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CELLULAR AND MOLECULAR

G Protein Coupling and Signaling Pathway Activation by M₁ Muscarinic Acetylcholine Receptor Orthosteric and Allosteric Agonists

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (R.L.T., R.M., R.A.J.C.); and Psychiatry Centre of Excellence in Drug Discovery, GlaxoSmithKline, Harlow, United Kingdom (C.J.L., M.D.W.)

The M₁ 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 M₁ 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 M₁ mACh receptor. Each agonist was able to activate G_q/11-dependent signaling, as demonstrated by an increase in guanosine 5'-O-(3-thiotriphosphate) ([³⁵S]GTPS) binding to G_q/11 proteins and total [³H]inositol phosphate accumulation assays in intact cells. All three orthosteric agonists caused significant enhancements in [³⁵S]GTPS binding to G_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 G_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 G_q/11- and G_s-dependent signaling; however, AC-42 and 77-LH-28-1, unlike oxo-M, arecoline, and pilocarpine, do not seem to promote M₁ mACh receptor-G_i1/2 coupling, suggesting that allosteric agonists have the potential to activate distinct subsets of downstream effectors.

Address correspondence to: Dr. R.A. John Challiss, Department of Cell Physiology and Pharmacology, University of Leicester, Room 4/04, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK. E-mail: jc36{at}leicester.ac.uk