinositide hydrolysis responses to the partial agonist arecoline were similar in CHO-M1 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 M1 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

Publisher: American Society for Pharmacology and Experimental Therapeutics (ASPET)
Year: 2009
DOI identifier: 10.1124/jpet.109.160242
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