An investigation of whether agonist-selective receptor conformations occur with respect to M 2 and M 4 muscarinic acetylcholine receptor signalling via G i/o and G s proteins

Abstract

A range of muscarinic acetylcholine (mACh) receptor agonists (methacholine (MCh), oxotremorine-M (OXO-M), oxotremorine (OXO), arecoline (AREC), bethanechol (BETH), pilocarpine (PILO)) have been investigated with respect to their binding to, and activation of, M 2 and M 4 mACh receptors, recombinantly expressed in Chinese hamster ovary cells, to explore the possibility that these agonists may differentially affect mACh receptor-G i/o and -G s coupling. M 2/M 4 mACh receptor coupling to the adenylyl cyclase/cyclic AMP signalling pathway has been explored in intact cells. G i/o-mediated negative coupling to adenylyl cyclase was explored functionally by assessing the ability of the mACh receptor agonists to inhibit forskolin-stimulated enzymic activity. Following pertussis toxin treatment (100 ng ml -1, 18-20 h) to inactivate G i/o proteins, each agonist caused a G s-mediated enhancement of forskolin-stimulated adenylyl cyclase activity. At both M 2 and M 4 mACh receptors, all agonists tested were more potent in mediating G i/o- versus G s-coupled responses. This difference (determined as the pIC 50 (G i/o coupling) minus pEC 50 (G s coupling) value) was greatest for AREC (65-75-fold) and least for BETH and PILO (≤10-fold). Using apparent binding affinities (pK B), and potency (EC 50/IC 50) and responsiveness (E max/I max) estimates, relative efficacy (e rel) values for each agonist with respect to M 2 and M 4 mACh receptor coupling to G i/o- and G s-mediated signalling were also calculated. While the e rel values obtained for MCh and OXO-M in CHO-m2 cells were similar, OXO-M behaved as a 'super-agonist' at the M 4 mACh receptor giving greater e rel values for both G i/o and G s coupling relative to MCh. The experimental data indicate that while interesting differences between agonists with respect to M 2/M 4 mACh receptor activation and receptor-G i/o and -G s coupling can be discerned, no clear examples of agonist trafficking of signal have emerged. © 2005 Nature Publishing Group All rights reserved.