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ACADIA Pharmaceuticals, Inc., San Diego, California
Received March 23, 2006; accepted August 31, 2006
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Abstract |
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; Bartolomeo et al., 2000; Weiss et al., 2000), making the M1 receptor an attractive therapeutic target for treating cognitive dysfunction in Alzheimer's disease and psychosis (Bymaster et al., 2002). Unfortunately, the M1 agonists that have been developed are not M1-selective and retain significant dose-limiting side effects such as sweating, vomiting, and nausea (Bodick et al., 1997; Bartolomeo et al., 2000; Thal et al., 2000). Work with knockout animals suggests that these side effects are likely to be caused by activation of the M2 and M3 muscarinic receptor subtypes (Bymaster et al., 2002; Wess, 2003).
Muscarinic M1 receptors bind their endogenous agonist, acetylcholine, through a binding site embedded in the transmembrane domains of the receptor and involving TM3, TM4, TM5, TM6, and TM7 (Hulme et al., 2003). This is termed the orthosteric site, because it binds the endogenous ligand for the receptor. TM3 is a crucial part of this orthosteric site and is believed to fulfill a central role in activation mechanism of muscarinic receptors and many other G-protein-coupled receptors (Gether, 2000). A series of residues in TM3 have been shown to participate in binding and activation by muscarinic agonists (Lu and Hulme, 1999). The primary feature of acetylcholine binding is a salt bridge believed to exist between the choline head group of acetylcholine and aspartate 105 (Asp105) in TM3. This residue reacted with the affinity label acetylcholine mustard, in which the onium head group of acetylcholine is replaced by a highly reactive aziridinium group (Spalding et al., 1994), demonstrating that this moiety was physically close to Asp105 when it bound the receptor. When Asp105 was replaced with the neutral amino acid alanine, thereby preventing the salt bridge from forming, the affinity of acetylcholine was reduced by 60-fold, and the compound no longer showed agonist activity (Lu and Hulme, 1999). Hulme et al. (2003) suggested that other residues in TM3 such as tryptophan 101 (Trp101), leucine 102 (Leu102), and tyrosine 106 (Tyr106), along with residues in TM6 and TM7 form a hydrophobic cage around Asp105 that closes around the acetylcholine molecule, thus triggering the isomerization of the receptor into an active conformation.
We recently identified a novel agonist, AC-42, that potently activates the M1 subtype but has no agonist activity on M2 to M5 subtypes. Using a series of chimeric receptors, we demonstrated that residues in the N terminus/TM1 and the third outer loop/TM7 domains are required for AC-42 to elicit agonist activity at the M1 receptor (Spalding et al., 2002). The residues in these regions are not conserved among the muscarinic subtypes, which probably accounts for the selectivity of AC-42. Recently, AC-42 was shown to act allosterically at M1 receptors based on the observations that it did not completely displace NMS from M1, that it retarded the dissociation of NMS from M1, and that atropine antagonism of AC-42-induced functional responses yielded Schild slopes less than unity (Langmead et al., 2006).
Moreover, it has been shown that the active metabolite of the atypical antipsychotic clozapine, N-desmethylclozapine, is a potent M1 receptor partial agonist (Sur et al., 2003; Weiner et al., 2004; Davies et al., 2005). We and others (Li et al., 2005) have proposed that the M1 agonist activity of N-desmethylclozapine may contribute to the procognitive benefits of clozapine therapy. N-desmethylclozapine has also been suggested to bind to M1 receptors at a site distinct from the acetylcholine-binding site (Sur et al., 2003).
We present data from a series of experiments examining whether the activation sites of AC-42, AC-260584 (a structurally related compound with substantially greater potency and efficacy than AC-42), N-desmethylclozapine, and clozapine overlap with the orthosteric binding site on TM3. We investigated the interactions of these ligands with a series of receptors mutated at residues in TM3 crucial for interaction with orthosteric agonists. We show that unlike carbachol, the binding affinity and agonist activity of each of these other ligands is generally maintained, and in some cases greatly increased, with two activation patterns apparent, one for AC-42 and AC-260584, and the other for N-desmethylclozapine and clozapine. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not of N-desmethylclozapine and clozapine. Together, these data suggest that M1 muscarinic receptors can be activated in at least three distinct ways: one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine.
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Materials and Methods |
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DNA Constructs. The rat M1 receptor and the W101A, L102A, D105A, Y106A, S109A, and N110A mutants were the kind gift of Dr. E. C. Hulme, MRC National Institute for Medical Research (London, UK). All constructs were sequence-verified.
RSAT. RSAT functional assays were carried out essentially as described in Spalding et al. (2002). NIH-3T3 cells were grown in 96-well tissue culture plates to 70 to 80% confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.3 mg/ml L-glutamine (Invitrogen, Carlsbad, CA), and 10% calf serum (Sigma-Aldrich). Cells were transfected for 18 h with DMEM containing 0.08 µg/ml receptor DNA and 0.3 µg/ml pSI-
-galactosidase (Promega, Madison WI) and 0.5% v/v Polyfect (QIAGEN, Valencia, CA). Medium was replaced with DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 0.3 mg/ml L-glutamine, 0.5% calf serum, 25% Ultraculture synthetic supplement (Cambrex Bio Science Walkersville, Inc., Walkersville, MD) instead of calf serum, and varying concentrations of ligand. Carbachol was tested at concentrations up to 100 µM, AC-42 was tested up to 5 µM, and AC-260584, N-desmethylclozapine, and clozapine were tested up to 10 µM. Higher concentrations of AC-42 have been shown to nonspecifically inhibit cell growth (data not shown). Cells were grown in a humidified atmosphere with 5% ambient CO2 for 5 days. Medium was removed from the plates, and -galactosidase activity was measured by the addition of o-nitrophenyl-D-galactopyranoside in phosphate-buffered saline with 5% Nonidet P-40. The resulting colorimetric reaction was measured in a spectrophotometric plate reader (Titertek, Huntsville, AL) at 420 nm. The data were fitted to the following equation using Prism software (Graph-Pad Software, Inc., San Diego, CA): Response = Basal Response + (Maximum Response - Basal Response) x [Ligand]/(EC50 + [Li-gand])
Phosphotidyl Inositol Hydrolysis Assays. Phosphatidyl inositol hydrolysis assays were performed essentially as follows: TsA cells (a human embryonic kidney 293 cell derivative) were seeded at 10,000 cells/well in DMEM (Invitrogen) supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) in a 37°C humidified atmosphere containing 5% CO2. Eighteen hours later, the cells were transfected as described above with the indicated plasmid DNAs (30 ng/well of a 96-well plate). Approximately 20 to 24 h after transfection, the cells were washed and labeled overnight with DMEM culture medium containing 0.2 µCi [myo-2-3H]inositol (NET1114, 37 MBq/ml; PerkinElmer Life and Analytical Sciences, Boston, MA) per well (0.1 ml). The cells were washed and incubated with Hanks' balanced salt solution (Invitrogen) supplemented with 1 mM CaCl2, 1 mM MgCl2, 10 mM LiCl, and 0.2% bovine serum albumin for 45 min. The buffer was removed, and the cells were incubated for another 45 min at 37°C in the same buffer with the concentrations of freshly made ligands indicated in the figure legends. The reaction was stopped by exchange with ice-cold 20 mM formic acid, and the total [3H]inositol phosphate (IP1,IP2, and IP3) formation was determined by ion-exchange chromatography on 1-ml minicolumns loaded with 200 µl of a 50% suspension of AG 1-X8 resin (200-400 mesh, formate form; Bio-Rad, Hercules, CA). The columns were washed with 1 ml of 40 mM ammonium hydroxide, pH 9, after loading the cell extracts and then eluted with 0.4 ml 2 M ammonium formate in 0.1 M formic acid. The eluates (0.1 ml) were loaded on LumaPlate-96 plates (Yttrium silicate scintillator coated; PerkinElmer Life and Analytical Sciences), air-dried overnight, and counted on a Microplate Scintillation and Luminescence Counter (TopCount NXT; PerkinElmer Life and Analytical Sciences).
Radioligand Binding Assays. Radioligand binding assays were carried out as described by Wess et al. (1991). To determine ligand potency, washed membranes were prepared from human embryonic kidney 293 cells transfected with 10 µg of plasmid DNA per 15-cm plate and stored at -80°C. Radioligand binding assays were carried out in 25 mM sodium phosphate, 5 mM magnesium chloride, and 0.01% bovine serum albumin (binding buffer). Incubations were for 2 h at room temperature, and reactions were stopped by rapid filtration onto GF/B filters. To determine the Kd of [3 H]NMS, membranes were incubated in 0.2 ml (Y106A), 1 ml (M1 wild-type and W101A), or 1.5 ml (S109A) buffer with eight [3H]NMS dilutions between 8 and 1000 pM (M1 wild-type), 18 and 2600 pM (W101A), 160 and 20,000 pM (Y106A), or 4 and 500 pM (S109A) in the presence or absence of 1 µM (M1 wild-type, W101A, S109A) or 30 µM (Y106A) atropine. The pKd values of [3H]NMS were the following (mean ± S.D., n = 2): M1 wild-type, 10.0 ± 0.3; W101A, 9.4 ± 0.2; Y106A, 8.2 ± 0.2; and S109A, 9.7 ± 0.1. To determine the IC50 value of AC-42, AC-260584, clozapine, N-desmethylclozapine, and carbachol, membranes were incubated with ligand in 0.2 ml buffer in the presence of [3H]NMS at up to three times its Kd value on that receptor. [3H]NMS concentrations were the following: M1 wild-type, 160 pM; W101A, 640 pM; Y106A, 1300 pM; and S109A, 160 pM. Expression levels for all receptors used were published by Lu and Hulme (1999). To examine whether or not compounds could simultaneously occupy M1 receptors, these equilibrium binding assays were repeated as described above using CHO-M1 cell membranes and [3H]NMS concentrations of 0.2 and 2 nM.
[3H]NMS Dissociation Rate Assays. For dissociation studies, 1 µg/well CHO-M1 cell membranes were preincubated with 200 pM [3H]NMS and binding buffer or atropine (1 µM) for at least 60 min. Binding buffer containing 1 µM atropine and the indicated ligands was then added. Total and nonspecific binding was determined as described above at the indicated time points. Data were fitted to the following equation using GraphPad Prism software: [3H]NMS bound = ([3H]NMS bound (-t = 0)e (-kt), where k is the dissociation rate constant.
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Results |
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) (Table 1 and Fig. 2). The wild-type rat M1 receptor gave a robust signal in this assay when exposed to carbachol with a maximum response typically 7-fold over basal (assigned a value of 100%, see Table 1). Compared with carbachol, AC-42 and N-desmethylclozapine were each partial agonists, and clozapine displayed weak but reproducible agonist activity at M1, as reported previously (Spalding et al., 2002; Sur et al., 2003; Weiner et al., 2004; Davies et al., 2005). AC-260584 displayed nearly full efficacy compared with carbachol and substantially increased potency and efficacy compared with AC-42 (Fig. 2, A and B; Table 1). Like AC-42, AC-260584 retains high selectivity for M1 over the other muscarinic receptor subtypes, with greater than 50-fold selectivity versus M2 and no significant agonist activity at M3 (S. R. Bradley, T. Son, N. Cook, J. Lameh, H. Schiffer, T. Spalding, J. Henderson, R. Davis, M. Brown, E. Burstein, et al., manuscript in preparation). As expected, all of the TM3 mutants tested were severely compromised in their responses to carbachol (Table 1 and Fig. 2). The potency of carbachol was reduced 33-fold for W101A, 51-fold for S109A, and more than 100-fold for L102A and N110A. Mutation of Y106 to alanine completely eliminated functional responses to carbachol at concentrations up to 100 µM. None of these mutated receptors showed significantly increased basal activity. The basal activities of the wild-type receptor and the W101A receptor were decreased slightly by atropine to 
7 and 6% of their maximal responses to carbachol, respectively, and no significant responses were seen of any other mutant to atropine (data not shown). In contrast to the results observed with carbachol, responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were maintained at many of the mutant receptors and were greatly increased at some.
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The most striking differences observed were at the W101A mutant (Fig. 2C), where 50-fold and 33-fold increases were seen in the potencies of AC-42 and AC-260584, respectively, whereas this same mutation caused a more than 20-fold decrease in the potency of carbachol (Fig. 2C and Table 1). The maximum response to AC-42 was also greatly increased at W101A to more than twice that observed at the wild-type receptor. Likewise, the maximum response to clozapine was increased almost 5-fold over that observed at the wild-type receptor to a level comparable with carbachol (Fig. 2D). In contrast to AC-42 and AC-260584, the potencies of clozapine and N-desmethylclozapine were not changed significantly.
Striking differences in the effects of mutations on carbachol and the other tested ligands were also seen on the Y106A and S109A mutants (Fig. 2, G-J; Table 1). On Y106A, the maximum responses of N-desmethylclozapine and clozapine were increased 1.4- and more than 7-fold, respectively, compared with their responses at the wild-type receptor, whereas no response to carbachol could be detected. Small but clear functional responses to AC-42 were observed, and robust functional responses to AC-260584 were observed at Y106A receptors. On S109A, the potencies of AC-42, AC-260584, and N-desmethylclozapine were hardly affected, whereas the potency of carbachol was reduced more than 50-fold. The maximal response to clozapine on S109A was not increased as it was at several of the other mutant receptors.
The Leu102 and Asn110 mutations caused significant impairment to responses induced by each of the tested ligands, but even here there were some apparent differences between carbachol and the other ligands (Fig. 2, E, F, K, and L; Table 1). For example, the maximum response to clozapine was increased over 2-fold on both L102A and N110A compared with wild-type receptor, whereas the maximal response to carbachol was reduced at these mutants compared with wildtype. In general, the potencies for all ligands were significantly reduced on L102A and N110A, though more for carbachol (>100-fold in each case) and less for the other ligands (typically 30-fold in most cases).
To confirm the differential effects of these mutations upon ligand activity, we tested several of the most interesting ligand-receptor combinations in conventional phosphatidyl inositol hydrolysis assays. The potencies of carbachol, AC-260584, and N-desmethylclozapine at wild-type M1 were very similar to those observed in RSAT (Table 2 and Fig. 3). AC-260584 displayed full activity relative to carbachol, N-desmethylclozapine was a partial agonist, and clozapine displayed minimal responses.
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On W101A, we again observed that the potency of AC-260584 and the maximal response to clozapine were each strongly increased, whereas the potency of carbachol was dramatically decreased (Table 2 and Fig. 3B). On Y106A, the maximal response to clozapine was equal to N-desmethylclozapine and greater than AC-260584, and carbachol was totally inactive (Fig. 3C). On S109A, the potency of carbachol was reduced more than 50-fold, whereas the potencies of AC-260584 and N-desmethylclozapine were unaffected (Fig. 3D). AC-42 displayed activities that were qualitatively similar to those of AC-260584 on wild-type M1 and these mutant receptors (data not shown). These results are highly consistent with the RSAT results.
To assess the effect of these mutations on receptor affinity, radioligand binding studies were carried out using the antagonist radioligand [3H]NMS (Table 3). The binding affinity of carbachol was significantly reduced on Y106A and S109A, although it was unchanged on W101A. Likewise, the binding affinities of the orthosteric antagonists NMS and atropine were strongly reduced at Y106A. In contrast, the affinities of AC-42, AC-260584, clozapine, and N-desmethylclozapine were only slightly affected on Y106A and S109A, and the affinities of AC-42 and AC-260584 were greatly increased at W101A.
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To directly examine whether N-desmethylclozapine, clozapine, AC-42, and AC-260584 act allosterically at M1 receptors, [3H]NMS-inhibition binding studies were performed using increased amounts of [3H]NMS (Fig. 4). N-Desmethylclozapine, clozapine, and AC-260584 were each able to completely displace [3H]NMS, with AC-260584 requiring significantly higher concentrations than either N-desmethylclozapine or clozapine to achieve this. In contrast, AC-42 was unable to completely displace [3H]NMS at concentrations of up to 300 µM. We cannot rule out the possibility that AC-42 would completely displace [3H]NMS at higher concentrations than 300 µM; however, the compound was not soluble above 300 µM. As expected, gallamine only partially displaced [3H]NMS, and this effect became much more pronounced as the concentration of [3H]NMS was increased.
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Discussion |
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; Hulme et al., 2003). Mutation of all of these residues profoundly reduces the ability of orthosteric ligands such as carbachol to bind and activate M1, confirming earlier findings (Fraser et al., 1989; Page et al., 1995; Lu and Hulme, 1999).
AC-42 and AC-260584 activity was affected by the mutations very differently. Most strikingly, mutation of Trp101 to alanine substantially increased the potency of AC-42 and AC-260584 (50- and 30-fold, respectively) and caused a greater than 20-fold decrease in carbachol potency. Likewise, the binding affinities of AC-42 and AC-260584 increased 50-fold, and more than 100-fold, respectively. Mutations to Tyr106 and Ser109 also had strikingly different effects on these ligands. M1-Y106A was not activated by carbachol, and its affinity for carbachol was reduced over 40-fold. In contrast, M1-Y106A retained the ability to be activated by AC-42 and AC-260584. Likewise, on M1-S109A, the potencies of AC-42 and AC-260584 were unchanged, and their maximal responses were increased, whereas the potency of carbachol for M1-S109A was decreased 50-fold in functional assays and 9-fold in radioligand binding, and its maximal responses decreased. Mutation of Leu102 and Asn110 impaired responses to AC-42, AC-260584, and carbachol, although the reduction in potency of AC-260584 (30-fold) was less than that of carbachol (>100-fold). Possibly AC-42 can activate M1-L102A and M1-N110A at doses that could not be tested because of dose-limiting cytotoxicity (unpublished observations).
A third pattern of activation was observed for clozapine and N-desmethylclozapine. In contrast to AC-42 and AC-260584, the potency of neither clozapine nor N-desmethylclozapine was increased at M1-W101A, although their maximum responses were, especially for clozapine. At M1-Y106A, the maximum responses to clozapine and N-desmethylclozapine were dramatically increased, whereas both the maximum response and potency of AC-42 and AC-260584 were reduced. Conversely, the maximum responses to N-desmethylclozapine and especially clozapine were reduced at M1-Y109A, whereas the maximum responses to AC-42 and AC-260584 were increased. Responses to N-desmethylclozapine and clozapine were impaired to similar degrees as AC-42 and AC-260584 at M1-L102A and M1-N110A, although responses to N-desmethylclozapine were slightly less affected than responses to AC-260584 at M1-N110A, and the maximum response to clozapine actually increased at M1-L102A and M1-N110A, in contrast to all the other ligands tested.
Mutations such as W101A that induce large potency increases are rare; frequently, they are caused by increases in constitutive activity (Burstein et al., 1995; Spalding et al., 1995, 1998; Lu and Hulme, 1999) as would be predicted by increasing J, the isomerization constant defining interconversion of receptors between active and inactive conformations (Samama et al., 1993). This is unlikely in this case, because the constitutive activity of the mutant and wild-type receptors were similar (see Results); thus, their J values are likely to be similar, and the receptors were expressed at similar levels (Lu and Hulme, 1999). We therefore suggest that the W101A mutation may directly strengthen interactions between AC-42 and AC-260584 and the M1 receptor. Possibly, steric constraints are removed upon replacement of a large tryptophan residue with a smaller alanine residue at this position, allowing AC-42 and AC-260584 to bind more tightly to the receptor. This strengthened interaction could involve other residues in TM3 or residues elsewhere in the receptor that are revealed by a change in receptor conformation.
According to modern models of receptor activation (Samama et al., 1993; Spalding et al., 1997; Christopoulos and Kenakin, 2002), agonists preferentially bind to active receptor conformations; inverse agonists preferentially bind to inactive receptor conformations; and neutral antagonists have equal affinity for active and inactive receptor conformations. Therefore, clozapine is likely to bind the inactive and active conformations of M1 with similar affinity because it is an extremely weak partial agonist at the wild-type M1 receptor. The large increase in maximal responses to clozapine caused by most mutations in TM3 suggests that the primary effect of these mutations is to increase the relative affinity of clozapine for an active conformation of M1. Another mechanism for increasing the maximal response of partial agonists is increased receptor reserve; however, given that the maximal response of the partial agonist AC-42 decreased at M1-Y106A and that both M1-W101A and M1-Y106A are expressed at similar levels to wild-type M1 (Lu and Hulme, 1999), this seems unlikely.
Current models of rhodopsin-like receptor activation propose movement of TM3 and TM6 as crucial to attaining an active conformation; in addition to acetylcholine, the positively charged head groups of dopamine, serotonin, histamine, epinephrine, and norepinephrine are all believed to interact directly with the aspartate analogous to Asp105 (AspIII.083.32) that is conserved in all biogenic amine receptors (Gether, 2000). We did not observe functional responses to any of the ligands tested here at the M1-D105A (Table 1). Possibly, interactions of AC-42, AC-260584, clozapine, or N-desmethylclozapine with Asp105 essential for receptor activation are lost when this residue is mutated to alanine. Alternatively, Asp105 may be essential for signaling by M1, and that mutation of this residue to alanine disrupts receptor activation regardless of where agonists bind the receptor, and/or that M1-D105A achieves insufficient cell surface expression to mediate functional responses (Lu and Hulme, 1999).
TM3 is believed to form an 
-helix based on mutagenesis and affinity labeling data (Javitch et al., 1995; Spalding et al., 1998; Lu and Hulme, 1999) and by inference from the 3D structure of rhodopsin (Palczewski et al., 2000; Hulme et al., 2003). To approximate their positions in the 3D structure of the M1 receptor, the residues tested in this study were mapped onto a helical net (Fig. 6). Trp101 is predicted to lie one turn above Asp105, Tyr106 is adjacent to Asp105, and Ser109 is predicted to lie one turn below Asp105. As described above, the side-chains of Tyr106 and Ser109 are essential for carbachol activity, suggesting that carbachol makes interactions well into the transmembrane domain of the receptor. In contrast, AC-42, AC-260584, and N-desmethylclozapine were substantially less affected by these mutations, and the activity of clozapine was dramatically increased on M1-Y106A, suggesting that these ligands bind closer to the extracellular space. This is consistent with data showing that carbachol, but not AC-42 or N-desmethylclozapine activity, is strongly impaired, and clozapine activity is greatly increased by mutations of tyrosine 381 and asparagine 382 in TM6 (Spalding et al., 2002; Sur et al., 2003), which are also believed to lie well into the transmembrane domain. The strong potentiating effect of W101A on AC-42 and AC-260584 activity could be explained as an allosteric effect propagated to an AC-42 binding site located elsewhere; however, it is more likely that AC-42 and AC-260584 interact with the extracellular regions of TM3. This is consistent with the observation that AC-42 agonist activity is dependent on M1 sequence in the extracellular parts of the receptor such as the N terminus and the third extracellular loop (Spalding et al., 2002).
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; this article), whereas we were unable to demonstrate that N-desmethylclozapine and clozapine bind allosterically [i.e., through a nonoverlapping (with respect to carbachol) site]. AC-42 and AC-260584 exhibit high functional selectivity for M1 over the other muscarinic subtypes (Spalding et al., 2002 and see above), possibly because these ligands act through nonconserved amino acid residues, consistent with an allosteric mechanism of action. In contrast, N-desmethylclozapine and clozapine are substantially less selective for M1 over the other muscarinic subtypes (Weiner et al., 2004; Davies et al., 2005), possibly reflecting interactions more similar to those used by orthosteric (and nonselective) ligands like carbachol. We conclude that although N-desmethylclozapine and clozapine contact different residues than carbachol to activate M1, they may occupy a substantially overlapping space with carbachol, whereas AC-42 and AC-260584 seem to occupy separable spaces.
These observations demonstrate that G-protein-coupled receptors do not have a single agonist binding site, where a ligand must bind to activate the receptor. Instead, receptors seem to spontaneously adopt active conformations, and ligands that stabilize one of these active conformations will act as agonists, irrespective of the site where they bind the receptor.
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Acknowledgements |
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Footnotes |
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Address correspondence to: Dr. Ethan S. Burstein, ACADIA Pharmaceuticals, Inc., 3911 Sorrento Valley Boulevard, San Diego, CA 92121. E-mail: eburstein{at}acadia-pharm.com
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), AC-260584 (
), N-desmethylclozapine (
), clozapine (
), and carbachol (
), clozapine (
