Abstract

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 Gα_q/11-dependent signaling, as demonstrated by an increase in guanosine 5′-O-(3-thiotriphosphate) ([³⁵S]GTPγS) binding to Gα_q/11 proteins and total [³H]inositol phosphate accumulation assays in intact cells. All three orthosteric agonists caused significant enhancements in [³⁵S]GTPγS binding to Gα_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 Gα_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 Gα_q/11- and Gα_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-Gα_i1/2 coupling, suggesting that allosteric agonists have the potential to activate distinct subsets of downstream effectors.