Mutational Analysis and Molecular Modeling of the Allosteric Binding Site of a Novel, Selective, Noncompetitive Antagonist of the Metabotropic Glutamate 1 Receptor

Abstract

A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F8016.51A, Y8056.55A, and T8157.39M caused complete loss of the [3H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K+ channel current and [Ca2+]i response. The mutation W7986.48F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC50value (4 versus 128 nm) compared with wild type. The V7575.47L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC50 value (1160 versus 128 nm). Two mutations, N74745.51A and N75045.54A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca2+]i response. We observed a striking conservation in the position of critical residues. The residues Val-7575.47, Trp-7986.48, Phe-8016.51, Tyr-8056.55, and Thr-8157.39 are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-7986.48, Phe-8016.51, and Tyr-8056.55, thereby blocking the movement of the TM6 helix, which is crucial for receptor activation. A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F8016.51A, Y8056.55A, and T8157.39M caused complete loss of the [3H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K+ channel current and [Ca2+]i response. The mutation W7986.48F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC50value (4 versus 128 nm) compared with wild type. The V7575.47L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC50 value (1160 versus 128 nm). Two mutations, N74745.51A and N75045.54A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca2+]i response. We observed a striking conservation in the position of critical residues. The residues Val-7575.47, Trp-7986.48, Phe-8016.51, Tyr-8056.55, and Thr-8157.39 are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-7986.48, Phe-8016.51, and Tyr-8056.55, thereby blocking the movement of the TM6 helix, which is crucial for receptor activation. metabotropic glutamate human mGlu transmembrane seven-transmembrane seven-transmembrane domain 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile G-protein-coupled receptor G-protein-coupled inwardly rectifying K+ channel wild type γ-aminobutyric acid, type B extracellular loop 7-(hydroxyimino)cyclopropan[b]chromen-1a-carboxylic acid ethyl ester 2-methyl-6-(phenylethynyl)pyridine [(3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on] (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)-pyrrolidine diphenylacetyl-carbamic acid ethyl ester (9H-xanthene-9-carbonyl)-carbamic acid butyl ester 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethylpropyl)-phenol N-[2-[(2-chloro-6-fluorobenzyl)thio]ethyl]2-thiophenecarboxamide Chinese hamster ovary 5-hydroxytryptamine type 4 The mGlu1 receptor family currently comprises eight receptors that are divided into three classes on the basis of their sequence similarities, signal transduction, and agonist rank order of potency. Group I (mGlu1 and -5) receptors are coupled to the stimulation of phosphoinositide hydrolysis; group II (mGlu2 and -3) and group III receptors (mGlu4, -6, -7, and -8) are negatively coupled to cAMP production (1Conn P.J. Pin J.P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Google Scholar, 2Pin J.P. De Colle C. Bessis A.S. Acher F. Eur. J. Pharmacol. 1999; 375: 277-294Google Scholar, 3De Blasi A. Conn P.J. Pin J.P. Nicoletti F. Trends Pharmacol. Sci. 2001; 22: 114-120Google Scholar). Many studies have demonstrated the involvement of mGlu receptors in the modulation of synaptic transmission, ion channel activity, and synaptic plasticity (4Holscher C. Gigg J. O'Mara S.M. Neurosci. Biobehav. Rev. 1999; 23: 399-410Google Scholar, 5Nakanishi S. Neuron. 1994; 13: 1031-1037Google Scholar), and dysfunction of these receptors has been implicated in psychiatric and neurological diseases (6Bordi F. Ugolini A. Prog. Neurobiol. 1999; 59: 55-79Google Scholar). The mGlu receptors belong to the family 3 of G-protein-coupled receptors (GPCRs). Other members of this family include the GABAB, Ca2+-sensing, vomeronasal, pheromone, and putative taste receptors (7Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Google Scholar). The family 3 GPCRs shares a low sequence similarity with the other families. In contrast to family 1, the family 3 receptors are characterized by two distinctly separated topological domains: an exceptionally long extracellular amino-terminal domain (500–600 amino acids), which forms the agonist-binding pocket (8O'Hara P.J. Sheppard P.O. Thogersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Google Scholar, 9Galvez T. Prezeau L. Milioti G. Franek M. Joly C. Froestl W. Bettler B. Bertrand H.O. Blahos J. Pin J.P. J. Biol. Chem. 2000; 275: 41166-41174Google Scholar, 10Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Google Scholar), and the 7TM helical segments involved in receptor activation and G-protein coupling (11Parmentier M.L. Prezeau L. Bockaert J. Pin J.P. Trends Pharmacol. Sci. 2002; 23: 268-274Google Scholar). Compounds acting at group I mGlu receptors can be grouped into two categories. Category one comprises competitive agonists and antagonists. These compounds are phenylglycine derivatives or rigidified analogs of glutamate (12Schoepp D.D. Jane D.E. Monn J.A. Neuropharmacology. 1999; 38: 1431-1476Google Scholar), which logically bind to the glutamate-binding domain. Competitive group I ligands have achieved only limited subtype selectivity and potency, perhaps due to the high sequence homology of the mGlu receptor family agonist-binding site supported by the three-dimensional structure of mGlu1 amino-terminal domain (10Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Google Scholar). However, recent development of more sensitive technologies for functional screening of GPCRs has resulted in the discovery of a second category of compounds. These novel compounds, which interact within the 7TMD of group I mGlu receptor, act as positive or negative allosteric modulators (13Gasparini F. Kuhn R. Pin J.P. Curr. Opin. Pharmacol. 2002; 2: 43-49Google Scholar). CPCCOEt was the first non-amino acid derivative, subtype-selective antagonist of the mGlu1 receptor (IC50 = 6.5 μm at hmGlu1b) to be described (14Annoura H. Fukunaga A. Uesugi M. Tatsuoka T. Horikawa Y. Bioorg. Med. Chem. Lett. 1996; 6: 763-766Google Scholar). Litschig et al. (15Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461Google Scholar) elucidated the site of action of CPCCOEt, which binds within the 7TMD of mGlu1, in close contact with the residues Thr-815 and Ala-818 of TM7. Similarly, methyl-6-(phenylethynyl)pyridine (MPEP), which was the first noncompetitive, highly potent, mGlu5-selective antagonist (IC50 = 36 nm at hmGlu5a) to be described (16Gasparini F. Lingenhohl K. Stoehr N. Flor P.J. Heinrich M. Vranesic I. Biollaz M. Allgeier H. Heckendorn R. Urwyler S. Varney M.A. Johnson E.C. Hess S.D. Rao S.P. Sacaan A.I. Santori E.M. Velicelebi G. Kuhn R. Neuropharmacology. 1999; 38: 1493-1503Google Scholar), was suggested to make close contact with the amino acid residues Ala-810 in TM7 and Pro-655 and Ser-658 in TM3 of the mGlu5 receptor. Moreover, it has been demonstrated that the CPCCOEt and MPEP interact with residues that appear to form overlapping binding pockets in homologous regions of the 7TMD of the mGlu1 and -5 receptors, respectively (17Pagano A. Ruegg D. Litschig S. Stoehr N. Stierlin C. Heinrich M. Floersheim P. Prezeau L. Carroll F. Pin J.P. Cambria A. Vranesic I. Flor P.J. Gasparini F. Kuhn R. J. Biol. Chem. 2000; 275: 33750-33758Google Scholar). Recently, BAY36-7620, another highly potent mGlu1-selective antagonist (IC50 = 160 nm at rmGlu1a) that interacts within the 7TMD, has been reported (18Carroll F.Y. Stolle A. Beart P.M. Voerste A. Brabet I. Mauler F. Joly C. Antonicek H. Bockaert J. Muller T. Pin J.P. Prezeau L. Mol. Pharmacol. 2001; 59: 965-973Google Scholar). Knoflach et al. (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar) have described a novel class of ligands RO 67-7476, RO 01-6128, and RO 67-4853 acting as positive allosteric modulators of the mGlu1 receptor. Interestingly, their binding pocket appears to be also located within the 7TMD of mGlu1. Furthermore, the mutational analysis revealed (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar) that RO 67-7476 binding site in the TM3 region of mGlu1 appears to overlap with that of the MPEP binding site in the homologous region of the mGlu5 receptor. Urwyler et al. (20Urwyler S. Mosbacher J. Lingenhoehl K. Heid J. Hofstetter K. Froestl W. Bettler B. Kaupmann K. Mol. Pharmacol. 2001; 60: 963-971Google Scholar) concomitantly reported on the identification of CGP7930 and its aldehyde analog CGP13501, as positive modulators of GABAB receptor function. In the present study, we have probed the allosteric antagonist-binding site of mGlu1 using molecular modeling, site-directed mutagenesis, [3H]1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) binding, Ca2+ imaging, and G-protein-coupled inwardly rectifying K+ channel (GIRK) current activation. [3H]EM-TBPC is a highly potent, subtype-selective, noncompetitive antagonist of rmGlu1 receptor (21.Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Scholar, F. Mutel V. Kew J.N.C. L. Vieira E. Jolidon S. Wichmann J. Malherbe P. Kemp J.A. Neurosci. 2001; Scholar). acid residues in the -6, and and extracellular loop from an of the 7TMD of rmGlu1 with demonstrated by mutational analysis to be determinants of the noncompetitive antagonist binding pocket of the mGlu1 receptor. A homology model on the crystal of rhodopsin K. Kumasaka T. T. H. B.A. I. T. Yamamoto M. M. 2000; Scholar) these and a binding of EM-TBPC at (21.Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Scholar). [3H]EM-TBPC was by P. at the and (21.Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Scholar). activity, was at was from The position of amino acid in the 7TMD of mGlu receptor is by its sequence the signal and by the by and J.A. H. Neurosci. Scholar) which is as In this amino acid residues in the 7TMD are two the first to the and the second its position to a highly of family GPCRs in that which is The amino in the extracellular loop are to their the 4 and The highly to be is the and the residues within the are to the of the seven-transmembrane of rmGlu1 the transmembrane of rhodopsin with of our using a of R. Scholar). from The sequence of rhodopsin was from the rhodopsin structure on a with a using our Muller K. J. Mol. Scholar, J. Mol. Scholar) on the at model of mGlu1 was by the rmGlu1 sequence the extracellular domain on the rhodopsin template with the In a a model was and for amino from the rhodopsin also from the rhodopsin structure or in of amino by using the value the J. Mol. Biol. Scholar). of the structure revealed regions with of amino acid that by the of the model was by in and only the to In a only in a position other to In a of applied to The of the model was with EM-TBPC was into the 7TMD region as template for amino acid in the rmGlu1 model that with the antagonist amino acid within a of the antagonist in a of the and receptors in II from S. Nakanishi receptor was from a human in using from the sequence using the site-directed The regions of from using an as described P. Knoflach F. C. S. C. G. H. Kemp J.A. Mutel V. Mol. Pharmacol. 2001; 60: Scholar). the and three with and at The was in and with a for at at for at 4 the was in and as The was in a of a for at the was using the with as the The was at the at for at 4 the in the binding to a of of by the of [3H]EM-TBPC 1, and nm) to these for at binding in the of the with in and three with binding binding was in the of μm a noncompetitive, subtype-selective antagonist of mGlu1 with = which is a from the class as (21.Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Scholar). The on the was on a with the of of by using the from the of a and the of = B is the of at B is the of binding is the of and is the The three in and the S.D. of the and are reported in I. binding was as described V. G. S. A. J. V. Malherbe P. J. 2000; of mGlu1 and in the in a at on with the with a of using the with μm ester for at with in at in with as μm separated by antagonist on an with a long A was used to at and to a The in was and a to the as described G. M. J. Biol. Chem. Scholar). to the = A Chinese hamster ovary human was with a of using channel using the of the as described (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar). for current the 3 to with and was to with The was applied to the by from a The of was The at and the in which K+ be = = by of of the to the 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile is a highly potent, subtype-selective, noncompetitive antagonist that binds only to mGlu1 = = nm). has a low affinity for human mGlu1 and for the mGlu5 F. Mutel V. Kew J.N.C. L. Vieira E. Jolidon S. Wichmann J. Malherbe P. Kemp J.A. Neurosci. 2001; Scholar). a of and receptors (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar), the binding pocket of EM-TBPC has been to the 7TMD of mGlu1 the binding of EM-TBPC an of the seven-transmembrane of the family the transmembrane of rhodopsin was The agonist of was as a template for the of in mGlu1 located within from in the crystal structure of rhodopsin K. Kumasaka T. T. H. B.A. I. T. Yamamoto M. M. 2000; Scholar, T. K. 2001; Scholar) as to binding of In the of these amino of the rmGlu1 and the family with rhodopsin is this amino in the -6, and and regions for the mutational in and in in the 7TMD region by site-directed mutagenesis. binding on from the with the and receptors using nm of The from the are in I. mutations, and the [3H]EM-TBPC affinity compared with the receptor Three mutations, and [3H]EM-TBPC binding, and the mutation led to a decrease in affinity = nm). [3H]EM-TBPC bind to our Interestingly, only one amino acid the and human receptor in the it is a at position in the and a in the human receptor. the of the at position of the receptor by a to the receptor a binding affinity for [3H]EM-TBPC with that of = nm for the nm the of the with a or an led to a reduction in [3H]EM-TBPC affinity by 13-fold = nm and = in the of the to a or a increased the binding affinity of the by and = nm and = In order to of the on the glutamate binding pocket and to for receptor on from receptors using The and of binding from the are in I. the have on and the of receptor was with that the antagonist binding In with receptors, glutamate a increase in as by = receptors an increase in [Ca2+]i of μm the of functional receptors. However, of EM-TBPC at with μm glutamate in the resulted in a inhibition of glutamate-evoked [Ca2+]i with IC50 = 128 nm The inhibition of glutamate-evoked in [Ca2+]i by EM-TBPC in the receptors is in and their IC50 are in of on inhibition of [Ca2+]i by in the and for the inhibition by EM-TBPC of glutamate-evoked [Ca2+]i in the with the and receptors. are of with in a IC50 and for the inhibition by EM-TBPC of glutamate-evoked [Ca2+]i in the with the and receptors. are of with from the binding the antagonist inhibition of [Ca2+]i was by the mutation In the and which bind EM-TBPC was to glutamate-evoked [Ca2+]i and resulted in the large in IC50 nm for and In with the binding the functional with the was by EM-TBPC with an increase in the IC50 value (1160 versus 128 nm) as compared with The which increased binding a marked decrease in the IC50 value 128 nm) to two mutations, and which on the binding affinity of resulted in a decrease in the antagonist IC50 and versus 128 and with with receptors. we have (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar), the on the activation of by group I mGlu receptors can be to the of the compounds. we the wild type and critical receptors in a human and we the subtype selectivity of EM-TBPC for receptor. of glutamate an current in the or receptors. of EM-TBPC at with μm glutamate in the resulted in a inhibition of glutamate-evoked current with IC50 = nm = = the antagonist MPEP Similarly, MPEP glutamate-evoked current in the with a = IC50 = = EM-TBPC on current 4 The of μm EM-TBPC on the μm glutamate-evoked current in the critical receptors is in 4 C. EM-TBPC its to block the glutamate-evoked from with the of or or inhibition by μm which led to complete loss of binding affinity for The inhibition by EM-TBPC of glutamate-evoked current in the is in D. In with the binding and the inhibition is to the a marked decrease in IC50 value for this mutation = IC50 = and = Although the mGlu receptor and rhodopsin are in sequence we have used the 7TM of rhodopsin as a model for a receptor to into the of action of a negative allosteric modulator, the agonist of as template for the of EM-TBPC within the of the in close to and as critical residues in family mutation studies J.A. L. J.A. Mol. Pharmacol. 2001; 60: Scholar), as for mutation the mutation we a three-dimensional model of the 7TMD of the mGlu1 receptor using the of rhodopsin the amino in the to the binding affinity of and a binding for this allosteric the that are located in -6, and and of we observed that the F8016.51A, Y8056.55A, and T8157.39M resulted in complete loss of the [3H]EM-TBPC binding affinity and also blocked the inhibition by EM-TBPC of glutamate-evoked current or [Ca2+]i response. In our model, the group of Thr-8157.39 is at to EM-TBPC to form a In an study, Litschig et al. (15Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461Google Scholar), the CPCCOEt binding pocket of demonstrated that the two residues Thr-8157.39 and are molecular determinants for the binding of the antagonist We have also that the mutation a on [3H]EM-TBPC of to or EM-TBPC binding affinity or functional the of the mutation which led to a decrease of in [3H]EM-TBPC binding the other residues in TM3 and on [3H]EM-TBPC In the three-dimensional structure of is in close contact to the residues in which and is also that the loop has an functional in the family J.A. L. J.A. Mol. Pharmacol. 2001; 60: Scholar). Interestingly, we two in the N74745.51A and N75045.54A, which on the binding affinity increased the efficacy of the EM-TBPC block of glutamate-evoked (IC50 of and IC50 of 128 nm for it that these residues are in contact with the might form an to the glutamate binding in the extracellular domain into the region in the mGlu [3H]EM-TBPC low affinity for human mGlu1 and for The sequence of the 7TMD with the of mGlu1, which a at position mGlu receptors have at this is the only amino acid in the 7TMD region and human mGlu1. was as the critical for the binding selectivity of [3H]EM-TBPC at the versus human mGlu1 receptor. The mutation in the affinity of this receptor for [3H]EM-TBPC to a value with that of rmGlu1 and nm for rmGlu1 and Moreover, mutation of rmGlu1 to or led to a decrease in [3H]EM-TBPC binding affinity by the of for selectivity for mGlu1. we have (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar), is also a critical for the of the positive allosteric of mGlu1, RO is to the critical residues involved in the EM-TBPC-binding site of rmGlu1 with of pocket of rhodopsin or in reported of other family GPCRs We observed a striking conservation in the position of critical residues. We that the residues Val-7575.47, Trp-7986.48, Phe-8016.51, Tyr-8056.55, and Thr-8157.39 are crucial in the EM-TBPC-binding pocket of validating the of rhodopsin three-dimensional structure K. Kumasaka T. T. H. B.A. I. T. Yamamoto M. M. 2000; Scholar, T. K. 2001; Scholar, Annu. Rev. 2002; Scholar) as a template for the family 3 Furthermore, conservation in the position of these critical residues was also observed in the reported for or receptors J.A. L. J.A. Mol. Pharmacol. 2001; 60: Scholar, A. D. Muller J. Biol. Chem. 2002; Scholar, M.L. M. B. M. M. L. J. Mol. 2001; Scholar, L. S. M. Bessis A.S. Bockaert J. A. J. Biol. Chem. 2002; Scholar) the allosteric binding site of mGlu1 shares a as rhodopsin-like family of pocket of mGlu1 allosteric antagonist with of rhodopsin-like in a The of mGlu1 is of it is highly in and in family In the of and studies C. K. 1996; Scholar, S.P. Nature. 1996; Scholar) demonstrated the for a movement of the of TM6 from TM3 rhodopsin activation and that the to the to TM6 The B. M.L. H. Y. Nakanishi K. 2000; Scholar) also that in the form the of the ring of is to Similarly, in the three-dimensional structure of the of within of K. Kumasaka T. T. H. B.A. I. T. Yamamoto M. M. 2000; Scholar, T. K. 2001; Scholar). of these studies are with a for the acting as a for the of the receptor allosteric In our model, the crucial residues and are located in to in the TM6 we that the aromatic cluster formed from Trp-7986.48, Phe-8016.51, and interacts with the aromatic ring of blocking the movement in TM6 helix, which is for receptor activation. In with this model, we observed that of with a led to a large decrease of EM-TBPC IC50 value for the inhibition of the due to the more with et al. L. S. M. Bessis A.S. Bockaert J. A. J. Biol. Chem. 2002; Scholar) have reported that three residues of receptor, and are molecular determinants for activity, mutation of these residues to blocked the of Interestingly, two of these residues are homologous to the critical residues and that we in the EM-TBPC-binding pocket of Although the agonist of EM-TBPC is has been reported for and the noncompetitive mGlu1 (18Carroll F.Y. Stolle A. Beart P.M. Voerste A. Brabet I. Mauler F. Joly C. Antonicek H. Bockaert J. Muller T. Pin J.P. Prezeau L. Mol. Pharmacol. 2001; 59: 965-973Google Scholar) and mGlu5 (17Pagano A. Ruegg D. Litschig S. Stoehr N. Stierlin C. Heinrich M. Floersheim P. Prezeau L. Carroll F. Pin J.P. Cambria A. Vranesic I. Flor P.J. Gasparini F. Kuhn R. J. Biol. Chem. 2000; 275: 33750-33758Google Scholar) In we have demonstrated for the first that the crystal structure of can be successfully applied as a template for the family 3 The allosteric binding pocket on mGlu receptors an the contact of allosteric antagonist with the receptor the of with subtype selectivity and potency. We are to and for