G(q/11) and G(i/o) activation profiles in CHO cells expressing human muscarinic acetylcholine receptors: dependence on agonist as well as receptor-subtype

Abstract

1. Profiles of G protein activation have been assessed using a [35S]-GTPgammaS binding/immunoprecipitation strategy in Chinese hamster ovary cells expressing either M1, M2, M3 or M4 muscarinic acetylcholine (mACh) receptor subtypes, where expression levels of M1 and M3, or M2 and M4 receptors were approximately equal. 2. Maximal [35S]-GTPgammaS binding to G(q/11)alpha stimulated by M1/M3 receptors, or G(i1-3)alpha stimulated by M2/M4 receptors occurred within approximately 2 min of agonist addition. The increases in G(q/11)alpha-[35S]-GTPgammaS binding after M1 and M3 receptor stimulation differed substantially, with M1 receptors causing a 2-3 fold greater increase in [35S]-GTPgammaS binding and requiring 5 fold lower concentrations of methacholine to stimulate a half-maximal response. 3. Comparison of M2 and M4 receptor-mediated G(i1-3)alpha-[35S]-GTPgammaS binding also revealed differences, with M2 receptors causing a greater increase in G(i1-3)alpha activation and requiring 10 fold lower concentrations of methacholine to stimulate a half-maximal response. 4. Comparison of methacholine- and pilocarpine-mediated effects revealed that the latter partial agonist is more effective in activating G(i3)alpha compared to G(i1/2)alpha for both M2 and M4 receptors. More marked agonist/partial agonist differences were observed with respect to M1/M3-mediated stimulations of G(q/11)alpha- and G(i1-3)alpha-[35S]-GTPgammaS binding. Whereas coupling to these Galpha subclasses decreased proportionately for M1 receptor stimulation by these agonists, pilocarpine possesses a greater intrinsic activity at M3 receptors for G(i)alpha versus G(q/11)alpha activation. 5. These data demonstrate that mACh receptor subtype and the nature of the agonist used govern the repertoire of G proteins activated. They also provide insights into how the diversity of coupling can be pharmacologically exploited, and provide a basis for a better understanding of how multiple receptor subtypes can be differentially regulated.

Figure 1

Expression of Gα proteins in CHO-m1, -m2, -m3, -m4, and untransfected CHO cell membranes. Cell membranes (20 μg protein) were solubilized, proteins separated and transferred to nitrocellulose for immunoblotting as described in Methods. Gα proteins were identified using 1 : 1000 dilutions of antisera to G_q/11α (a), G_i1/2α (b), G_i3/0α (c), G₀α (d), or G_sα (e). All Gα proteins migrated on electrophoresis consistently with the expected molecular weight (40 – 45 kDa) for each Gα protein compared to M_r standards. For each blot, lane 1 (brain)=crude rat brain homogenate, lanes 2 – 5=CHO-m1 to CHO-m4 membranes respectively and lane 6 (CHO-wt)=untransfected CHO cell membranes.

Figure 2

Time-course of methacholine-stimulated [³⁵S]-GTPγS binding to G_i1-3α in CHO-m2 and CHO-m4 cell membranes. Cell membranes prepared from CHO-m2 (a) or CHO-m4 (b) cells were incubated ([GDP]=10 μM) in the absence (basal) or presence of methacholine (MCh, 1 mM) for the times indicated at 30°C. Insert panels illustrate the net change in [³⁵S]-GTPγS bound-over-basal. Data are shown as means±s.e.mean for five separate experiments carried out in duplicate. Agonist-stimulated [³⁵S]-GTPγS binding was significantly greater than basal binding (P<0.05) for both CHO-m2 and CHO-m4 membranes for all time-points beyond 0.25 min.

Figure 3

Time-course of methacholine-stimulated [³⁵S]-GTPγS binding to G_q/11α in CHO-m1 and CHO-m3 cell membranes. Cell membranes prepared from CHO-m1 (a) or CHO-m3 (b) cells were incubated ([GDP]=1 μM) in the absence (basal) or presence of methacholine (MCh, 1 mM) for the times indicated at 30°C. Insert panels illustrate the net change in [³⁵S]-GTPγS bound-over-basal. Data are shown as means±s.e.mean for five separate experiments carried out in duplicate. Agonist-stimulated [³⁵S]-GTPγS binding was significantly greater than basal binding (P<0.05) for both CHO-m1 and CHO-m3 membranes for all time-points.

Figure 4

Quantitation of agonist-stimulated [³⁵S]-GTPγS binding to specific Gα protein subtypes in CHO-m2 and CHO-m4 cell membranes by immunoprecipitation with subtype-specific G protein antisera. Cell membranes prepared from CHO-m2 or CHO-m4 cells were incubated ([GDP]=10 μM) in the absence or presence of methacholine (1 mM, panel a) or pilocarpine (1 mM, panel b) for 2 min at 30°C. Data are shown as means±s.e.mean for five separate experiments carried out in duplicate. Statistically significant increases in [³⁵S]-GTPγS binding caused by agonist addition are indicated as ^*P<0.05.

Figure 5

Quantitation of agonist-stimulated [³⁵S]-GTPγS binding to specific Gα protein subtypes in CHO-m1 and CHO-m3 cell membranes by immunoprecipitaion with subtype-specific G protein antisera. Cell membranes prepared from CHO-m1 or CHO-m3 cells were incubated ([GDP]=1 μM) in the absence or presence of methacholine (1 mM, panel a) or pilocarpine (1 mM, panel b) for 2 min at 30°C. Data are shown as means±s.e.mean for five separate experiments carried out in duplicate. Statistically significant increases in [³⁵S]-GTPγS binding caused by agonist addition are indicated as ^*P<0.05.

Figure 6

Concentration-dependencies of methacholine-stimulated [³⁵S]-GTPγS binding to specific Gα proteins in CHO-m1, -m2, -m3 and -m4 cell membranes. CHO cell membranes were incubated with methacholine (10⁻⁹ – 10⁻³ M) under optimal assay conditions. (a) shows concentration-response curves for [³⁵S]-GTPγS-G_i3/oα binding in CHO-m2 and CHO-m4 cell membranes, while (b) shows similar data for [³⁵S]-GTPγS-G_q/11α binding in CHO-m1 and CHO-m3 cell membranes. Data are shown as means±s.e.mean for 3 – 5 separate experiments carried out in duplicate.