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CELLULAR AND MOLECULAR

Constitutive Activity and Inverse Agonism at the M₂ Muscarinic Acetylcholine Receptor

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom

Received for publication August 19, 2005
Accepted September 26, 2005.

	Abstract

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Introduction of a single-point mutation (Asn to Tyr) at position 410 at the junction between transmembrane domain 6 and the third extracellular loop of the human M₂ muscarinic acetylcholine (mACh) receptor generated a mutant receptor (N410Y) that possesses many of the hallmark features of a constitutively active mutant receptor. These included enhanced agonist binding affinity and potency, in addition to agonist-independent accumulation of [³H]inositol phosphates in cells coexpressing the chimeric G_qi5 protein and the N410Y mutant M₂ mACh receptor. Constitutive activity was sensitive to inhibition by a range of muscarinic ligands, including those used clinically in the management of overactive bladder (oxybutynin, tolterodine, and darifenacin), indicating that these ligands behave as inverse agonists at the M₂ mACh receptor. Long-term (24-h) treatment of Chinese hamster ovary cells expressing the N410Y mutant M₂ mACh receptor with certain mACh receptor inverse agonists (atropine, darifenacin, and pirenzepine) elicited a concentration-dependent up-regulation of cell surface receptor expression. However, not all ligands possessing negative efficacy in the [³H]inositol phosphate accumulation assays were capable of significantly up-regulating receptor expression, perhaps indicating a spectrum of negative efficacies among ligands traditionally defined as mACh receptor antagonists. Finally, structurally distinct agonists exhibited differences in their relative potencies for the activation of G_i/o versus G_s, consistent with agonist-directed trafficking of signaling at the N410Y mutant, but not at the wild-type M₂ mACh receptor. This indicates that the N410Y mutation of the M₂ mACh receptor alters receptor-G-protein coupling in an agonist-dependent manner, in addition to generating a constitutively active receptor phenotype.

One of the most powerful tools used by researchers in this area has been the development of GPCRs harboring specific mutations known to enhance the agonist-independent coupling of receptor and G-protein [so-called constitutively active mutant (CAM) receptors] (Seifert and Wenzel-Seifert, 2002). Mutations in a number of well conserved domains, including the D/ERY motif at the intracellular interface of the third transmembrane domain (TM3) and the BBXXB motif (where B is Arg or Lys) toward the C-terminal end of the third intracellular loop, have been reported to enhance agonist-independent signaling of a wide range of GPCRs (Parnot et al., 2002).

In the case of the muscarinic acetylcholine (mACh) receptors, a number of studies have identified constitutively activating mutations, particularly in the predominantly G_q/11-coupled M₁, M₃, and M₅ receptor subtypes (Spalding et al., 1995, 1997; Ford et al., 2002). Spalding et al. (1995) first identified that mutation of adjacent serine (Ser465) and threonine (Thr466) residues at the junction between TM6 and the third extracellular loop results in a CAM-M₅ mACh receptor. Mutation of these two conserved residues has since been demonstrated to enhance constitutive activity of all five mACh receptor family subtypes (Ford et al., 2002). The mutant receptors displayed many of the characteristic properties of CAM-GPCRs, including enhanced agonist affinity and potency, in addition to an elevated basal functional activity (proportional to the receptor expression level), which was sensitive to the inverse agonist atropine (Ford et al., 2002).

Huang et al. (1998) had earlier investigated both double (³⁸⁸Ser/³⁸⁹Thr to Tyr/Pro) and single (³⁸⁸Ser to Tyr, and ³⁸⁹Thr to Pro) mutations in the M₁ mACh receptor subtype and found that mutation of ³⁸⁸Ser alone was sufficient to generate a mutant receptor displaying many of the common properties exhibited by a CAM receptor (enhanced agonist potency and binding affinity). In contrast, mutation of ³⁸⁹Thr seemed to influence receptor-G-protein coupling fidelity, introducing multiple apparent affinity binding states in agonist competition binding experiments (Huang et al., 1999). Moreover, Spalding et al. (1997) reported that mutation of the homologous residue (⁴⁶⁵Ser) in the M₅ subtype, particularly to large (Phe or Val) or basic (Arg or Lys) residues, generated receptors with significantly enhanced constitutive activity, relative to the wild type. Taken together, these data suggest that the conserved serine residue at the boundary between TM6 and the third extracellular loop is implicated in constraining the M₁ and M₅ mACh receptors in the inactive state.

The primary aim of the present study was to generate a CAM-M₂ mACh receptor by the targeted mutation of the conserved asparagine residue at position 410 of the human M₂ receptor (homologous to ³⁸⁸Ser in M₁ and ⁴⁶⁵Ser in M₅ mACh receptors) to tyrosine (to generate the N410Y mutant). To date, there have been surprisingly few reports of CAM-M₂ mACh receptors. Liu et al. (1996) reported that insertion of one to four alanine residues into TM6, three residues C-terminal to the BBXXB motif of the M₂ mACh receptor, significantly enhanced the constitutive inhibition of adenylate cyclase activity. More recently, Ford et al. (2002) demonstrated that the double-mutant (⁴¹⁰Asn/⁴¹¹Thr to Tyr/Pro) CAM-M₂ receptor displayed approximately 4- to 5-fold higher affinity for agonist and 62% constitutive activity relative to wild-type M₂ mACh receptors expressed in a COS-7 cell background. However, Ford et al. (2002) observed no additional agonist-mediated functional response above the level of constitutive activity at the CAM-M₂ receptor, and, as in the earlier study by Liu et al. (1996), further characterization of the mutant receptor beyond the establishment of constitutive activity has not been reported. Therefore, the present study aims to provide a more thorough analysis of the ^N410YM₂ mACh receptor mutant and, in particular, to define the inverse agonist properties of a number of clinically relevant ligands previously classified as mACh receptor antagonists.

	Materials and Methods

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Materials. [³H]NMS, myo-[³H]inositol, and [³H]cAMP were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Darifenacin and tolterodine were synthesized in the laboratories of Pfizer Central Research (Sandwich, Kent, UK). All other reagents were purchased from Sigma Chemical (Poole, Dorset, UK) or Fisher Scientific Co. (Pittsburgh, PA). G_qi5 in pCDN was a generous gift from Dr. S. Rees (GlaxoSmithKline, Uxbridge, Middlesex, UK).

Generation of ^N410YM₂ Mutant Receptor. Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the wild-type M₂ mACh receptor gene in pcDNA3 as a template. The following oligonucleotide primer and its complement were used to incorporate a single amino acid change (Asn to Tyr) at position 410 (via substitution of T for A, shown in bold): 5'-GCC CCA TAC AAT GTC ATG GTC CTC ATT TAC ACC TTT TGT GCA CCT-3'. The underlined nucleotide represents a silent mutation leading to the incorporation of an additional restriction site for the enzyme AvaII, allowing for initial confirmation of the successful mutagenesis.

Cell Culture and Transient Transfection of Chinese Hamster Ovary Cells. CHO-K1 cells were grown in minimum essential medium- supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B. CHO-K1 cells stably expressing cloned human wild-type M₂ or ^N410YM₂ receptors generated in this project were grown in an identical medium to that used in the culture of CHO-K1 cells, supplemented with 500 µg/ml G418 (Geneticin) selection reagent. Cells were maintained at 37°C in a humidified atmosphere of O₂/CO₂ (19:1) and were routinely split 1:5 every 3 to 4 days using trypsin-EDTA. Cells were transfected 48 h before experimentation using GeneJuice transfection reagent, and the medium was replaced approximately 6 h after transfection.

CHO Cell Membrane Preparation and Radioligand Binding. Confluent monolayers of CHO cells were briefly washed with HEPES-buffered saline (10 mM HEPES and 0.9% NaCl, pH 7.4) and cells lifted from the flask by the addition of HEPES-buffered saline-EDTA (10 mM HEPES, 0.9% NaCl, and 0.2% EDTA, pH 7.4) for approximately 15 min. A cell pellet was recovered by centrifugation at 1000g for 5 min. The cell pellet was homogenized on ice in lysis buffer (10 mM HEPES and 10 mM EDTA, pH 7.4) using a Polytron homogenizer (2 x 20-s bursts at 4°C; Kinematica, Basel, Switzerland). The homogenate was then centrifuged (40,000g, 15 min, 4°C), rehomogenized, and recentrifuged as described above in 10 mM HEPES and 0.1 mM EDTA, pH 7.4. The final membrane pellet was resuspended in the same buffer at a concentration of 2 mg of protein/ml and stored at -80°C until required.

Saturation binding was performed using a range of concentrations of [³H]NMS (0.03-6.0 nM; specific activity, 81 Ci/mmol) in the absence and presence of atropine (10 µM) to define nonspecific binding. Binding assays were performed in a final volume of 500 µl of 20 mM HEPES, pH 7.4, containing 25 to 100 µg of membrane protein for 60 min at 37°C. When monitoring the receptor expression levels after transient transfections, a single high concentration (3-4 nM) of [³H]NMS (performed in duplicate) was generally used to approximate the mACh receptor expression level. Competition binding experiments were performed using a single concentration of [³H]NMS (0.3-0.5 nM) in the absence and presence of a range of antagonist concentrations. Bound radioligand was separated from free by rapid vacuum filtration through GF/B filters (Whatman, Maidstone, UK) on a 24-well cell harvester (Brandel Inc., Gaithersburg, MD), and radioactivity was quantified by liquid scintillation counting (Nelson et al., 2004). Intact cell [³H]NMS binding assays were performed on cell monolayers on 24-well plates, as described previously (Nelson et al., 2004).

Long-Term Antagonist Treatment of CHO Cells. Where indicated, cells were incubated with putative inverse agonist ligands in culture medium for 24 h before assaying. At this point, cells were thoroughly washed three times with 1 ml of KHB before being incubated in 1 ml of KHB at 37°C for 20 min. After this time, KHB was aspirated, and cells were washed with 1 ml of KHB before assaying. Preliminary experiments determined that binding of atropine (300 nM) to the M₂ receptor could be fully reversed using this washing protocol, so this treatment was applied to cells before all subsequent [³H]inositol phosphate ([³H]IP_x) accumulation experiments.

Cyclic AMP and [³H]Inositol Phosphate Accumulation Assays. For cyclic AMP experiments, cells in 24-well multiwells were stimulated with forskolin (10 µM) for 10 min in the presence of agonist (agonist added 10 min before forskolin addition). Assays were stopped by aspiration and addition of ice-cold 0.5 M trichloroacetic acid (400 µl). Samples were neutralized as described previously (Nelson et al., 2004), and cyclic AMP was determined using the method of Brown et al. (1971). Where indicated, cell monolayers approaching confluence in 24-well multiwells were treated with pertussis toxin (PTx; 100 ng/ml) for 20 to 24 h before experimentation.

For [³H]IP_x assays, cDNAs encoding N410Y mutant or wild-type M₂ mACh receptors and the chimeric G-protein G_qi5 (Conklin et al., 1993) were cotransfected into CHO-K1 cells 24 h before experimentation. [³H]inositol (3 µCi/ml) was also added during this 24-h period. Putative inverse agonists were preincubated with [³H]inositol-prelabeled cell monolayers for 15 min before the addition of LiCl (10 mM) and continuation of the incubation for a further 15 min. [³H]inositol phosphate accumulation in the presence of various ligands was calculated as a percentage of that in the absence of ligand, after subtraction of the Li⁺-independent accumulation (i.e., the [³H]inositol phosphate accumulation over the same time course but in the absence of Li⁺).

Immunoblot Analysis. Cells were lysed with a Triton X-100-based buffer (20 mM HEPES, 200 mM NaCl, 10 mM EDTA, and 1% Triton X-100, pH 7.4) and added to an equal volume of 2x sample buffer (125 mM Tris/HCl, 4% SDS, 20% glycerol, 50 µM dithiothreitol, and 0.01% bromphenol blue, pH 6.8) before boiling at 90°C for 5 min. Samples were subjected to electrophoresis on 10% SDS-polyacrylamide gel electrophoresis minigels with 5% stacking gels and run at 120 V for 90 min (running buffer: 25 mM Tris, 250 mM glycine, and 0.1% SDS, pH 8.0). Transfer to nitrocellulose was achieved using a semidry apparatus (transfer buffer: 48 mM Tris, 39 mM glycine, 0.037% SDS, and 20% methanol, pH 8.3). Nitrocellulose membrane washing steps were performed using a modified high-stringency TBS-Tween buffer (20 mM Tris/HCl, 1 M NaCl, and 1% Tween 20, pH 7.5) and blocking with 20% milk in TBS-Tween buffer. Immunoblotting was performed using a rabbit polyclonal G_q anti-serum (IQB) (1:1000) raised against amino acids 119 to 134 of G_q (Mullaney et al., 1993). Immunoreactive proteins were detected using a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, St. Louis, MO) and enhanced chemiluminescence reagents.

Data and Statistical Analysis. Data are shown as mean ± S.E.M. for the indicated number of experiments. Saturation binding data were fitted with hyperbolae (one-site binding) using GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA). B_max and K_d values were derived from these curves. Competition binding curves and functional concentration-response curves were fitted to the four-parameter logistic equation: Y = bottom + (top - bottom)/[1 + 10 (logEC₅₀ - X) x n_H] using GraphPad Prism 3.0, where n_H is the Hill coefficient. The best fit between a variable Hill coefficient and a Hill coefficient fixed to unity was determined using an F test. IC₅₀ values, generated by these inhibition curves, were corrected to give binding constant (K_i) values for each test compound, using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

The statistical significance of differences between data were determined using either Student's t test, with K_i values first being converted to the respective normally distributed negative logarithm (pK_i), or one-way analysis of variance with Dunnett's post test for multiple comparisons.

	Results

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Measurement of Agonist-Mediated Responses at Transiently Expressed Wild-Type and N410Y Mutant M₂ mACh Receptors. Initial attempts to characterize pharmacologically the ^N410YM₂ mACh receptor by measuring cyclic AMP accumulation in HEK293 or CHO cells transiently expressing either the wild type or mutant receptor proved unsuccessful. Although increases in cyclic AMP could be generated in response to forskolin (1, 3, or 10 µM) in either cell background, a range of concentrations of MCh (up to 1 mM) failed to elicit significant, reproducible inhibitions of the forskolin-stimulated cyclic AMP responses in cells expressing either wild-type or ^N410YM₂ mACh receptors at levels >1 pmol/mg of protein (data not shown). These data contrast with the ability of MCh to cause >90% inhibition (IC₅₀, 230 nM) of forskolin-stimulated cyclic AMP accumulation in CHO cells stably expressing M₂ mACh receptors (Mistry et al., 2005).

The chimeric G_qi5 protein [G_q containing the C-terminal five amino acids (DCGLF) of G_i1-3; Conklin et al., 1993] has been shown previously to couple a variety of G_i/o-linked GPCRs, including the M₂ mACh receptor, to phospholipase C- activation and phosphoinositide hydrolysis in recombinant cell systems (Conklin et al., 1993; Liu et al., 1995). Therefore, cotransfection of cDNAs coding for G_qi5 and either the wild-type or ^N410YM₂ mACh receptor into CHO-K1 cells allowed the measurement of [³H]IP_x accumulation (under Li⁺ block) as an index of M₂ mACh receptor activation. Figure 1A shows the concentration-response relationship for MCh in CHO cells coexpressing G_qi5 and either the wild-type M₂ (2.74 ± 0.22 pmol/mg of protein) or ^N410YM₂ (1.95 ± 0.09 pmol/mg of protein) mACh receptor. Both basal and MCh-stimulated [³H]IP_x accumulations were linear over the time course of these experiments (data not shown), and no significant [³H]IP_x response to MCh was observed in untransfected CHO cells (Fig. 1A). Maximal [³H]IP_x responses to MCh were similar in wild-type and ^N410YM₂ mACh receptor-expressing cells (53,015 ± 6283 versus 52730 ± 5123 dpm/mg of protein, respectively). However, MCh was significantly more potent (>10-fold) at the N410Y mutant than at the wild-type M₂ mACh receptor (pEC₅₀ values, 7.58 ± 0.09 versus 6.44 ± 0.11, respectively; P < 0.05). G_qi5 protein overexpression relative to endogenous G_q levels (determined by Western blotting using a G_q antibody (IQB; Mullaney et al., 1993) was found to be similar in CHO cells coexpressing wild-type M₂ mACh receptors (5.1 ± 0.2-fold) versus ^N410YM₂ mACh receptor-expressing cells (4.9 ± 0.3-fold) (P > 0.05; n = 3).

View larger version (17K): [in this window] [in a new window]   Fig. 1. A, MCh-stimulated [3H]inositol phosphate accumulation in untransfected CHO-K1 cells (dashed line) or CHO-K1 cells transiently coexpressing Gqi5 and either wild-type (closed symbols) or N410Y mutant (open symbols) M2 receptor. Data are expressed as mean percentage of [3H]inositol phosphate accumulation over basal ± S.E.M., n = 3. B, basal [3H]inositol phosphate accumulation in the absence (WT-, MUT-) and presence (WT+, MUT+) of atropine (1 µM) in CHO-K1 cells transiently coexpressing Gqi5 and either wild-type (WT) or N410Y mutant (MUT) M2 mACh receptor. Results are expressed as means ± S.E.M., n 3. Statistically significant differences between WT and MUT are indicated as , P < 0.05, and between-basal and plus-atropine values as *, P < 0.05.

Constitutive Activity and Inverse Agonism at Wild-Type and Mutant ^N410YM₂ mACh Receptors Coexpressed with G_qi5

In cells coexpressing either wild-type or ^N410YM₂ mACh receptor with G_qi5, addition of atropine 15 min before and throughout a 15-min incubation with Li⁺ significantly reduced constitutive [³H]IP_x accumulation [Fig. 1B; atropine-induced decrease in basal [³H]IP_x accumulation: wild type (WT)-M₂, 2247 ± 548; ^N410YM₂, 9171 ± 1128 dpm/mg of protein], whereas in untransfected CHO cells, atropine had no effect upon [³H]IP_x accumulation (data not shown). In these experiments, therefore, atropine behaves as an inverse agonist, reducing constitutive mACh receptor activity. A number of other mACh receptor antagonists were assayed for inverse agonist activity. A maximal concentration of each ligand was selected (a concentration approximately 100-fold greater than the binding affinity for the ligand at the M₂ mACh receptor) and the effect on agonist-independent [³H]IP_x accumulation assessed. In CHO cells expressing the ^N410YM₂ mACh receptor, all antagonists tested significantly reduced the basal [³H]IP_x accumulation (Fig. 2B), whereas all ligands tested, barring darifenacin, significantly inhibited basal M₂ mACh receptor-dependent activity (Fig. 2A). The concentration dependence of the atropine-mediated reduction in basal [³H]IP_x accumulation was also investigated in both wild-type and ^N410YM₂ mACh receptor-expressing cells (Fig. 2C). The mean pEC₅₀ values for atropine at wild-type and mutant M₂ receptors were 9.04 ± 0.17 and 8.42 ± 0.20. Therefore, atropine was approximately 4-fold more potent in reducing constitutive [³H]IP_x accumulation in cells expressing wild-type M₂ than ^N410YM₂ receptors (P < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Inhibition of basal [3H]inositol phosphate accumulation, in CHO-K1 cells transiently coexpressing Gqi5 and either wild-type (A) or N410Y mutant (B) M2 mACh receptor, by a range of mACh receptor antagonists: atropine (Atr), darifenacin (Dari), oxybutynin (Oxy), tolterodine (Tolt), pirenzepine (Pirenz), methoctramine (Methoc), and 4-diphenylacetoxy-N-methylpiperidine. C, concentration-dependent inhibition of basal [3H]IPx accumulation, in CHO-K1 cells transiently coexpressing Gqi5 and wild-type M2 mACh receptor, by atropine. Results are expressed as means ± S.E.M., n 3. Statistically significant differences from control basal values are indicated as *, P < 0.05.

Effect of Long-Term Inverse Agonist Treatment on the Expression of Transiently Transfected Wild-Type and ^N410YM₂ mACh Receptors. Initial observations indicated that long-term treatment (24 h) with inverse agonist (atropine) up-regulated receptor expression levels, particularly of the ^N410YM₂ mACh receptor. Because this was consistent with previous reports on CAM-GPCRs (see Milligan and Bond, 1997), the effect of long-term treatment of CHO cells transiently expressing either wild-type or mutant receptor was investigated for a range of putative mACh receptor inverse agonists (Fig. 3). In CHO cells expressing the wild-type M₂ mACh receptor atropine, darifenacin, pirenzepine, and methoctramine significantly enhanced receptor expression levels (P < 0.05), whereas in cells expressing the ^N410YM₂ mACh receptor, only atropine, darifenacin, and pirenzepine exerted this effect (P < 0.05) (Fig. 3, A and B). It is notable that the maximal effects of atropine and darifenacin were more pronounced in cells expressing ^N410YM₂ compared with wild-type receptor (approximately 60 versus <40%, respectively). The concentration dependence of the up-regulatory effect was determined for atropine at the ^N410YM₂ mACh receptor, yielding an EC₅₀ value of 19 nM (pEC₅₀ value, 7.72 ± 0.18; Fig. 3C); unfortunately, the smaller maximal effect of atropine at the wild-type M₂ mACh receptor precluded analysis of the concentration dependence of this response.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effect of long-term (24-h) treatment of CHO-K1 cells transiently coexpressing Gqi5 and either wild-type (A) or N410Y mutant (B) M2 mACh receptor with a range of mACh receptor ligands on M2 receptor expression. C, concentration-dependent up-regulation of M2 mACh receptor expression by atropine in CHO-K1 cells transiently coexpressing Gqi5 and N410Y mutant M2 mACh receptor. Results are expressed as means ± S.E.M., n 3. Statistically significant changes from control receptor expression levels are indicated as *, P < 0.05.

In contrast to the up-regulatory effects mediated by a subset of mACh receptor inverse agonists, in both wild-type and ^N410YM₂ mACh receptor-expressing cells, 24-h treatment with the muscarinic agonist carbachol (100 µM) produced significant decreases (by 57 ± 5 and 62 ± 1%, respectively) in the receptor expression, relative to vehicle-treated cells (data not shown).

Stable Expression of Wild-Type and ^N410YM₂ mACh Receptors in CHO Cells: Radioligand Binding Assays. A number of CHO cell clones stably expressing either wild-type or ^N410YM₂ mACh receptors under G418 selection were next created. Single wild-type (CHO-m2 WT) and ^N410YM₂ [CHO-m2 mutant (MUT)] mACh receptor-expressing clones (receptor expression levels, 627 ± 86 and 247 ± 51 fmol/mg of protein, respectively) were selected for further study. Data for these (and at least one other) wild-type and ^N410YM₂ mACh receptor-expressing clone indicated that there was a trend toward [³H]NMS binding affinity (K_d) estimates being lower in cell membranes prepared from wild type (0.36 ± 0.02 nM) compared with ^N410YM₂ (0.55 ± 0.11 nM) receptor-expressing cells, although this trend failed to achieve significance (n 3).

The binding affinities of a number of mACh receptor antagonists, demonstrated to act as inverse agonists at the ^N410YM₂ receptor (see Fig. 3B), were determined in competition binding assays in membrane preparations from the wild-type and mutant receptor-expressing CHO cells. The mean binding affinity constants (pK_i) and Hill slopes are shown in Table 1. Corrected pK_i values were not significantly different between CHO-m2 WT- and MUT-derived membranes for atropine, tolterodine, pirenzepine, and methoctramine. However, darifenacin (6.2-fold) and, to a lesser extent, oxybutynin (2.2-fold), each exhibited significantly lower affinity for the ^N410YM₂ mACh receptor compared with wild type (P < 0.05) (see Table 1). In membranes prepared from each cell line, competition binding curves for methoctramine were characterized by Hill slopes significantly greater than 1 (P < 0.05). However, for all other competition binding curves, Hill slopes did not differ significantly from unity.

View this table: [in this window] [in a new window]   TABLE 1 Competition [3H]NMS binding data for a range of mACh receptor antagonists in membrane homogenates prepared from CHO cells stably expressing either the wild-type or N410Y mutant M2 mACh receptor Data are expressed as mean (S.E.M.) values from n 3 experiments.

Apparent binding affinity constant (pK_i) estimates for four mACh receptor agonists were also determined in [³H]NMS competition binding assays at 4°C (see Table 2). MCh, oxotremorine-M (Oxo-M), and oxotremorine (Oxo) all bound with significantly higher affinity to CHO cells expressing the ^N410YM₂ mACh receptor compared with wild-type receptor, whereas pilocarpine (Pilo) displayed similar affinities in each cell line (Table 2). In all cases, Hill slopes derived from agonist competition binding curves did not differ significantly from unity (data not shown).

View this table: [in this window] [in a new window]   TABLE 2 Intact cell apparent binding affinity constant (pKi) values and inhibition of (-PTx) and enhancement of (+PTx) forskolin-stimulated cyclic AMP accumulation in CHO cells stably expressing either wild-type or N410Y mutant M2 mACh receptors Data are expressed as mean (S.E.M.) values from n 3 experiments.

Functional Characterization of Wild-Type and ^N410YM₂ mACh Receptor Populations Stably Expressed in CHO Cells: Cyclic AMP Accumulation. Basal cyclic AMP accumulation was similar in CHO-m2 WT and MUT (1.4 ± 0.2 versus 1.3 ± 0.2 pmol/mg of protein, respectively; n = 6) cell lines. Cyclic AMP accumulation in response to forskolin (10 µM) was significantly lower in MUT than in WT CHO-m2 (220 ± 16 versus 855 ± 126 pmol/mg of protein, respectively; P < 0.05; n = 5). However, pretreatment with, or simultaneous addition of, a range of putative mACh receptor inverse agonists failed to enhance significantly cyclic AMP accumulation in response to either 3 or 10 µM forskolin in either CHO cell line (data not shown).

The stable CHO cell lines provide a model system in which to compare agonist pharmacology of the wild-type and ^N410YM₂ mACh receptors. The abilities of a range of mACh receptor agonists to inhibit forskolin-stimulated cyclic AMP accumulation through the G_i/o-coupled M₂ mACh receptors were assessed. In addition, after PTx pretreatment, mACh receptor agonists concentration-dependently increase forskolin-stimulated cyclic AMP accumulation via a G_s-dependent mechanism (Michal et al., 2001; Mistry et al., 2005). Therefore, it is possible to investigate whether differences exist in the ability of agonists to cause wild-type or ^N410YM₂ mACh receptors to couple via either G_i/o or G_s proteins (Fig. 4). Mean maximal inhibitory [E_max^(Gi/o)] and stimulatory [E_max^(Gs)] responses (expressed as a percentage of the response to the reference agonist MCh), as well as potencies for both inhibitory [pEC₅₀^(Gi/o)] and stimulatory [pEC₅₀^(Gs)] responses for each agonist, are summarized in Table 2.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Concentration-response curves for agonist-mediated inhibition or stimulation of forskolin-stimulated cyclic AMP accumulation in CHO-m2 WT (closed symbols) and CHO-m2 MUT (open symbols) cells after pretreatment without (inhibition) or with (stimulation) PTx (100 ng/ml) for 20 to 24 h. A, MCh; B, Oxo-M; C, Oxo; D, Pilo. Results are expressed as percent maximal inhibitions or stimulations of cyclic AMP accumulation relative to the maximal responses to the reference agonist MCh. Data are expressed as means ± S.E.M.., n 3.

Forskolin-stimulated cyclic AMP accumulation was significantly attenuated in both WT and MUT (129 ± 16 versus 56 ± 8 pmol/mg of protein; n = 5-8) CHO-m2 cell lines after PTx pretreatment (P < 0.05). In PTx-treated cells, EC₅₀ values for enhancements of the forskolin-stimulated response by agonists were much closer to apparent affinity (K_i) estimates. MCh (11.2-fold) and Oxo-M (6.8-fold) (but not Oxo, 2.0-fold) were significantly more potent with respect to EC₅₀^(Gs) values in MUT versus WT CHO-m2 cells, and both were full agonists in these cell lines with respect to this response (Table 2). In contrast, Oxo and Pilo behaved as partial agonists causing maximal responses that were significantly lower than those to MCh in both cell lines (Fig. 4, C and D). Comparison of stimulatory potencies on the cyclic AMP response with apparent binding affinities for the agonists [expressed as EC₅₀^(Gs)/K_i ratios] revealed values greater than unity for MCh (1.32 and 2.14 in WT and MUT CHO-m2 cells) and Oxo-M (1.10 and 1.38 in WT and MUT CHO-m2 cells), whereas those for Oxo were less than unity (0.31 and 0.45 in WT and MUT CHO-m2 cells) and could not be determined for Pilo.

Table 2 also summarizes the EC₅₀^(Gs)/EC₅₀^(Gi/o) ratios for MCh, Oxo-M, and Oxo, highlighting the generally lower potency observed for the stimulatory responses. In WT CHO-m2 cells, EC₅₀^(Gs)/EC₅₀^(Gi/o) ratios were comparable for the three agonists. In contrast, Oxo displayed a substantially larger difference in potency between stimulatory and inhibitory responses in MUT compared with WT CHO-m2 cells, whereas Oxo-M exhibited a smaller EC₅₀^(Gs)/EC₅₀^(Gi/o) ratio in MUT compared with WT CHO-m2 cells (Table 2).

Effect of PTx Pretreatment on Expression Levels of Wild-Type M₂ and ^N410YM₂ mACh Receptors in CHO Cells. In light of the observed differences in potency of, and maximal response to, mACh receptor agonists between cells in the absence and presence of PTx pretreatment, the binding affinities of MCh and Oxo in CHO-m2 MUT cells pretreated with PTx were also determined (data not shown). Saturation radioligand binding analyses were also performed in both WT and MUT CHO-m2 cells pretreated with PTx. PTx pretreatment had no effect on either [³H]NMS binding affinity (see Table 3) or pK_i estimates for MCh or Oxo in CHO-m2 MUT cells (data not shown). However, it is clear from the representative saturation binding curves shown in Fig. 5A (CHO-m2 WT) and Fig. 5B (CHO-m2 MUT) that PTx pretreatment significantly reduced the observed B_max in CHO-m2 MUT cells but had no effect upon the maximal binding in CHO-m2 WT cells. B_max estimates are summarized in Fig. 5C and Table 3.

View this table: [in this window] [in a new window]   TABLE 3 [3H]NMS binding affinity constant (Kd) and receptor expression (Bmax) estimates at intact CHO cells expressing either WT or N410Y mutant (MUT) M2 mACh receptors, with or without PTx (100 ng/ml; 20-24 h) and atropine (300 nM; 24 h) pretreatment Data are expressed as mean (S.E.M.) values from n 3 experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Effect of PTx pretreatment (100 ng/ml; 20-24 h) on mACh receptor expression levels in WT (A) and MUT (B) CHO-m2 cells, assessed by [3H]NMS saturation binding analysis in intact cells. Data points were performed in duplicate and curves shown are representative of three or more experiments. C, effect on mACh receptor expression of incubating WT and MUT CHO-m2 cells with atropine (300 nM) alone or in combination with PTx (100 ng/ml) for 24 h. Results are expressed as means ± S.E.M., n 3. Statistically significant differences between plus and minus atropine conditions are indicated as *, P < 0.05.

Cell surface receptor expression in wild-type and ^N410YM₂ mACh receptor-expressing CHO cells was also measured in cells incubated in the absence and presence of atropine (300 nM), with and without concurrent PTx treatment (see Fig. 5C; Table 3). Atropine per se did not significantly alter ^N410YM₂ mACh receptor expression levels in CHO cells, whereas PTx alone significantly reduced (by 33%) the cell surface expression of the mutant receptor (P < 0.05). Inclusion of atropine (300 nM) during the PTx treatment failed to attenuate the PTx-mediated reduction in the ^N410YM₂ mACh receptor expression level. No significant effect on wild-type receptor expression in CHO-m2 cells was observed for any treatment. In contrast to findings for transiently expressed wild-type and mutant M₂ mACh receptors (see above), stably expressed ^N410YM₂ mACh receptors exhibited a small but significantly greater affinity for [³H]NMS compared with the wild-type M₂ receptor (Table 3).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Some of the earliest examples of agonist-independent GPCR activity and its pharmacological reversal by receptor antagonists (subsequently reclassified as inverse agonists) were reported for the M₂ mACh receptor. Thus, the ability of atropine, and a subset of other mACh receptor antagonists, to suppress constitutive activity has been reported in membrane (Hilf and Jakobs, 1992) and intact (Jakubík et al., 1995) cell preparations endogenously or recombinantly expressing M₂ mACh receptors. Any constitutive activity exhibited by the wild-type receptor can often be enhanced by mutagenesis of key domains within the GPCR (Parnot et al., 2002; Seifert and Wenzel-Seifert, 2002). For the M₁-M₅ mACh receptor family, a single or double mutation at the TM6-e3 junction has been shown to promote constitutive activity (Spalding et al., 1995; 1997; Huang et al., 1999; Ford et al., 2002). Here, wild-type and N410Y mutant M₂ mACh receptors have been compared with respect to agonist and antagonist (inverse agonist) actions using both transient and stable receptor expression in CHO cells.

Cotransfection of wild-type or N410Y mutant M₂ mACh receptors with the chimeric G-protein G_qi5 allowed [³H]IP_x accumulation to be used as a readout of receptor activity. In this system, both the N410Y mutant and wild-type M₂ mACh receptors exhibited atropine-inhibited constitutive activity, with the ^N410YM₂ receptors exhibiting a 4-fold higher level of atropine-sensitive constitutive activity. The mutant receptor also displayed an enhanced agonist potency (>10-fold) relative to wild-type receptor, consistent with previous reports on the CAM ^N410Y,T411PM₂ mACh receptor (Ford et al., 2002). Although G-protein overexpression has been shown to increase agonist-independent signaling for G_q/11-coupled mACh receptors (Burstein et al., 1997), G_qi5 was expressed at similar levels in wild-type and N410Y mutant receptor-expressing cells in our experiments. Therefore, G-protein overexpression per se cannot account for the differences between wild-type and mutant responses seen.

The constitutive receptor activity present in the recombinant M₂ mACh receptor/G_qi5 coexpression system allowed us to identify inverse agonism and to investigate its short- and long-term consequences. Of the antagonists studied, we were particularly interested in comparing tolterodine, oxybutynin, and darifenacin, which are used in the management of overactive bladder (Moreland et al., 2004; Nelson et al., 2004). Recent estimates suggest that as many as 85% of GPCR antagonists actually exhibit negative efficacy when tested in constitutively active systems (Kenakin, 2004), and the data presented here are consistent with this notion, because all seven of the mACh receptor antagonists assayed possessed properties consistent with inverse agonism.

Although the maximal inhibition of constitutive [³H]IP_x accumulation did not generally differ between wild-type and mutant receptor-expressing cells, atropine did display a substantially higher potency at the wild-type (pEC₅₀ 9.04) compared with the N410Y mutant (pEC₅₀ 8.42) M₂ mACh receptor. According to the extended ternary complex model, inverse agonists may exert their effects via a selectively higher affinity for the inactive (R_i) than for the active (R_a) receptor species and/or by reducing the affinity of the ligand-bound receptor for its cognate G-protein (Samama et al., 1993; Strange, 2002). Thus, any perturbation of the system in favor of R_a and/or R_aG will result in a reduction in the apparent binding affinity of an inverse agonist for the receptor population (Costa and Herz, 1989; Samama et al., 1993; Huang et al., 1998; Wade et al., 2001).

Given the significant level of inverse agonism observed in the [³H]IP_x assays, it is surprising that only darifenacin and oxybutynin displayed significantly lower affinities at the CAM receptor, whereas all other inverse agonists exhibited equivalent affinity for wild-type and CAM receptors. Previous studies have also failed to observe differences in the binding affinity of inverse agonists between wild-type and CAM receptors (Kjelsberg et al., 1992; Ren et al., 1993; Ford et al., 2002). Theoretical analysis predicts that even when 50% of the total receptor population is present in the active state, a shift of only 2-fold might be observed in the binding affinity of an inverse agonist at a CAM receptor (Wade et al., 2001; Strange, 2002); therefore, it is possible that the differences in the magnitude of constitutive activity between wild-type and CAM receptors are insufficient for differences in antagonist affinities to be detected in radioligand binding assays. However, it remains unclear why darifenacin (and to a lesser extent oxybutynin) exhibits such a reduced affinity for the CAM receptor (6-fold).

The observation that long-term (24-h) treatment of ^N410YM₂ mACh receptor-expressing CHO-cells with certain inverse agonists caused a significant, concentration-dependent up-regulation of cell surface receptor number is consistent with numerous previous reports for CAM GPCRs (see Milligan and Bond, 1997). However, not all of the ligands that behaved as inverse agonists in the [³H]IP_x assays (with comparable negative efficacies) were capable of similarly facilitating an up-regulation of M₂ mACh receptor expression. If receptor up-regulation is related to the negative efficacy of the ligand, these data would suggest that the ligands investigated may possess a range of different negative efficacies toward this signaling pathway. As an alternative, properties distinct from negative efficacy might also contribute to the regulation of receptor expression levels. The ability of the ligand to cross the plasma membrane and stabilize newly synthesized receptors at the endoplasmic reticulum, facilitating their maturation and endoplasmic reticulum export, might be important, as has been suggested for CAM µ-opioid receptors (Li et al., 2001). As an alternative, the enrichment of a conformational state(s) that is uncoupled from G-proteins might not necessarily stabilize the receptor at the cell surface, particularly if internalization and G-protein activation are mediated by distinct receptor conformations, as suggested by the ability of antagonists or inverse agonists to elicit internalization (Barker et al., 1994; Roettger et al., 1997).

A further intriguing difference between wild-type and N410Y mutant M₂ mACh receptors emerged from studies initiated to assess G_i/o protein involvement in trafficking. PTx pretreatment of cells had no effect on the cell surface expression level of the wild-type receptor but caused a highly significant decrease in ^N410YM₂ mACh receptor expression. A possible cause of this down-regulation of the CAM receptor could be through destabilization caused by a decreased availability of G_i/o proteins. However, the failure of an inverse agonist to reverse this effect suggests that the down-regulation may not be related to the constitutive activity of the ^N410YM₂ mACh receptor. There is some evidence that G_i/o proteins play a role in the endocytosis/intracellular trafficking of proteins (Lang et al., 1995; Valenti et al., 1998), but there is little precedent for an effect of PTx treatment upon the expression of wild-type or CAM GPCRs. Indeed, Roseberry et al. (2001) reported that pretreatment of HEK293 cells stably expressing M₂ mACh receptors with PTx caused a modest increase in receptor expression.

Previous studies of mACh receptor mutants (e.g., Huang et al., 1999) have found that mutations within the TM6-e3 region can have substantial effects on agonist-mediated responses. Here, the creation of wild-type or N410Y mutant M₂ mACh receptor-expressing CHO cell lines has allowed agonist pharmacology also to be assessed. Consistent with the behavior of a CAM receptor, the partial agonists Pilo and Oxo displayed enhanced maximal responses at the ^N410YM₂ mACh receptor, whereas all four agonists tested exhibited greater potencies for inhibition of forskolin-stimulated cyclic AMP accumulation at the CAM receptor. However, when the signaling of the M₂ receptors through G_s (after PTx-mediated inactivation of G_i/o proteins) was investigated, only the full agonists MCh and Oxo-M displayed a greater potency at the CAM receptor.

In addition, there was some evidence for agonist-directed trafficking of signaling (ADTS) (Kenakin, 1995) at the N410Y mutant M₂ receptor, where Oxo was relatively weak at activating G_s compared with wild-type M₂ receptor. In contrast, Oxo-M displayed an unexpectedly high potency for the activation of G_s through the CAM M₂ receptor, as illustrated by its relatively small EC₅₀^(Gs)/EC₅₀^(Gi/o) ratio at the mutant receptor. The EC₅₀^(Gs)/EC₅₀^(Gi/o) ratio has been used previously to investigate ADTS at a variety of other GPCRs (e.g., Berg et al., 1998), and agonist-specific trafficking has also been previously demonstrated at the wild-type M₂ mACh receptor (Akam et al., 2001). However, in the present study, we found no evidence of ADTS for G_i/o versus G_s at the wild-type receptor, suggesting that the N410Y mutation alters the agonist-dependent signaling profile of the receptor. Malmberg and Strange (2000) similarly observed ligand-dependent alterations in receptor-G-protein coupling at the 5-HT_1A receptor after mutation of sites within the i3 loop. Work with the M₁ mACh receptor has indicated that mutation of the conserved threonine residue at the TM6-e3 junction influences G-protein coupling, whereas enhanced potency, affinity, and efficacy of agonists arise from mutation of the adjacent serine/asparagine residues (Huang et al., 1999). Our findings suggest that for the M₂ mACh receptor, the N410Y mutation alters G-protein coupling in an agonist-dependent manner, in addition to enhancing constitutive activity and agonist affinity, potency, and maximal response.

In summary, we report that the N410Y mutation, at the TM6-e3 junction, significantly enhances agonist-independent activity of the M₂ mACh receptor. Evidence has also been presented that long-term treatment with a subset of ligands, including those used in the clinical management of overactive bladder, can facilitate an increase in cell surface receptor expression. It has been proposed that inverse agonist-mediated receptor up-regulation might contribute to the development of tolerance upon long-term treatment (Smit et al., 1996). The potential for up-regulation of mACh receptors to occur in vivo, after long-term treatment with inverse agonists, therefore requires consideration in the clinical use of these ligands.

	Acknowledgements

 
We thank Stephen Rees (GlaxoSmithKline, Stevenage, UK) for generously providing the G_qi5 cDNA and Graeme Milligan (University of Glasgow, UK) for donating the anti-G_q antibody. We gratefully acknowledge the assistance of Mark R. Dowling in performing and validating the M₂ mACh receptor mutagenesis.

	Footnotes

 
Financial support for this work was provided in part by Novartis.

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

doi:10.1124/jpet.105.094383.

ABBREVIATIONS: GPCR, G-protein-coupled receptor; CAM, constitutively active mutant; TM, transmembrane; mACh, muscarinic acetylcholine; NMS, N-methyl-scopolamine methyl chloride; CHO, Chinese hamster ovary; KHB, Krebs-Henseleit buffer; IP_x, inositol phosphate; PTx, pertussis toxin; MCh, methacholine; WT, wild type; MUT, mutant; Oxo-M, oxotremorine-M; Oxo, oxotremorine; Pilo, pilocarpine; ADTS, agonist-directed trafficking of signaling.

Address correspondence to: R. A. J. Challiss, Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, UK. E-mail: jc36{at}le.ac.uk

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