Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells.

1. We have examined possible mechanisms of cross-talk between the G(q/11)-linked M(3) muscarinic acetylcholine (mACh) receptor and the G(i/o)-linked M(2) mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P(3)) and mitogen-activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO-m2m3 cells and compared to those stimulated in CHO-m2 and CHO-m3 cell-lines, expressing comparable levels of M(2) or M(3) mACh receptors. 2. Based on comparisons between cell-lines and pertussis toxin (PTx) pretreatment to eliminate receptor-G(i/o) coupling, evidence was obtained for (i) an M(2) mACh receptor-mediated contribution to the predominantly M(3) mACh receptor-mediated Ins(1,4,5)P(3) response and (ii) a facilitation of the inhibitory effect of M(2) mACh receptor on forskolin-stimulated cyclic AMP accumulation by M(3) mACh receptor coactivation at low agonist concentrations (MCh 10(-9)-10(-6) M). 3. The most profound cross-talk effects were observed with respect to ERK activation. Thus, while MCh stimulated ERK activation in both CHO-m2 and CHO-m3 cells (pEC(50) values: 5.64+/-0.09 and 5.57+/-0.16, respectively), the concentration-effect relation was approx 50-fold left-shifted in CHO-m2m3 cells (pEC(50): 7.17+/-0.07). In addition, the ERK response was greater and more sustained in CHO-m2m3 cells. In contrast, only minor differences were seen in the time-courses and concentration-dependencies of JNK activation in CHO-m3 and CHO-m2m3 cells. 4. Costimulation of endogenous P2Y(2) purinoceptors also caused an approx 10-fold left-shift in the MCh-stimulated ERK response in CHO-m2 cells, suggesting that the G(q/11)/G(i/o) interaction to affect ERK activation is not specific to muscarinic receptors. 5. PTx pretreatment of cells had unexpected effects on ERK activation by MCh in both CHO-m2m3 and CHO-m3 cells. Thus, in CHO-m3 cells PTx pretreatment caused a marked left-shift in the MCh concentration-effect curve, while in PTx-treated CHO-m2m3 cells the maximal responsiveness was decreased, but the potency of MCh was only slightly affected. 6. The data presented here strongly suggest that cross-talk between M(2) and M(3) mACh receptors occurs at the level of both second messenger and ERK regulation. Further, these data provide novel insights into the involvement of G(i/o) proteins in both positive and negative modulation of ERK responses evoked by G protein-coupled receptors.