title: G protein coupling and signaling pathway activation by m1 muscarinic acetylcholine receptor orthosteric and allosteric agonists.
abstract: The M(1) muscarinic acetylcholine (mACh) receptor is among a growing number of G protein-coupled receptors that are able to activate multiple signaling cascades. AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine) is an allosteric agonist that can selectively activate the M(1) mACh receptor in the absence of an orthosteric ligand. Allosteric agonists have the potential to stabilize unique receptor conformations, which may in turn cause differential activation of signal transduction pathways. In the present study, we have investigated the signaling pathways activated by AC-42, its analog 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone), and a range of orthosteric muscarinic agonists [oxotremorine-M (oxo-M), arecoline, and pilocarpine] in Chinese hamster ovary cells recombinantly expressing the human M(1) mACh receptor. Each agonist was able to activate Galpha(q/11)-dependent signaling, as demonstrated by an increase in guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to Galpha(q/11) proteins and total [(3)H]inositol phosphate accumulation assays in intact cells. All three orthosteric agonists caused significant enhancements in [(35)S]GTPgammaS binding to Galpha(i1/2) subunits over basal; however, neither allosteric ligand produced a significant response. In contrast, both orthosteric and allosteric agonists are able to couple to the Galpha(s)/cAMP pathway, enhancing forskolin-stimulated cAMP accumulation. These data provide support for the concept that allosteric and orthosteric mACh receptor agonists both stabilize receptor conformations associated with Galpha(q/11)- and Galpha(s)-dependent signaling; however, AC-42 and 77-LH-28-1, unlike oxo-M, arecoline, and pilocarpine, do not seem to promote M(1) mACh receptor-Galpha(i1/2) coupling, suggesting that allosteric agonists have the potential to activate distinct subsets of downstream effectors.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Room 4/04, Henry Wellcome Building, Lancaster Road, Leicester LE19HN, UK.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Thomas
authors.forename: Rajendra
authors.initials: R
authors.lastname: Mistry
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: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D018721:Muscarinic Agonists / Q000494:pharmacology*; D019832:N-Methylscopolamine / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology*; D015363:Quinolones / Q000494:pharmacology*; D043643:Receptor, Muscarinic M1 / Q000819:agonists*; D015398:Signal Transduction
chemical_list: C456520:4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)-piperidine hydrogen chloride; C531940:77-LH-28-1; D018721:Muscarinic Agonists; D010880:Piperidines; D015363:Quinolones; D043643:Receptor, Muscarinic M1; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs; D019832:N-Methylscopolamine