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CELLULAR AND MOLECULAR

G Protein Coupling and Signaling Pathway Activation by M₁ Muscarinic Acetylcholine Receptor Orthosteric and Allosteric Agonists

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (R.L.T., R.M., R.A.J.C.); and Psychiatry Centre of Excellence in Drug Discovery, GlaxoSmithKline, Harlow, United Kingdom (C.J.L., M.D.W.)

Received for publication June 3, 2008
Accepted July 28, 2008.

	Abstract

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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 G_q/11-dependent signaling, as demonstrated by an increase in guanosine 5'-O-(3-thiotriphosphate) ([³⁵S]GTPS) binding to G_q/11 proteins and total [³H]inositol phosphate accumulation assays in intact cells. All three orthosteric agonists caused significant enhancements in [³⁵S]GTPS binding to G_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 G_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 G_q/11- and G_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-G_i1/2 coupling, suggesting that allosteric agonists have the potential to activate distinct subsets of downstream effectors.

Spalding et al. (2002) have reported that a novel compound, AC-42, is able to activate selectively the M₁ mACh receptor. By constructing chimeric receptors, this group was able to demonstrate that AC-42 binds to a site (proposed to consist of residues in the N terminus and transmembrane 1 along with residues in transmembrane 7 and the third extracellular loop) distinct from the orthosteric site (Spalding et al., 2002). The suggested "ectopic" location of the binding site was further supported by the discovery that an Y381A point mutation in the acetylcholine binding site was sufficient to abolish receptor activation by an orthosteric agonist but had no effect on the functional response to AC-42 (Spalding et al., 2002). Subsequent work has demonstrated that AC-42 acts allosterically (Langmead et al., 2006); consequently, we use the term "allosteric agonist" here to describe AC-42.

Although it is well recognized that ligand binding to M₁ mACh receptors initiates G_q/11-dependent activation of phospholipase C and consequent generation of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, the ability of this receptor subtype to influence additional downstream effector pathways (via distinct G protein subtypes) has also been reported. Thus, the M₁ mACh receptor has been reported to activate G_i/o and G_q/11 proteins (Offermanns et al., 1994; Akam et al., 2001) and to activate adenylyl cyclase activity via a G_s protein-dependent mechanism (Burford and Nahorski, 1996). The ability of the M₁ mACh receptor to activate multiple downstream effectors via distinct signaling pathways allows the possibility that allosteric agonists like AC-42 may not only display functional mACh receptor subtype selectivity but also may activate all or a subset of the pathways activated by orthosteric agonists. This phenomenon, referred to variously as "agonist-directed trafficking of receptor stimulus," "functional selectivity," and most recently as "ligand-induced differential signaling" (Kenakin, 2003; Urban et al., 2007), has been reported for a number of receptors including pituitary adenylyl cyclase-activating protein receptors (Spengler et al., 1993), 5-hydroxytryptamine_2C receptors (Berg et al., 1998), and CB₁ cannabinoid receptors (Bonhaus et al., 1998). Previous work exploring G_i/o coupling through the M₁ and M₃ mACh receptors revealed differences between methacholine- and pilocarpine-stimulated [³⁵S]GTPS binding to G_i/o proteins (Akam et al., 2001), and this orthosteric agonist-specific activation was also observed at M₂ and M₄ mACh receptors, where pilocarpine seems to selectively activate G_i3 over G_i1 and/or G_i2 proteins (Akam et al., 2001).

AC-42 has been shown to stimulate both phosphoinositide hydrolysis and a Ca²⁺ response consistent with activation of G_q/11 subunits (Spalding et al., 2002, 2006; Langmead et al., 2006, 2008). However, investigation of alternative signaling pathways activated by allosteric agonists at the M₁ mACh receptor remains largely uncharacterized. Therefore, in this study, we have investigated the actions of AC-42, its analog 77-LH-28-1 (Langmead et al., 2008), and orthosteric mACh receptor agonists [oxotremorine-M (oxo-M), arecoline, and pilocarpine] to determine whether these compounds differentially affect receptor-G protein coupling, assessing these effects at the level of receptor recognition, G protein coupling, and signal transduction pathway activation.

	Materials and Methods

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Materials. All chemicals and reagents were purchased from Sigma Chemical (Poole, Dorset, UK) unless otherwise stated. Tissue culture reagents and Lipofectamine2000 transfection reagent were from Invitrogen (Paisley, UK). [³⁵S]GTPS, [³H]cAMP, [³H]NMS, myo-[³H]inositol, and protein A-Sepharose CL-4B were from GE Healthcare (Chalfont St. Giles, UK). Charcoal and borosilicate coverslips were from BDH (Poole, Dorset, UK). Primary antibody for G_q/11 was generated against the common 11-mer C terminus sequence LQLNLKEYNLV as described previously (Akam et al., 2001). Primary antibody for G_i was generated by Cambridge Research Biochemicals (Northwich, UK) against the common G_i1/2 C-terminal 10-mer sequence KENLKDCGLF. AC-42 and 77-LH-28-1 were synthesized "in-house" by GlaxoSmithKline (Harlow, UK).

Cell Culture. Chinese hamster ovary cells stably expressing the recombinant human M₁ mACh receptor (CHO-M₁ cells), originally obtained from Dr. Noel Buckley (then at National Institutes for Medical Research, London, UK), were grown in minimal essential medium- supplemented with fetal bovine serum (10%), penicillin (50 units/ml), streptomycin (50 µg/ml), and amphotericin B (2.5 µg/ml). Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air.

Cell Membrane Preparation. Confluent monolayers of CHO-M₁ cells were rapidly washed with 10 mM HEPES, 0.9% NaCl, and 0.2% EDTA, pH 7.4 (HBS-EDTA) before incubation with HBS-EDTA for 15 min to lift cells. Cells were centrifuged (400g, 4 min), and the pellet was resuspended in 10 mM HEPES and 10 mM EDTA, pH 7.4, homogenized using a Polytron homogenizer (3 x 5-s bursts; Kinematica AG, Littau, Switzerland) and centrifuged (40,000g, 15 min, 4°C). The cell pellet was resuspended in 10 mM HEPES and 0.1 mM EDTA, pH 7.4, and rehomogenized and centrifuged as described above. The final pellet was resuspended in the 10 mM HEPES and 10 mM EDTA, pH 7.4, buffer to give a final protein concentration of 2 mg/ml and either used immediately or snap-frozen and stored at -80°C.

[³H]NMS Binding. [³H]NMS inhibition binding assays were carried out as described previously (Mistry et al., 2005). In general, CHO-M₁ cells were seeded at a density of 75,000 cells/well in 24-well plates. The next day, cells were washed three times with warmed KHB (composition: 118 mM NaCl, 8.5 mM HEPES, 4.7 mM KCl, 4 mM NaHCO₃, 1.3 mM CaCl₂, 1.2 mM MgSO₄.7H₂O, 1.2 mM KH₂PO₄, and 11.7 mM glucose, pH 7.4) before being maintained on ice in a total assay volume of 1 ml of ice-cold KHB containing appropriate concentrations of agonists and approximately 0.2 nM [³H]NMS. After a 5-h incubation at 4°C cells were washed three times with ice-cold KHB before the addition of 0.1 M NaOH (500 µl). After cell solubilization, SafeFluor scintillation fluid was added, and samples were counted.

[³⁵S]GTPS Binding and Immunoprecipitation of G Subunits. [³⁵S]GTPS binding and immunoprecipitation of G subunits was performed as described previously (Akam et al., 2001). In general, CHO-M₁ membranes were diluted in assay buffer (10 mM HEPES, 100 mM NaCl, and 10 mM MgCl₂, pH 7.4) to give a final protein concentration of 75 µg per 50 µl. Membranes were then added to assay buffer containing final concentrations of 1 nM [³⁵S]GTPS and either 1 or 10 µM GDP (see Results). Membranes were incubated at 30°C for 2 or 5 min (see Results) with agonists before reactions were terminated by the addition of 1 ml of ice-cold assay buffer and immediate transfer to an ice bath. Samples were centrifuged (20,000g, 6 min, 4°C), and membrane pellets were solubilized by the addition of 50 µl of ice-cold solubilization buffer (100 mM Tris/HCl, 200 mM NaCl, 1 mM EDTA, 1.25% Igepal, and 0.2% SDS, pH 7.4) and regular vortex mixing over 30 min. After complete protein resolubilization, 50 µl of solubilization buffer without SDS was added. Solubilized protein was precleared using normal rabbit serum at a dilution of 1:100 and 3% (w/v) protein A-Sepharose beads in TE buffer (10 mM Tris/HCl and 10 mM EDTA, pH 8.0) added for 60 min at 4°C. Protein A-Sepharose beads and insoluble material were collected by centrifugation (20,000g, 6 min, 4°C), and 100 µl of the supernatant was transferred to fresh tubes containing G protein-specific anti-serum. Samples were vortex-mixed and rotated at 4°C for 90 min before being centrifuged (20,000g, 6 min, 4°C). Supernatants were aspirated, and the protein A-Sepharose beads were washed three times with ice-cold solubilization buffer (without SDS). Recovered beads were then mixed with 1 ml of FloScint-IV scintillation cocktail and counted by liquid scintillation spectrometry.

cAMP Accumulation Assay. CHO-M₁ cells seeded at 100,000 cells/well in 24-well plates 2 days before assay were washed twice before the addition of 450 µl of Ca²⁺-free KHB containing 100 µM EGTA. Cells were incubated at 37°C with agonists for 10 min before termination by aspiration of buffer and addition of ice-cold 0.5 M trichloroacetic acid (400 µl). After addition of 50 µl of 10 mM EDTA, samples were mixed with 500 µl of 1,1,2-trichlorofluoroethane:tri-n-octylamine (1:1, v/v). Samples were centrifuged at 16,000g for 4 min, and 200 µl of the upper phase was removed. Sample pH was brought to approximately 7.5 by the addition of 50 µl of 60 mM NaHCO₃. cAMP concentrations were determined using a binding assay as described previously (Mistry et al., 2005).

Total [³H]Inositol Phosphate Accumulation Assay. CHO-M₁ cells were seeded at 100,000 cells/well in 24-well plates and incubated in fresh medium containing 2.5 µCi/ml [³H]inositol for 48 h. Confluent cell monolayers were washed twice in KHB, and then 275 µl of KHB containing 10 mM LiCl was added. Cells were incubated for 30 min at 37°C before the addition of agonist or vehicle for 15 min. Reactions were terminated by the addition of ice-cold trichloroacetic acid (1 M), and samples were neutralized using the freon/tri-n-octylamine method as described above. The [³H]inositol mono-, bis-, and trisphosphate ([³H]InsP_x) fraction was recovered by anion exchange chromatography. Dowex-1 (formate form) columns were regenerated with 10 ml of ammonium formate (2 M)/formic acid (0.1 M) before being washed extensively with distilled water. Samples were washed onto the columns with 5 ml of distilled water. Columns were initially washed with 10 ml of ammonium formate (60 mM)/sodium tetraborate (5 mM). The [³H]InsP_x fraction was then eluted in 10 ml of ammonium formate (0.75 M)/formic acid (0.1 M). A 5-ml aliquot of the collected eluate was mixed with 10 ml of SafeFluor scintillation cocktail, and radioactivity was determined by liquid scintillation counting.

Ins(1,4,5)P₃ Mass Assay. CHO-M₁ cells seeded at 100,000 cells/well in 24-well plates 2 days before assay were washed twice, and 450 µl of warmed KHB was added for 30 min at 37°C before the addition of maximal agonist concentrations for the times indicated. Reactions were terminated by the addition of ice-cold trichloroacetic acid (0.5 M) and extracted using the freon/tri-n-octylamine method as described above. Determination of Ins(1,4,5)P₃ concentrations was by a competition binding assay described previously (Challiss et al., 1988).

Single-Cell Imaging of Ins(1,4,5)P₃. CHO-M₁ cells were seeded onto 25-mm glass coverslips and transiently transfected with the enhanced green fluorescent protein tagged to the pleckstrin homology domain of phospholipase C (eGFP-PH) construct (Stauffer et al., 1998) using Lipofectamine2000 according to the manufacturer's instructions. After 48 h, translocation of the eGFP-PH biosensor was visualized using an Olympus FV500 confocal microscope (Olympus, Tokyo, Japan), with cells maintained at 37°C and perfused with KHB (5 ml/min). Changes in the cell localization of eGFP-PH were expressed as a change in cytosolic fluorescence relative to basal as described previously (Nash et al., 2002). Drugs were applied through a perfusion line for the times indicated.

Data Analysis. All data are expressed as mean ± S.E.M. for the indicated number of experiments or as a representative trace of at least three independent experiments. Radioligand binding data and agonist concentration-response curves were analyzed using Prism 4.0 (GraphPad Software Inc., San Diego, CA). IC₅₀ values derived from agonist displacement of [³H]NMS binding were converted to K_b values using the method of Cheng and Prusoff (1973). For calculation of the initial rate of Ins(1,4,5)P₃ accumulation in eGFP-PH fluorescent imaging experiments, basal and peak fluorescence values were defined within an area of interest in the cytoplasm (see Bartlett et al., 2005); the time taken for the fluorescence to increase from 10 to 90% of the (peak - basal) value was determined, as was the magnitude of the 10 90% fluorescence change; this allowed the calculation of an initial rate as arbitrary fluorescence units (AFU) per second. Statistical differences between multiple data sets were assessed by one-way analysis of variance followed by Bonferroni's multiple-range test at P < 0.05 using Prism 4.0 (GraphPad Software Inc.). For agonist potency ranking, > represents a statistically significant difference, and represents no difference.

	Results

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Both AC-42 and 77-LH-28-1, a structural homolog, have been reported previously to have an allosteric mechanism of action (Langmead et al., 2006; Spalding et al., 2006; May et al., 2007). In preliminary experiments, both ligands were shown to possess similar allosteric properties. Thus, they each significantly slowed [³H]NMS dissociation from the M₁ mACh receptor in CHO-M₁ membranes, and analysis of the abilities of these compounds to displace specific [³H]NMS binding at different concentrations of [³H]NMS (0.2, 2, and 5 nM) was consistent with an allosteric mechanism of action (see Langmead et al., 2006; Spalding et al., 2006).

Agonist Affinity Estimates for M₁ mACh Receptors. M₁ mACh receptor binding affinities for oxo-M, AC-42, 77-LH-28-1, arecoline, and pilocarpine were determined by [³H]-NMS competition binding in intact CHO-M₁ cell monolayers. Initial [³H]NMS saturation binding analysis determined a B_max value of 4.84 ± 0.31 pmol/mg protein and a K_D value of 0.29 ± 0.05 nM in intact CHO-M₁ cells (n = 4; data not shown). CHO-M₁ cell monolayers were incubated with an approximate K_d value of [³H]NMS in the presence of varying agonist concentrations at 4°C for 5 h (to achieve equilibrium binding without causing receptor internalization). The apparent binding affinity (pK_B) and Hill slope for each agonist are summarized in Table 1. 77-LH-28-1 displayed a significantly higher binding affinity than AC-42, oxo-M, arecoline, and pilocarpine (P < 0.05).

Analysis of M₁ mACh Receptor-G_q/11 Coupling. Agonist stimulation in the presence of [³⁵S]GTPS and subsequent immunoprecipitation of specific G-subunits allows examination of a proximal signaling event in membrane preparations from a number of cells/tissues (Friedman et al., 1993; Lazareno and Birdsall, 1993; Akam et al., 2001). This technique has been optimized in a CHO cell background to show that the concentration-dependent increase in [³⁵S]-GTPS-G_q/11 binding, stimulated by the human M₁ mACh receptor, is optimal in the presence of 1 µM GDP after 2-min incubations at 30°C (Akam et al., 2001). Maximal activation of the M₁ mACh receptor by oxo-M caused an approximate 30-fold increase in [³⁵S]GTPS binding to G_q/11 with an EC₅₀ value of approximately 1 µM (Fig. 1; Table 2). The M₁-selective allosteric agonists, AC-42 and 77-LH-28-1, were partial agonists with respect to oxo-M, eliciting approximately 30 and 60% of the oxo-M response, respectively (Fig. 1A). 77-LH-28-1 was more potent than AC-42, causing an increase in [³⁵S]GTPS-G_q/11 binding with an EC₅₀ value of approximately 0.4 compared with 1.6 µM for AC-42 (Table 2). Pilocarpine and arecoline also behaved as partial agonists with respect to oxo-M, causing approximately 35 and 60% of the maximal response (Fig. 1B). Thus, AC-42 displays comparable relative efficacy to the orthosteric agonist pilocarpine in activating G_q/11 subunits, whereas 77-LH-28-1 is comparable with the orthosteric agonist arecoline. Time courses for agonist-stimulated increases in G_q/11-[³⁵S]GTPS binding are shown as insets to Fig. 1, A and B. In agreement with the previous observation of Akam et al. (2001), 2-min incubations are sufficient to cause maximal increases in G_q/11-[³⁵S]GTPS binding over basal values.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Concentration dependencies of agonist-stimulated [35S]GTPS binding to Gq/11 subunits in CHO-M1 cell membranes. CHO-M1 cell membranes were incubated with the indicated concentrations of oxo-M, AC-42, and 77-LH-28-1 (A) or oxo-M, arecoline, and pilocarpine (B) in the presence of 1 µMGDP and 1 nM[35S]GTPS for 2 min at 30°C. Incubations were terminated, and Gq/11 subunits were immunoprecipitated as described under Materials and Methods. Inset figures, A and B, time courses for increases in Gq/11-[35S]GTPS binding stimulated by agonist or vehicle additions: oxo-M (100 µM), AC-42 (30 µM), 77-LH-28-1 (10 µM), arecoline (1 mM), or pilocarpine (1 mM). Concentration dependence and time course data are presented as means ± S.E.M. for at least three separate experiments performed in duplicate (see Table 2).

Characterization of [³H]InsP_x Accumulation in CHO-M₁ Cells. As an index of PLC activation, agonist-stimulated accumulation of total [³H]InsP_x was assessed in the presence of Li⁺. Maximal stimulation with oxo-M caused a 15-fold increase in [³H]InsP_x accumulation (1,198,704 ± 293,374 dpm/mg protein over a basal value of 77,064 ± 18,779 dpm/mg protein) with an EC₅₀ of 0.1 µM (Fig. 2; Table 2). This represents a 10-fold leftward shift in the concentration dependence for the [³H]InsP_x accumulation versus G_q/11-[³⁵S]GTPS binding for the full, orthosteric agonist (Table 2). Both AC-42 and 77-LH-28-1 behaved as partial agonists in this assay with respect to the oxo-M response, causing approximately 55 and 87% of the full agonist response, respectively. The respective relative efficacy (R_max) values for arecoline and pilocarpine were 101 and 90% (Fig. 2; Table 2). Thus, R_max values for all of the orthosteric and allosteric partial agonists increased with respect to [³H]InsP_x accumulation compared with G_q/11-[³⁵S]GTPS binding; however, significant differences between EC₅₀ values for these two readouts were only observed for the full agonist (Table 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Concentration dependencies of agonist-stimulated [3H]inositol phosphate accumulation in CHO-M1 cells. CHO-M1 cells labeled with [3H]inositol (2.5 µCi/ml; 48 h) were preincubated at 37°C for 30 min with 10 mM LiCl before agonist addition. Incubations were terminated after 15 min by medium removal and the rapid addition of ice-cold trichloroacetic acid (0.5 M) and analyzed to determine [3H]InsPx accumulation as described under Materials and Methods. Data for AC-42 and 77-LH-28-1 (A) and arecoline and pilocarpine (B) are expressed as a percentage of the maximal oxo-M response (1,198,704 ± 293,374 dpm/mg protein) over basal values (77,064 ± 18,779 dpm/mg protein) and are presented as means ± S.E.M. for at least three separate experiments performed in duplicate (see Table 2).

Characterization of Ins(1,4,5)P₃ Responses in CHO-M₁ Populations and in Single Cells. Although the Li⁺-blocked, [³H]InsP_x accumulation assay provides an index of PLC activity, we also conducted experiments to investigate the time course of Ins(1,4,5)P₃ accumulation in CHO-M₁ cells to compare further the activation profiles of allosteric and orthosteric agonists with respect to G_q/11-coupled downstream signaling. Basal levels of Ins(1,4,5)P₃ were 44 ± 3 pmol/mg protein (n = 12) in cell population experiments. oxo-M (100 µM) caused a biphasic rise in Ins(1,4,5)P₃ levels, with an initial peak of 703 ± 37 pmol/mg protein after 15 s, falling to a plateau level (278 ± 20 pmol/mg protein) sustained from 60 s onwards (Fig. 3). In contrast, activation of CHO-M₁ cells by either arecoline (1 mM) or pilocarpine (1 mM) caused monophasic Ins(1,4,5)P₃ accumulation profiles, with maxima being reached at around 60 s (Fig. 3B). It was notable that the "steady-state" Ins(1,4,5)P₃ accumulations caused by oxo-M, arecoline, and pilocarpine between 60 and 300 s were similar (approximately 300 pmol/mg protein). The allosteric agonist 77-LH-28-1 (10 µM) caused a similar monophasic profile of Ins(1,4,5)P₃ accumulation to the orthosteric partial agonists, achieving a similar steady-state value (Fig. 3A). In contrast, stimulation with AC-42 (100 µM) produced a modest, monophasic increase in Ins(1,4,5)P₃ accumulation (reaching 122 ± 9 pmol/mg protein at 120 s; Fig. 3A), which was significantly lower than steady-state values for all of the other agonists examined (P < 0.01).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Time courses of agonist-stimulated Ins(1,4,5)P3 accumulation in CHO-M1 cells. Confluent monolayers of CHO-M1 cells were stimulated for the times indicated with oxo-M (100 µM), AC-42 (30 µM), or 77-LH-28-1 (10 µM) (A) or oxo-M (100 µM), arecoline (1 mM), or pilocarpine (1 mM) (B). Incubations were terminated at the indicated time points, and samples were analyzed for Ins(1,4,5)P3 levels as described under Materials and Methods. Data are presented as means ± S.E.M. for four (AC-42, 77-LH-28-1, pilocarpine) or five (oxo-M, arecoline) separate experiments performed in duplicate.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Assessment of changes in Ins(1,4,5)P3 in response to orthosteric and allosteric agonists in single CHO-M1 cells using confocal fluorescence imaging. CHO-M1 cells were transfected with the fluorescent biosensor eGFP-PH, and after 48 h, Ins(1,4,5)P3 production was measured by profiling the translocation of the eGFP-PH probe from the plasma membrane to the cytoplasm. Representative images and time course traces from single CHO-M1 cells stimulated for 30 s with oxo-M (100 µM), arecoline (1 mM), or pilocarpine (A) or oxo-M (100 µM), AC-42 (30 µM), or 77-LH-28-1 (10 µM) (B), followed by washout. In each panel, images are shown corresponding to a to d labels on the time course traces. Data are expressed as the change in fluorescence intensity from the basal fluorescence level (F/F0). The traces shown are representative of five to 10 cells analyzed per coverslip over three separate experiments.

Analysis of Receptor-G_i1/2 Coupling. Although the M₂ and M₄ mACh receptor subtypes couple preferentially to G_i/o proteins (Caulfield and Birdsall, 1998), it has been demonstrated that the M₁ and M₃ mACh receptor subtypes are also capable of activating G_i subunits in addition to G_q/11 subunits (Offermanns et al., 1994; Akam et al., 2001). To investigate whether the M₁ mACh receptor-selective allosteric agonists can cause the M₁ mACh receptor to activate multiple G protein-dependent pathways, we first assessed the agonist-dependent activation of G_i subunits in CHO-M₁ membranes.

In initial experiments designed to optimize the [³⁵S]-GTPS-G_i1/2 immunoprecipitation assay, oxo-M caused a marked (272 ± 74% over basal) and concentration-dependent [pEC₅₀ (M) = 7.14 ± 0.06] increase in [³⁵S]GTPS-G_i1/2 binding in CHO-M₂ membranes. Similar optimal binding conditions were obtained for agonist-stimulated increases in [³⁵S]GTPS-G_i1/2 binding in CHO-M₁ membranes (10 µM GDP, 2-min incubations at 30°C; see Fig. 5A, inset). Under these conditions, oxo-M stimulated a more modest [basal binding, 15,120 ± 707 (15); +oxo-M, 23,240 ± 1280 (15) cpm/mg protein] yet highly significant (P < 0.001) increase in [³⁵S]GTPS binding. The increase in [³⁵S]GTPS-G_i1/2 binding stimulated by oxo-M was concentration-dependent [pEC₅₀ (M) = 5.13 ± 0.08 M] and lay approximately 10-fold to the right of the oxo-M-stimulated [³⁵S]GTPS-G_q/11 binding curve (see Fig. 5B; Table 2). Arecoline and pilocarpine both stimulated significant increases in [³⁵S]GTPS-G_i1/2 binding over basal (Fig. 5, A and B), with their respective concentration-response curves also lying approximately 10-fold to the right of their respective [³⁵S]GTPS-G_q/11 binding responses (Table 2). In contrast, neither allosteric agonist [at concentrations up to 30 µM (77-LH-28-1) or 300 µM (AC-42)] was able to cause a significant increase in [³⁵S]GTPS-G_i1/2 binding in CHO-M₁ membranes (Fig. 5A; Table 2). These data suggest that the allosteric agonists are less able to facilitate M₁ mACh receptor-dependent GTP/GDP exchange on G_i1/2 proteins compared with orthosteric partial agonists that are equiefficacious with respect to other signaling readouts.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Concentration-dependent agonist-stimulated [35S]GTPS binding to Gi1/2 subunits in CHO-M1 cell membranes. CHO-M1 cell membranes were incubated with oxo-M (100 µM; n = 7), arecoline (1 mM; n = 5), pilocarpine (1 mM; n = 7), AC-42 (100 µM; n = 8), or 77-LH-28-1 (30 µM; n = 7) in the presence of 10 µMGDP and 1 nM [35S]GTPS for 2 min at 30°C (A). Incubations were terminated after this time, and specific G subunits were immunoprecipitated as described under Materials and Methods. Statistically significant differences are indicated as follows: *, P < 0.05; and **, P < 0.01. A, inset, time course of change in [35S]GTPS-Gi1/2 binding in the absence and presence of oxo-M (10 µM). Concentration-dependent increases in [35S]GTPS-Gi1/2 binding stimulated by oxo-M (n = 5), arecoline (n = 4), or pilocarpine (n = 6) are shown in B. All data are presented as means ± S.E.M. for the indicated number of separate experiments (see Table 2) performed in duplicate.

Analysis of Receptor-G_s Coupling.

As an alternate index of mACh receptor-G_s activation, we measured cAMP accumulation in intact CHO-M₁ cells. Addition of oxo-M (100 µM) caused a robust 10- to 20-fold increase in cAMP accumulation (basal, 11 ± 3; +oxo-M, 183 ± 8 pmol/mg protein; pEC₅₀, 5.05 ± 0.08); however, none of the partial agonists caused a significant increase over basal (data not shown). In contrast, arecoline, pilocarpine, AC-42, and 77-LH-28-1 significantly enhanced the increase in cAMP accumulation caused by forskolin (1 µM). Previous studies have found that under these conditions, receptor activation in CHO cells can indirectly modulate forskolin-stimulated adenylyl cyclase activity through activation of Ca²⁺ entry and Ca²⁺-dependent effects on isoforms of adenylyl cyclase (Cooper et al., 1995). Therefore, in these assays, Ca²⁺ was omitted from the medium, and EGTA (100 µM) was added to remove the possibility of Ca²⁺ entry-driven effects on M₁ mACh receptor-mediated cAMP responses. Under these conditions, all agonists induced concentration-dependent, monophasic enhancements of forskolin-stimulated cAMP accumulation (Fig. 6). oxo-M caused a 7.5-fold enhancement of the forskolin-stimulated response (+forskolin, 151 ± 10; +forskolin/oxo-M, 1130 ± 66 pmol/mg protein). Arecoline, pilocarpine, AC-42, and 77-LH-28-1 all behaved as partial agonists with respect to the maximal oxo-M response (Fig. 6, A and B; Table 2). The rank order of potency with respect to cAMP accumulation was 77-LH-28-1 (6.01) oxo-M (5.71) > AC-42 (5.06) arecoline (4.61) pilocarpine (4.41).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Concentration-dependent effects of mACh receptor agonists on forskolin-stimulated cAMP accumulation. Confluent CHO-M1 cell monolayers were washed twice in Ca2+-free KHB before incubation in the presence of the indicated concentrations of mACh receptor agonists for 10 min before the addition of forskolin (1 µM) for 10 min. After this time, incubations were terminated, and cAMP levels were determined as described under Materials and Methods. Data are shown for AC-42 and 77-LH-28-1 (A) and arecoline and pilocarpine (B); oxo-M data are reproduced in both panels. Data are presented as means ± S.E.M. for the indicated number of separate experiments (see Table 2) performed in duplicate.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present study provides a profile of the proximal signaling pathways coupled to an allosteric agonist of the M₁ mACh receptor, AC-42 (Spalding et al., 2002), and a closely related compound, 77-LH-28-1 (May et al., 2007; Langmead et al., 2008). In general, we have focused on the ability of orthosteric and allosteric mACh receptor agonists to activate G_q/11-dependent signaling events and explored the ability of these agonists to stimulate M₁ mACh receptor coupling to alternative G_i/o and G_s protein-dependent signaling pathways. Although orthosteric and allosteric agonists share many properties, we have demonstrated that there exist subtle differences in the signaling outcomes these two subclasses of agonist are able to activate.

Initial assessment of the abilities of orthosteric and allosteric agonists to promote M₁ mACh receptor-G_q/11 protein coupling in the CHO-M₁ membranes used the [³⁵S]GTPS binding assay (Hilf et al., 1989; Lazareno and Birdsall, 1993) adapted to distinguish [³⁵S]GTPS binding to specific G protein subpopulations (Friedman et al., 1993; Akam et al., 2001). Consistent with previous work (Spalding et al., 2002, 2006; Langmead et al., 2006, 2008), AC-42 and 77-LH-28-1 were able to activate the M₁ mACh receptor in the absence of an orthosteric ligand, causing robust increases in [³⁵S]-GTPS-G_q/11 binding, with 77-LH-28-1 exhibiting a greater intrinsic activity and higher potency than AC-42 with respect to this proximal signaling readout. Nevertheless, recent data indicate that AC-42 is sufficiently efficacious to stimulate significant M₁ mACh receptor-stimulated [³⁵S]-GTPS-G_q/11 binding in native systems (Salah-Uddin et al., 2008).

A consequence of M₁ mACh receptor-G_q/11 coupling is the activation of PLC. In the presence of lithium, it is possible to assess [³H]InsP_x accumulation as an index of PLC activity in cells labeled to equilibrium with [³H]inositol. Using this assay, it was observed that the EC₅₀ for the full agonist oxo-M was approximately 10-fold left-shifted for [³H]InsP_x accumulation in intact cells compared with the membrane-based [³⁵S]GTPS-G_q/11 binding response. In contrast, EC₅₀ values for these responses were similar for both orthosteric and allosteric partial agonists, but increases in relative efficacy for [³H]InsP_x accumulation compared with [³⁵S]GTPS-G_q/11 binding responses were seen in all cases (Table 2).

To gain a more kinetic perspective on the actions of the different mACh receptor agonists on PLC activity, temporal profiles for Ins(1,4,5)P₃ accumulation were also investigated. Ins(1,4,5)P₃ response profiles vary both with respect to the initiating GPCR and the pharmacological properties of the stimulating agonist (Willars and Nahorski, 1995; Nash et al., 2002; Bartlett et al., 2005). Here, only the full agonist oxo-M stimulated a rapid, peak-and-plateau Ins(1,4,5)P₃ response. Monophasic increases in Ins(1,4,5)P₃, reaching similar plateau levels to those seen for oxo-M after 60 to 120 s, were seen for arecoline, pilocarpine, and 77-LH-28-1. In contrast, AC-42, which yielded similar pEC₅₀ and R_max values to pilocarpine with respect to stimulating [³⁵S]GTPS-G_q/11 binding (see Table 2), stimulated a monophasic increase in Ins(1,4,5)P₃ that was <50% of that achieved by the other partial agonists. Further differences between the agonists used were illustrated by single-cell studies of Ins(1,4,5)P₃ generation, using eGFP-PH (Stauffer et al., 1998; Nash et al., 2002). Application of oxo-M caused a rapid increase in eGFP-PH translocation from the plasma membrane to the cytoplasm, which rapidly reversed on agonist washout. Arecoline, pilocarpine, and 77-LH-28-1 gave broadly similar responses to each other, with slower increases, lower maxima, and slower reversibility on washout than oxo-M. Greater variation in individual responses was seen with respect to eGFP-PH translocation after AC-42 application (delivered either by perfusion or direct bath application). Although the eventual extent of eGFP-PH translocation caused by AC-42 in some cells approached that caused by 77-LH-28-1, the rate of rise was always slow (>10-fold slower relative to oxo-M). These data clearly indicate kinetic differences between the agonists, but these seem to be more related to efficacy differences between agents than whether they bind at an orthosteric or allosteric site on the receptor.

Agonists can bind to GPCRs and stabilize or induce specific conformations that allow the activated receptor to regulate downstream signaling cascades, a phenomenon referred to variously as agonist-directed trafficking of receptor stimulus, functional selectivity, and "ligand bias" (Kenakin, 2003; Urban et al., 2007; Violin and Lefkowitz, 2007). Unambiguous determination of agonist-selective conformations can best be deduced at the level of the receptor-G protein interaction.

We initially investigated agonist-stimulated M₁ mACh receptor-G_i coupling because in a previous report, we showed that the M₁ mACh receptor is capable of activating G_i/o subunits in a CHO cell background, and the degree of coupling to G_i/o is agonist-dependent (Akam et al., 2001). Here, we were able to confirm the activation of M₁ mACh receptor-stimulated G_i protein GTP/GDP exchange using a G_i1/2 antibody to immunoprecipitate [³⁵S]GTPS-G_i1/2 complexes. The orthosteric agonists oxo-M, arecoline, and pilocarpine all stimulated significant increases in [³⁵S]-GTPS-G_i1/2 binding, and pEC₅₀ values could be obtained for these agonists, which were approximately 10-fold right-shifted relative to those determined for receptor-stimulated [³⁵S]GTPS-G_q/11 binding (see Table 2). In contrast, both allosteric agonists failed to cause a significant increase in [³⁵S]GTPS-G_i1/2 binding, and as a consequence, pEC₅₀ values could not be determined. These data suggest that M₁ mACh receptor conformations favoring coupling to G_i1/2 proteins are better stabilized by oxo-M, arecoline, and pilocarpine compared with AC-42 and 77-LH-28-1.

G_q/11-coupled receptors have often been reported to stimulate cAMP accumulation in a variety of cell types. Although in some cases this has been shown to involve G_s protein-independent coupling via a Ca²⁺- and/or PKC-dependent mechanism (Felder et al., 1989; Cooper et al., 1995), in the CHO cell background, direct G_s coupling has been demonstrated for the _1B-adrenergic (Horie et al., 1995) and M₁ mACh (Burford and Nahorski, 1996) receptors. Assessment of receptor-G_s coupling using a [³⁵S]GTPS-G_s binding/immunoprecipitation assay (Carruthers et al., 1999) was not possible because significant agonist-dependent increases in [³⁵S]GTPS-G_s binding were not observed in CHO-M₁ membranes challenged with any of the mACh receptor agonists. This apparent lack of M₁ mACh receptor-G_s coupling should be interpreted cautiously, however, because isoprenaline only elicited a maximal 4-fold increase in [³⁵S]GTPS-G_s binding in CHO-β₂ membranes. In intact CHO-β₂ cells, isoprenaline causes a profound increase in cAMP accumulation (>100-fold), which is at least an order of magnitude greater that that elicited by oxo-M in intact CHO-M₁ cells (see Results). To compare M₁ mACh receptor-G_s coupling stimulated by the orthosteric and allosteric agonists, we looked at the ability of each agonist to enhance cAMP accumulation stimulated by a submaximal concentration of forskolin (1 µM). Indirect effects on adenylyl cyclase isoforms because of activation of a G_q/11/PLC/Ca²⁺ pathway can be abolished by performing experiments under Ca²⁺-free conditions (omission of Ca²⁺ from the KHB and addition of EGTA; see Materials and Methods). Under these conditions, cAMP accumulation is unaffected by pertussis toxin treatment or protein kinase C inhibitors (R. Mistry and R. A. J. Challiss, unpublished data) and is a result of G_s activation. All agonists studied were able to stimulate monophasic enhancements of forskolin-stimulated cAMP accumulation. oxo-M stimulated the greatest increase, with a pEC₅₀ value 10-fold right-shifted relative to that for the [³H]InsP_x response (Table 2). In contrast to the data obtained for M₁ mACh receptor-G_i coupling, 77-LH-28-1 and AC-42 were able to increase cAMP accumulation with EC₅₀ values only 4- to 5-fold right-shifted compared with those obtained for [³H]InsP_x accumulation, suggesting they are able to stabilize an M₁ mACh receptor conformation capable of interacting with the G_s subunits.

Although much of the relevant literature focuses on M₁ mACh receptor signaling via G_q/11, there are reports of M₁ mACh receptors linking to distal cellular outputs via G_i and G_s. For example, although it is well established that M₁ mACh receptors inhibit the M-type potassium current via G_q/11 (Delmas and Brown, 2005), a pertussis toxin-sensitive component to M-current inhibition has also been reported (Haley et al., 2000; Lechner et al., 2003). Likewise, it has been reported that M₁ mACh receptor stimulation of cAMP response element-driven luciferase reporter gene expression in JEG-3 cells occurs via a PLC/Ca²⁺-independent, G_s-dependent pathway (Migeon and Nathanson, 1994). Thus, it is possible that G_i-, G_s-, and G_q/11-dependent signaling contribute to at least a subset of M₁ mACh receptor-mediated responses in different cell backgrounds and under particular (patho)physiological conditions.

In conclusion, although AC-42 and its structural homolog 77-LH-28-1 clearly interact with the M₁ mACh receptor differently from conventional orthosteric agonists (Spalding et al., 2002, 2006; Langmead et al., 2006), they nevertheless cause activation of G_q/11 signaling that is functionally indistinguishable from orthosteric agonists. In addition, AC-42 and 77-LH-28-1 are able to enhance forskolin-induced cAMP stimulation, as are orthosteric agonists. In contrast, AC-42 and 77-LH-28-1 are much less able to facilitate receptor-G_i1/2 coupling than are orthosteric agonists of comparable intrinsic activity. Thus, in addition to the potential for much greater mACh receptor subtype selectivity of allosteric agonists, it is possible that these agents will activate only a subset of signaling pathways permitting further specificity of action.

	Acknowledgements

 
We thank Tobias Meyer (Stanford University, Palo Alto, CA) for providing the eGFP-PH biosensor and S.R. Nahorski (University of Leicester, Leicester, UK) for wise counsel during the initial stages of the project.

	Footnotes

 
This work was supported by the Biotechnology and Biological Sciences Research Council/GlaxoSmithKline (CASE studentship to R.L.T.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.141788.

ABBREVIATIONS: mACh, muscarinic acetylcholine; GPCR, G protein-coupled receptor; AC-42, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine; 77-LH-28-1, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone; oxo-M, oxotremorine-M; GTPS, guanosine 5'-O-(3-thiotriphosphate); NMS, N-methylscopolamine; CHO, Chinese hamster ovary; KHB, Krebs-Henseleit buffer; [³H]InsP_x, [³H]inositol mono-, bis-, and trisphosphate; Ins(1,4,5)P₃, inositol-1,4,5-trisphosphate; eGFP-PH, enhanced green fluorescent protein tagged to the pleckstrin homology domain of phospholipase C; AFU, arbitrary fluorescence unit(s); PLC, phospholipase C.

Address correspondence to: Dr. R.A. John Challiss, Department of Cell Physiology and Pharmacology, University of Leicester, Room 4/04, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK. E-mail: jc36{at}leicester.ac.uk

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