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

Abstract

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.

Figure 1

Effects of PTx pretreatment on basal and MCh-stimulated [³⁵S]-GTPγS binding to G_q/11α in membranes prepared from CHO-m2, -m3 and -m2m3 cells. CHO cells were preincubated in the presence of PTx (100 ng ml⁻¹; 24 h) or vehicle before preparation of membranes. MCh (1 mM) or buffer additions to membranes were for 2 min at 30°C and, following solubilization, [³⁵S]-GTPγS-G_q/11α complexes were immunoprecipitated and radioactivity assessed as described in the Methods section. Data are shown as means±s.e. mean for four separate experiments performed in duplicate. For both CHO-m3 and CHO-m2m3 membranes, a significant (^*P<0.05) increase in G_q/11α-associated [³⁵S]-GTPγS binding was stimulated by MCh irrespective of PTx pretreatment. In addition, following PTx pretreatment a greater agonist-stimulated G_q/11α-[³⁵S]-GTPγS binding was observed (^#P<0.05) in CHO-m2m3, but not CHO-m2 or -m3 membranes.

Figure 2

Comparison of time- and concentration-dependent agonist-stimulated Ins(1,4,5)P₃ accumulations in CHO-m3 and -m2m3 cells. Cell monolayers were stimulated with either MCh (1 mM) for the times indicated (a), or for 15 s with different concentrations of MCh (b). CHO-m3 (c) or CHO-m2m3 (d) cells were pretreated with PTx (100 ng ml⁻¹, 24 h) or vehicle before challenge with the indicated concentrations of MCh for 15 s. Incubations were terminated and extracts prepared for Ins(1,4,5)P₃ mass determination as described in the Methods section. Data are shown as means±s.e. mean for three (panels a and c) or four (panels b and d) separate experiments performed in duplicate.

Figure 3

Effects of PTx pretreatment on time- and concentration-dependencies of MCh-stimulated cyclic AMP accumulations in CHO-m2m3 cells. CHO-m2m3 cell monolayers were pretreated with PTx (100 ng ml⁻¹, 24 h) or vehicle before challenge with either 1 mM MCh for the times indicated (a), or with different concentrations of MCh for 5 min (b). Incubations were terminated and extracts prepared for cyclic AMP mass determination as described in the Methods section. Data are shown as means±s.e. mean for three separate experiments performed in duplicate.

Figure 4

Effects of M₂ and/or M₃ mACh receptor activation on forskolin-stimulated cyclic AMP accumulations in CHO-m2, -m3 and -m2m3 cells. The indicated concentrations of MCh were added to cell monolayers for 10 min before challenge with forskolin (10 μM). After a further period of 10 min incubations were terminated and cyclic AMP mass determined as described in the Methods section. Data are shown as means±s.e. mean for three (CHO-m2) or four (CHO-m3 and -m2m3) separate experiments performed in duplicate. The inset focuses on the inhibitory effects of 10⁻⁹–10⁻⁶ M MCh in the three cell-lines.

Figure 5

Effects of PTx pretreatment on the concentration-dependent modulatory actions of MCh on forskolin-stimulated cyclic AMP accumulations in CHO-m3 and -m2m3 cells. (a) CHO-m3 and (b) CHO-m2m3 cell monolayers were pretreated with PTx (100 ng ml⁻¹, 24 h) or vehicle before challenge with the indicated concentrations of MCh for 10 min before addition of forskolin (10 μM). After a further period of 10 min, incubations were terminated and cyclic AMP mass determined as described in the Methods section. Results are expressed relative to the cyclic AMP responses to forskolin alone (CHO-m3: −PTx, 1008±41; +PTx, 433±50; CHO-m2m3: −PTx, 908±110; +PTx, 526±51 pmol mg⁻¹ protein), which are set to 100% for each condition. Data are shown as means±s.e. mean for four (CHO-m3) or five (CHO-m2m3) separate experiments performed in duplicate. PTx did not significantly affect the modulatory effect of MCh in CHO-m3 cells, but significant differences were seen in toxin-treated CHO-m2m3 cells (^*P<0.05).

Figure 6

Time- and concentration-dependent increases in extracellular signal-regulated kinase (ERK) activity stimulated by MCh in CHO-m2, -m3 and -m2m3 cells. (a) Confluent monolayers of CHO cells were stimulated with MCh (100 μM) for the times indicated, (b) or with different concentrations of MCh for 5 min. Incubations were terminated, cell lysates prepared and kinase assays performed as described in the Methods section. Data are expressed as either a ‘fold' increase over basal ERK activity (a) or as an enzymic activity (expressed as fmol phosphate incorporated into the EGF receptor fragment substrate per min per mg of cell protein (b)). Data are presented as means±s.e. mean for four to eight separate experiments (panel a), or, for data shown in panel b, four (CHO-m2), five (CHO-m3) or six (CHO-m2m3) separate experiments.

Figure 7

Concentration-dependent increases in c-Jun N-terminal kinase (JNK) activity stimulated by MCh in CHO-m3 and -m2m3 cells. Confluent monolayers of CHO cells were stimulated with the indicated concentrations of MCh for 30 min. Incubations were then terminated, cell lysates prepared and JNK assays performed as described in the Methods section. Data are expressed as enzymic activities (expressed as fmol phosphate incorporated into c-Jun fusion protein per min per mg of cell protein) and represent means±s.e. mean for four (CHO-m3) or six (CHO-m2m3) separate experiments. The inset shows a representative experiment to illustrate a typical time-course of MCh (100 μM)-stimulated JNK activation in the two cell-lines.

Figure 8

Effects of coincident P2Y/M₂ receptor stimulation on ERK activity in CHO-m2 cells. Confluent CHO-m2 monolayers were challenged with the indicated concentrations of MCh in the absence or presence of UTP (10 μM) for 5 min. Incubations were terminated, cell lysates prepared and kinase assays performed as described in the Methods section. Data are expressed as a ‘fold' increase over basal ERK activity and are presented as means±s.e. mean for three separate experiments. The inset shows the concentration-dependency of ERK activation by UTP (n=3 for all data, except 300 μM UTP point where n=2).

Figure 9

Effects of PTx pretreatment on the concentration-dependencies of ERK activation by MCh in CHO-m2, -m3 and -m2m3 cells. (a) CHO-m2, (b) CHO-m2m3 and (c) CHO-m3 cell monolayers were pretreated with PTx (100 ng ml⁻¹, 24 h) or vehicle before challenge with the indicated concentrations of MCh (panel a; 100 μM). Incubations were terminated, cell lysates prepared and kinase assays performed as described in the Methods section. Data are expressed either as a ‘fold' increase over basal ERK activity (panels a and b), or as a percentage of the response to 100 μM MCh in vehicle-treated CHO-m3 cells (panel c). In all cases, data are shown as means±s.e. mean for at least three separate experiments.