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

Pari MalherbeDagger §, Nicole Kratochwil, Frédéric KnoflachDagger , Marie-Thérèse ZennerDagger , James N. C. KewDagger ||, Claudia KratzeisenDagger , Hans P. Maerki, Geo Adam, and Vincent MutelDagger **

From the Pharma Division, Discovery Research Dagger  CNS and  Chemistry, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland

Received for publication, November 19, 2002, and in revised form, December 23, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 IC50 value (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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-3). Many studies have demonstrated the involvement of mGlu receptors in the modulation of synaptic transmission, ion channel activity, and synaptic plasticity (4, 5), and dysfunction of these receptors has been implicated in psychiatric and neurological diseases (6). 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 (7). 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 (8-10), and the 7TM helical segments involved in receptor activation and G-protein coupling (11).

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 (12), 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 (10). 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 (13). CPCCOEt was the first non-amino acid derivative, subtype-selective antagonist of the mGlu1 receptor (IC50 = 6.5 µM at hmGlu1b) to be described (14). Litschig et al. (15) 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 (16), 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 (17). Recently, BAY36-7620, another highly potent mGlu1-selective antagonist (IC50 = 160 nM at rmGlu1a) that interacts within the 7TMD, has been reported (18). Knoflach et al. (19) 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 (19) 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. (20) 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, 22). Amino acid residues in the TM3, -5, -6, and -7 and extracellular loop 2 (EC2) regions, initially identified from an alignment of the 7TMD of rmGlu1 with bovine rhodopsin, were demonstrated by mutational analysis to be important determinants of the noncompetitive antagonist binding pocket of the mGlu1 receptor. A homology model constructed based on the x-ray crystal of bovine rhodopsin (23) visualizes these findings and suggests a possible binding mode of EM-TBPC.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

EM-TBPC and N-[2-[(2-chloro-6-fluorobenzyl)thio]ethyl]2-thiophenecarboxamide (CFBTE-TPC) were synthesized at Hoffmann-La Roche Ltd. (21). [3H]EM-TBPC (specific activity 33.4 Ci/mmol) was synthesized by Dr. P. Huguenin at the Roche chemical and isotope laboratories (21). [3H]Quisqualate (specific activity, 32-35 Ci/mmol; TRK 1070) was synthesized at Amersham Biosciences. 2-MPEP was obtained from Sigma.

Residue Numbering Scheme

The position of each amino acid residue in the 7TMD of mGlu receptor is identified both by its sequence number (including the signal peptide) and by the generic numbering system proposed by Ballesteros and Weinstein (24) which is shown as superscript. In this numbering system, amino acid residues in the 7TMD are given two numbers; the first refers to the TM number, and the second indicates its position relative to a highly conserved residue of family 1 GPCRs in that TM which is arbitrarily assigned 50. The amino acids in the extracellular loop EC2 are labeled 45 to indicate their location between the helix 4 and 5. The highly conserved cysteine, thought to be disulfide-bonded, is given the index number 45.50 (SWISS-PROT: opsd_bovin C187), and the residues within the EC2 are then indexed relative to the "50" position.

Molecular Modeling

Alignment-- An alignment of the seven-transmembrane helices of rmGlu1 toward the transmembrane helices of bovine rhodopsin (Protein Data Bank code 1f88) were obtained with help of our in-house program Xsae,2 using a modified version of ClustalV (25). Sequences were obtained from SWISS-PROT: rmGlu1, P23385. The sequence of bovine rhodopsin was read directly from the rhodopsin structure (Protein Data Bank code 1f88).

Model Building-- All modeling calculations were made on a Silicon Graphics Octane with a single R12000 processor using our in-house modeling package Moloc (26, 27) (available on the World Wide Web at www.Moloc.ch). An initial C-alpha model of rat mGlu1 was built by fitting the aligned rmGlu1 sequence lacking the extracellular domain on the bovine rhodopsin template C-alpha structure. Loops were optimized with the Moloc C-alpha force field. In a next step, a full atom model was generated. phi  and psi  angles were obtained for aligned amino acids from the rhodopsin template. chi  angles were also adopted from the bovine rhodopsin structure where possible or in case of nonidentical amino acids generated by using the most probable value applying the Ponder-Richards method (28). An energy calculation of the initial full peptide structure revealed regions with bad van der Waals contacts of amino acid side chains that were subsequently improved by manually adjusting the relevant chi  angles. Repulsive van der Waals interactions were removed manually where necessary. Refinement of the model was done by keeping all backbone atoms in fixed positions and allowing only the side chains to move.

In a following optimization step, only C-alpha atoms were kept in a fixed position while all other atoms were allowed to move. In a third round of optimization, no atoms were kept stationary, but constraints were applied to C-alpha atoms. The quality of the model was then checked with Moloc internal programs. EM-TBPC was manually docked into the 7TMD region taking cis-retinal as template for location. Where necessary, nonconserved amino acid side chains in the rmGlu1 model were rotated such that no van der Waals conflicts with the antagonist occurred. All amino acid side chains reaching within a 6-Å distance of the antagonist were subsequently included in a round of optimization.

Plasmids, Cell Culture, and Membrane Preparation

cDNAs encoding the rmGlu1a and rmGlu5a receptors in pBlueScript II were obtained from Prof. S. Nakanishi (Kyoto, Japan). hmGlu1a receptor cDNA was amplified from a human fetal brain cDNA library in pCMV.SPORT2 (Invitrogen) using primers derived from the hmGlu1a sequence (AC:U31216). All point mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene). The entire coding regions of all point mutants were sequenced from both strands using an automated cycle sequencer (Applied Biosystems).

HEK-293 cells were transfected as previously described (29). 48 h posttransfection, the cells were harvested and washed three times with cold PBS and frozen at -80 °C. The pellet was suspended in cold 20 mM HEPES-NaOH buffer containing 10 mM EDTA (pH 7.4) and homogenized with a Polytron homogenizer (Kinematica, AG) for 10 s at 10,000 rpm. After centrifugation at 48,000 × g for 30 min at 4 °C, the pellet was resuspended in cold 20 mM HEPES-NaOH buffer containing 0.1 mM EDTA (pH 7.4), homogenized, and respun as above. The pellet was resuspended in a smaller volume of a cold 20 mM HEPES-NaOH buffer containing 0.1 mM EDTA (pH 7.4). After homogenization for 10 s at 10,000 rpm, the protein content was measured using the BCA method (Pierce) with bovine serum albumin as the standard. The membrane homogenate was frozen at -80 °C before use.

[3H]EM-TBPC and [3H]Quisqualate Bindings

After thawing, the membrane homogenates were centrifuged at 48,000 × g for 10 min at 4 °C, the pellets were resuspended in the 20 mM HEPES-NaOH (pH 7.4) binding buffer to a final assay concentration of 20 µg of protein/ml. Saturation isotherms were determined by the addition of various [3H]EM-TBPC concentrations (0.3, 1, 3, 10, 30, 100, and 300 nM) to these membranes for 1 h at room temperature (equilibrium binding conditions determined in kinetic experiments). At the end of the incubation, membranes were filtered onto Filtermate (unitfilter Packard: 96-well white microplate with bonded GF/B filter preincubated 1 h in 0.1% polyethyleneimine) and washed three times with cold binding buffer. Nonspecific binding was measured in the presence of 100 µM CFBTE-TPC, a noncompetitive, subtype-selective antagonist of mGlu1 with Ki = 390 nM, which is a compound from the same class as EM-TBPC, Fig. 1 (21). The radioactivity on the filter was counted on a Packard Top-count microplate scintillation counter with quenching correction after the addition of 40 µl of microscint 40 (Canberra Packard S.A.). Saturation experiments were analyzed by Prism 3.0 (GraphPad Software, San Diego, CA) using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action, B = (Bmax * [F])/(KD + [F]), where B is the amount of ligand bound at equilibrium, Bmax is the maximum number of binding sites, [F] is the concentration of free ligand, and KD is the ligand dissociation constant. The experiments were performed three times in triplicate, and the mean ± S.D. of the individual KD values were calculated and are reported in Table I. [3H]Quisqualate binding was performed as previously described (30).

Single Cell Calcium Imaging

HEK-293 cells were plated at 5 × 104 cells on glass coverslips (diameter 15 mm) coated with 100 µg/ml poly-D-lysine. After 24 h, the cells were co-transfected with a 2:1 (w/w) mixture of mGlu/enhanced green fluorescent protein plasmids using LipofectAMINE 2000 (Invitrogen). 48 h later, the transfected cells were incubated with 20 µM fura-2 acetoxymethyl ester plus 0.5% Pluronic F-127 (Molecular Probes, Inc., Eugene, OR) for 40 min at room temperature with 20-min postincubation in balanced salt solution. Cells were stimulated at room temperature in artificial cerebrospinal fluid with drug as indicated. Glutamate applications (drug + 30 µM glutamate, 30-s exposure) were separated by 10-min intervals (3-min wash, 7-min antagonist incubation). Imaging measurements were made on an inverted microscope with a long distance ×40 objective (Axiovert 405M; Zeiss). A cooled CCD camera (CH-250; Photometrics) was used to acquire image pairs at 340- and 380-nm excitation wavelengths (with dark correction) to a computer. Exposure times were 400 ms. The intrinsic fluorescence in cells not dye-loaded was less than 5% and did not contribute a significant error to the measurements. Fluorescence ratio values were calculated as previously described (31). Inhibition curves were fitted according to the Hill equation: y = 100/(1 + (x/IC50)nH), where nH represents slope factor.

Electrophysiology

A Chinese hamster ovary (CHO) cell line stably expressing human GIRK1-GIRK2c dimer was co-transfected with a 1:1 (w/w) mixture of mGlu/enhanced green fluorescent protein plasmids using LipofectAMINE 2000. GIRK channel currents were recorded 24-96 h after transfection using the whole-cell configuration of the patch clamp technique as described previously (19). Briefly, for GIRK current recordings, the pipette solution contained 130 mM KCl, 1 mM MgCl2, 10 mM HEPES, 5 mM K4BAPTA, 3 mM Na2ATP, 0.3 mM Na2GTP, adjusted to pH 7.2 with KOH, and osmolarity was adjusted to 310 mosM with sucrose. The drug was applied locally to the cell by fast perfusion from a double-barreled pipette assembly. The rate of solution exchange was around 20 ms. The CHO cells were held at -70 mV, and the recordings were made under conditions in which K+ currents would be inward ([K+]i = 150 mM, [K+]o = 30 mM). Concentration-response curves were obtained by applying 20-s pulses of varying concentrations of the compound every 90 s to the cells.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Modeling of 7TM Domain of the rmGlu1 Receptor-- [3H]EM-TBPC, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (Fig. 1) is a highly potent, subtype-selective, noncompetitive antagonist that binds only to rat mGlu1 (KD = 6.6 ± 0.5 nM, Ki = 11 ± 2 nM). It has a low affinity for human mGlu1 and none for the rat mGlu5 (22). Using a series of chimeric rmGlu1/5a and rmGlu5/1alpha receptors (19), the binding pocket of EM-TBPC has been localized to the 7TMD of mGlu1 (data not shown). To elucidate the binding mode of EM-TBPC further, an alignment of the seven-transmembrane helices of the whole rmGlu family toward the transmembrane helices of bovine rhodopsin (Protein Data Bank code 1f88) was made. The inverse agonist of rhodopsin, cis-retinal, was employed as a template for the location of EM-TBPC. Amino acids in mGlu1 located within 6.0 Å away from retinal in the x-ray crystal structure of rhodopsin (23, 32) were considered as likely candidates to affect binding of EM-TBPC. In Fig. 2, the alignment of these amino acids of the rmGlu1 and the rmGlu family with rhodopsin is shown. From this preselection, amino acids in the TM3, -4, -5, -6, and -7 and EC2 regions (Fig. 2, boldface type) were chosen for the mutational studies.


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Fig. 1.   Structures of [3H]EM-TBPC, CFBTE-TPC, (-)-CPCCOEt, BAY36-7620, RO 67-7476, and MPEP.


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Fig. 2.   Alignment of amino acid residues of 7TMD and the EC2 loop in the rmGlu sequences relative to bovine rhodopsin. The amino acids shown were found to be in close proximity to the inverse agonist cis-retinal in the x-ray crystal structure of the bovine rhodopsin (6 Å, Protein Data Bank code 1f88) and were considered as guidance for mutational studies. The Ballesteros Weinstein numbering scheme of the amino acids is given to facilitate the comparison with other GPCRs (see "Experimental Procedures"). The conserved residue in each TM of rhodopsin that is assigned as 50 is shown in the bottom row. The asterisks indicate identical residues among mGlu and rhodopsin. Colons indicate identical residues among mGlus. The residues that have been point-mutated in rmGlu1 are shown in boldface type.

Generation of Point Mutations and the [3H]EM-TBPC Binding-- 20 mutations (19 in rmGlu1alpha and 1 in hmGlu1alpha ) were accordingly introduced in the 7TMD region by site-directed mutagenesis. Saturation binding analyses were performed on membranes isolated from the HEK-293 transfected with the WT and mutated receptors using 0.3-300 nM of [3H]EM-TBPC. The dissociation constants (KD) derived from the saturation isotherms are given in Table I. Eleven mutations, A699V, Y672F, T723, N747A, S749T, S749A, N750A, N750Q, V802M, A818S, and A818I, did not significantly affect the [3H]EM-TBPC affinity compared with the WT rmGlu1alpha receptor (Table I). Three mutations, F801A, Y805A, and T815M, completely abolished [3H]EM-TBPC binding, and the mutation Y672V led to a 5-fold decrease in affinity (KD = 32 ± 5.4 nM). [3H]EM-TBPC does not bind to hmGlu1 under our experimental conditions (Table I). Interestingly, only one amino acid differs between the rat and human receptor in the TM domain; it is a valine at position 757 in the rat and a leucine in the human receptor. As expected, the replacement of the leucine residue at position 757 of the hmGlu1 receptor by a valine (L757V) gave to the mutant receptor a binding affinity for [3H]EM-TBPC comparable with that of WT rmGlu1alpha (KD = 9.9 ± 2.9 nM for the hmGlu1alpha L757V versus 6.6 ± 0.5 nM rmGlu1alpha ). Conversely, the replacement of the valine 757 with a leucine (V757L) or an alanine (V757A) led to a dramatic, although not complete, reduction in [3H]EM-TBPC affinity by 13-fold (KD = 84 ± 39 nM and KD = 85 ± 19 nM), respectively, in both mutants.

                              
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Table I
Binding properties of WT mGlu1 and mutants
Saturation binding isotherms of [3H]EM-TBPC and [3H]quisqualate were performed on membrane preparations from HEK-293 cells transiently transfected with the WT and point-mutated receptors as described under "Experimental Procedures." Values are mean ± S.D. of the KD values, calculated from two and three independent experiments (each performed in triplicate) for [3H]quisqualate and [3H]EM-TBPC, respectively. The mutations that affected the [3H]EM-TBPC binding affinity are shown in boldface type.

Finally, the conversion of the tryptophan 798 to a phenylalanine (W798F) or a tyrosine (W798Y) significantly increased the binding affinity of the radioligand by 10- and 5-fold (KD = 0.7 ± 0.2 nM and KD = 1.3 ± 0.2 nM), respectively.

In order to examine effects of the mutations on the glutamate binding pocket and to check for receptor expression, saturation analyses were performed on membranes from selected mutated receptors using [3H]quisqualate. The KD values and maximum number of binding sites (Bmax) derived from the saturation isotherms are given in Table I. As seen, the mutations have no significant effect on quisqualate affinity, and the level of receptor expression was comparable even with mutants that completely lost the antagonist binding (Table I).

Effect of Mutations on Inhibition of Glutamate-induced [Ca2+]i Response by EM-TBPC as Measured by Single-cell Ca2+ Imaging-- In HEK-293 cells transiently transfected with rmGlu1alpha receptors, glutamate elicited a concentration-dependent increase in intracellular free calcium [Ca2+]i, as assayed by single-cell fura-2 imaging (EC50 = 6.8 µM). All mutated receptors elicit an increase in [Ca2+]i response upon application of 30 µM glutamate, indicating the presence of functional receptors. However, co-application of EM-TBPC at various concentrations with 30 µM glutamate in the cells expressing WT rmGlu1alpha resulted in a concentration-dependent inhibition of glutamate-evoked [Ca2+]i response with IC50 = 128 nM (Fig. 3). The concentration-dependent inhibition of glutamate-evoked increases in [Ca2+]i by EM-TBPC in the cells expressing various mutated receptors is shown in Fig. 3, A and B, and their derived IC50 and nH values are shown in Table II.


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Fig. 3.   The effect of mutations on inhibition of glutamate-stimulated increases in [Ca2+]i by EM-TBPC. Concentration-dependent inhibition of 30 µM glutamate-stimulated increases in [Ca2+]i by EM-TBPC as assayed by single cell fura-2 imaging of HEK-293 transiently transfected with the rmGlu1alpha WT and mutated receptors. In A, the mutations that shifted the dose-response curve to the left, and in B, the mutations that shifted the dose-response curve to the right or have no effect are shown. Data are mean ± S.E. of four separate experiments with 15-20 cells/experiment. Responses are normalized to the first control response.

                              
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Table II
Effect of mutations on inhibition of glutamate-induced [Ca2+]i response by EM-TBPC
IC50 and Hill coefficient (nH) values for the inhibition by EM-TBPC of glutamate-evoked [Ca2+]i response in the HEK-293 cells transiently transfected with the mGlu1alpha WT and mutated receptors. Data are means ± S.E. of four separate experiments with 15-20 cells/experiment.

As expected from the binding studies, the antagonist inhibition of glutamate-induced [Ca2+]i response was not affected by the mutation A818S. In cells expressing the mutants Y805A and T815M, which did not bind [3H]EM-TBPC, EM-TBPC was not able to efficiently inhibit glutamate-evoked [Ca2+]i response and thus resulted in the large increases in IC50 values (>10,000 nM for Y805A and T815M). In agreement with the binding experiments, the glutamate-induced functional response with the mutant V757L was inhibited by EM-TBPC with an increase in the IC50 value (1160 versus 128 nM) as compared with WT.

The mutant W798F, which showed increased radioligand binding affinity, exhibited a marked decrease in the IC50 value (4 versus 128 nM) relative to WT. Surprisingly, two mutations, N747A and N750A, which had no effect on the binding affinity of [3H]EM-TBPC, resulted in a decrease in the antagonist IC50 values (30 and 10 versus 128 nM WT, respectively) and with Hill coefficient values somewhat lower than found with WT receptors.

Effect of Point Mutation on the Inhibition of the GIRK Channel Current by EM-TBPC-- As we have shown previously (19), the electrophysiological assay based on the activation of GIRKs by group I mGlu receptors can be employed to assess the pharmacology of the compounds. Therefore, we transiently expressed the wild type and selected critical mutated rmGlu1alpha receptors in a CHO line stably expressing concatenated human Kir3.1 and Kir3.2c GIRK subunits. First, we examined the subtype selectivity of EM-TBPC for rmGlu1alpha receptor. Rapid application of glutamate (10 µM) induced an inward current in GIRK-CHO cells expressing the rmGlu1alpha or rmGlu5a receptors. Co-application of EM-TBPC at various concentrations with 10 µM glutamate in the GIRK-CHO cell expressing rmGlu1alpha resulted in a dose-dependent inhibition of glutamate-evoked GIRK current with IC50 = 15.3 nM (pIC50 = 7.81 ± 0.08, nH = 1.69 ± 0.17), whereas the mGlu5a-selective antagonist MPEP had no effect (Fig. 4A). Similarly, MPEP inhibited glutamate-evoked GIRK current in the GIRK-CHO cells expressing rmGlu5a with a dose-dependent manner (pIC50 = 8.1 ± 0.1, IC50 = 8 nM, and nH = 0.92 ± 0.13), whereas EM-TBPC had no effect on GIRK current (Fig. 4B). The effect of 1 µM EM-TBPC on the 10 µM glutamate-evoked GIRK current in the critical point-mutated mGlu1alpha receptors is shown in Fig. 4C. EM-TBPC lost its ability to block the glutamate-evoked GIRK currents from rmGlu1alpha with the point-mutations of F801A, Y805A, or T815M (<45%, <35%, or <10% inhibition by 1 µM EM-TBPC, respectively), which all led to complete loss of binding affinity for [3H]EM-TBPC. The concentration-response inhibition curve by EM-TBPC of glutamate-evoked current in GIRK-CHO cell expressing the mutant W798F is shown in Fig. 4D. In good agreement with the binding and Ca2+ imaging studies, the inhibition curve is shifted to the left, indicating a marked decrease in IC50 value for this mutation (pIC50 = 8.69 ± 0.08, IC50 = 2 nM, and nH = 1.87 ± 0.41).


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Fig. 4.   Effect of allosteric antagonists on glutamate-evoked GIRK current in WT and mutated receptors. Concentration-response curves for the effect of the mGlu1 receptor antagonist EM-TBPC and the mGlu5 receptor antagonist MPEP on 10 µM glutamate-evoked GIRK current in GIRK-CHO cells transiently transfected with either rmGlu1alpha (A) or rmGlu5a (B) receptor cDNA. C, block of the 10 µM glutamate-evoked GIRK current by 1 µM EM-TBPC in GIRK-CHO expressing WT and mutated rmGlu1alpha receptors. D, concentration-dependent inhibition of 10 µM glutamate-induced GIRK current by EM-TBPC in GIRK-CHO cells transiently transfected with the WT and W798F mutated rmGlu1alpha receptors. The values are normalized to the control responses obtained with glutamate alone (10 µM, 100%). Each point represents the mean ± S.E. of five cells. The sigmoidal curves were generated with the mean IC50 and Hill coefficient.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although the mGlu receptor and rhodopsin are distant in sequence identity, we have used the 7TM domains of rhodopsin as a general model for a GPCR receptor to gain further insight into the mechanism of action of a negative allosteric modulator, EM-TBPC. cis-Retinal, the inverse agonist of rhodopsin, served as template for the location of EM-TBPC within the TM domains. Amino acids of the TM regions, in close proximity to cis-retinal and very often identified as critical residues in family 1 GPCR mutation studies (33), were selected as candidates for mutation studies. To visualize the mutation data, we constructed a three-dimensional model of the 7TMD of the mGlu1 receptor using the atomic coordinates of bovine rhodopsin (Protein Data Bank code 1f88). Fig. 5 shows the amino acids in the TM region, found to affect the binding affinity of EM-TBPC, and suggests a possible binding mode for this allosteric modulator.


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Fig. 5.   Molecular modeling of the rmGlu1 complexed with EM-TBPC. Shown is a homology model of rmGlu1 based on the high resolution structure of bovine rhodopsin. A three-dimensional side view of the 7TM binding pocket of the rmGlu1 receptor is shown with a suggested binding mode of EM-TBPC. C-alpha traces are represented as ribbons: TM1 (cyan), TM2 (yellow), TM3 (red), TM4 (blue), TM5 (orange), TM6 (gray), and TM7 (magenta). The important residues that were mutated in the current work are labeled in one-letter code. Color coding of Tyr-672, Asn-747, Asn-750, Val-757, Trp-798, Phe-801, Tyr-805, and Thr-815 (blue) and EM-TBPC (magenta) atoms is as follows: red, oxygen; blue, nitrogen; white, carbon.

Among the 19 point mutations that are located in TM3, -4, -5, -6, and -7 and EC2 of rmGlu1alpha , we observed that the F8016.51A, Y8056.55A, and T8157.39M mutations resulted in complete loss of the [3H]EM-TBPC binding affinity and also blocked the inhibition by EM-TBPC of glutamate-evoked GIRK current or [Ca2+]i response. In our model, the aliphatic hydroxyl group of Thr-8157.39 is positioned at sufficient proximity to EM-TBPC to form a hydrogen bond. In an earlier study, Litschig et al. (15), probing the CPCCOEt binding pocket of hmGlu1alpha , demonstrated that the two nonconserved residues Thr-8157.39 and Ala-8187.42 are important molecular determinants for the binding of the antagonist CPCCOEt. We have also found that the mutation Thr8157.39M had a detrimental effect on [3H]EM-TBPC affinity; however, conversion of Ala-8187.42 to Ser or Ile did not affect EM-TBPC binding affinity or functional IC50.

With the exception of the mutation Y6723.40V, which led to a decrease of 5-fold in [3H]EM-TBPC binding affinity, the other mutated residues in TM3 and TM4 had no significant effect on [3H]EM-TBPC affinity. In the three-dimensional structure of rhodopsin, cis-retinal is in close contact to the residues in EC2, which connects TM4 and TM5. There is also evidence that the EC2 loop has an important functional role in the entire GPCR family 1 (33). Interestingly, we identified two mutations in the EC2 loop, N74745.51A and N75045.54A, which had no significant effect on the binding affinity but increased the efficacy of the EM-TBPC block of glutamate-evoked [Ca2+]i response (IC50 of 30 and 10 nM, respectively, versus IC50 of 128 nM for WT). Thus, it seems that although these residues are not in direct contact with the antagonist, they might nevertheless form an important linker to transduce the glutamate binding event in the extracellular domain into the TM region in the mGlu family.

[3H]EM-TBPC had very low affinity for human mGlu1 and none for mGlu5. The sequence comparison of the 7TMD showed that, with the exception of rat mGlu1, which carries a valine at position 757, all mGlu receptors have leucine at this position. This is the only amino acid difference found in the 7TMD region between rat and human mGlu1. Therefore, Val-7575.47 was identified as the critical residue for the binding selectivity of [3H]EM-TBPC at the rat versus human mGlu1 receptor. The mutation L7575.47V in hmGlu1 converted the affinity of this receptor for [3H]EM-TBPC to a value comparable with that of WT rmGlu1 (KD values 6.6 and 9.9 nM for rmGlu1 and hmGlu1 L757V, respectively). Moreover, mutation of rmGlu1 Val-7575.47 to leucine or alanine both led to a similar decrease in [3H]EM-TBPC binding affinity by 13-fold, indicating the importance of valine for selectivity for rat mGlu1. As we have shown previously (19), Val-7575.47 is also a critical residue for the enhancing effect of the positive allosteric modulator of mGlu1, RO 67-7476.

It is interesting to compare the critical residues involved in the EM-TBPC-binding site of rmGlu1 with those of the cis-retinal-binding pocket of rhodopsin or in reported ligand recognition sites of other family 1 GPCRs (Table III). We observed a striking conservation in the position of critical residues. We found that the five 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 rmGlu1, thus validating the application of rhodopsin three-dimensional structure (23, 32, 34) as a template for the family 3 GPCRs. Furthermore, conservation in the position of these critical residues was also observed in the previously reported ligand recognition sites for hbeta 2 adrenergic, hA3 adenosine, or h5HT4 receptors (33, 35-37) (Table III). Therefore, the allosteric binding site crevice of mGlu1 shares a similar structural feature as rhodopsin-like family 1 GPCRs.

                              
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Table III
Comparison of ligand-binding pocket of mGlu1 allosteric antagonist with those of rhodopsin-like GPCRs
The 17 residues located at a distance of 4.5 Å from cis-retinal in the three-dimensional structure of rhodopsin are shown (23,32,34). The generic numbering system proposed by Ballesteros and Weinstein (24) was used to compare residues in the 7TMD of the different GPCRs. The residues that have been experimentally determined to be located in the binding sites are as follows: epinephrine of hbeta 2-adrenergic receptor (R) (33), hA3 receptor ligand recognition (35), antagonist GR113808 ([1-[2-(methylsulfonylamino)ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate) of h5HT4 receptor (36), and inverse agonist of h5HT4 receptor (37).

The residue Trp-7986.48 of mGlu1 is of special interest, because it is highly conserved in all mGlus and in family 1 GPCRs. In the case of rhodopsin, spin labeling and Cys cross-linking studies (38, 39) demonstrated the requirement for a rigid body movement of the cytoplasmic end of TM6 away from TM3 upon rhodopsin activation and that the Trp-2656.48 serves to transmit the chromophore motion to TM6 helix. The cross-linking experiment (40) also showed that in the inactivated form (in the dark) of rhodopsin, the beta -ionone ring of cis-retinal is linked to Trp-2656.48. Similarly, in the three-dimensional structure of rhodopsin, the indole side chain of Trp-2656.48 comes within about 3.8 Å of the cis-retinal C20 (23, 32). Taken together, all of these studies are consistent with a pivotal role for the Trp6.48, acting as a switch for the transition of the receptor between different allosteric states. In our model, the crucial residues Phe-8016.51 and Tyr-8056.55 are located in proximity to Trp-7986.48 in the TM6 helix. Thus, we propose that the aromatic cluster formed from Trp-7986.48, Phe-8016.51, and Tyr-8056.55 interacts with the aromatic ring of EM-TBPC, blocking the Trp-7986.48 movement in TM6 helix, which is required for receptor activation. In good agreement with this model, we observed that replacement of Trp-7986.48 with a phenylalanine led to a large decrease of EM-TBPC IC50 value for the inhibition of the glutamate-mediated signal, due to the more preferable pi -interaction with Phe side chain. Joubert et al. (37) have recently reported that three residues of h5HT4 receptor, Asp-1003.32, Trp-2726.48, and Phe-2756.51, are important molecular determinants for agonist-independent activity, since mutation of these residues to alanine completely blocked the effects of inverse agonists. Interestingly, two of these residues are homologous to the critical residues Trp-7986.48 and Phe-8016.51 that we identified in the EM-TBPC-binding pocket of rmGlu1. Although the relative inverse agonist activity of EM-TBPC is presently unknown, such activity has been previously reported for BAY36-7620 and MPEP, the selective noncompetitive mGlu1 (18) and mGlu5 (17) antagonists, respectively.

In conclusion, we have demonstrated for the first time that the x-ray crystal structure of rhodpsin can be successfully applied as a template for the family 3 GPCRs. The allosteric binding pocket on mGlu receptors represents an attractive drug target. Hence, detailed information about the contact sites of allosteric antagonist with the receptor should further facilitate the rational design of drugs with improved subtype selectivity and potency.

    ACKNOWLEDGEMENTS

We are grateful to Birgit Molitor, Rachel Fimbel, Michael Weber, and Klaus Christensen for excellent technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 41-61-688-6286; Fax: 41-61-688-1720; E-mail: parichehr.malherbe@roche.com.

|| Present address: Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Ave., Harlow, Essex CM19 5AW, UK.

** Present address: Addex Pharmaceuticals SA, 12 Chemin Des Aulx, CH-1228 Plan Les Ouates, Switzerland.

Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211759200

2 C. Broger, unpublished work.

    ABBREVIATIONS

The abbreviations used are: mGlu, metabotropic glutamate; hmGlu, human mGlu; TM, transmembrane; 7TM, seven-transmembrane; 7TMD, seven-transmembrane domain; EM-TBPC, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile; GPCR, G-protein-coupled receptor; GIRK, G-protein-coupled inwardly rectifying K+ channel; WT, wild type; GABAB, gamma -aminobutyric acid, type B; EC, extracellular loop; CPCCOEt, 7-(hydroxyimino)cyclopropan[b]chromen-1a-carboxylic acid ethyl ester; MPEP, 2-methyl-6-(phenylethynyl)pyridine; BAY36-7620, [(3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on]; RO 67-7476, (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)-pyrrolidine; RO 01-6128, diphenylacetyl-carbamic acid ethyl ester; RO 67-4853, (9H-xanthene-9-carbonyl)-carbamic acid butyl ester; CGP7930, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethylpropyl)-phenol; CFBTE-TPC, N-[2-[(2-chloro-6-fluorobenzyl)thio]ethyl]2-thiophenecarboxamide; CHO, Chinese hamster ovary; 5HT4, 5-hydroxytryptamine type 4.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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