Interdomain movements in metabotropic glutamate receptor activation

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Sep 13, 2011 - metabotropic glutamate receptors activate the seven transmem- brane (7TM) ... activated by amino acids, i.e., the receptors for the two major.
Interdomain movements in metabotropic glutamate receptor activation Siluo Huanga,b,c,d, Jianhua Caod, Ming Jiangd, Gilles Labessec,e,f, Jianfeng Liud,1, Jean-Philippe Pina,b,c,1, and Philippe Rondarda,b,c,1 a

Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France; bInstitut National de la Santé et de la Recherche Médicale (INSERM), U661, F-34000 Montpellier, France; cUniversités de Montpellier 1 and 2, F-34000 Montpellier, France; dSino-France Laboratory for Drug Screening, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China; eCNRS, UMR 5048, Centre de Biochimie Structurale, F-34000 Montpellier, France; and fINSERM U1054, F-34000 Montpellier, France Edited by Robert J. Lefkowitz, Duke University Medical Center/Howard Hughes Medical Institute, Durham, NC, and approved August 12, 2011 (received for review May 16, 2011)

Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation. transmembrane signaling allosteric modulation

| G protein-coupled receptor |

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ost cell surface receptors are multimeric complexes of which each subunit is produced through the association of different domains throughout evolution (1–5). The activation of such receptor complexes is a result of coordinated conformational changes or movement of these different domains. Although an increasing amount of 3D crystal structures are becoming available, there is still limited information available on the structural basis of interdomain communication. Class C G protein-coupled receptors (GPCRs) represent key examples of such receptor complexes (6, 7). These receptors are obligatory dimers, either homo- or heterodimers, made by the association of two domains over evolutionary time; an extracellular bilobate venus flytrap (VFT) domain associated with a G protein activating 7 transmembrane (7TM) domain (Fig. 1A) (8). The VFTs are evolved from certain types of bacterial periplasmic binding proteins, especially those of the leucine-isoleucinevaline binding protein family involved in the transport of amino acids, sugars, or ions. Not surprisingly, class C GPCRs are activated by amino acids, i.e., the receptors for the two major neurotransmitters, the eight metabotropic glutamate receptors (mGluRs), and the GABAB receptor; sugar (the sweet taste receptors); or ions [the calcium-sensing receptor (CaSR)]. Accordingly, class C GPCRs represent exciting new targets for drug development for both the pharmaceutical and food industries, as illustrated by the number of drugs targeting these receptors already on the market (the GABAB receptor agonist baclofen, umami compounds, and various sweeteners such as aspartame, 15480e15485 | PNAS | September 13, 2011 | vol. 108 | no. 37

and cinacalcet, a positive allosteric modulator of the CaSR), and those in clinical trials (9–11). The precise mechanisms of how agonist binding in the VFTs leads to the 7TM conformational changes that are required for G protein activation remain unknown. Structural and mutagenesis studies indicated that the closure of the VFT resulting from agonist binding in the cleft represents a key step in receptor activation (12–16). It is assumed that this VFT conformational change is associated with a reorientation of the two VFTs in these dimeric receptors (15, 16), leading to a relative movement of the two 7TMs and the activation of one of them (17). In most class C GPCRs, the VFT is linked to the 7TM through a 70-amino-acid-long cysteine-rich domain (CRD) containing nine perfectly conserved Cys (18) (Fig. 1B). Although absent in the GABAB receptor, several lines of evidence highlight the crucial role of the CRD in the allosteric connection between the VFT and the 7TM in the other class C GPCRs. First, genetic mutations of cysteines in mGlu6 and CaSR CRDs have been identified in human diseases, such as “night blindness” (19) and “hypocalciuric hypercalcemia” (20–22), respectively. Second, deletion of the CRD in mGluRs results in a loss of function (23). Third, mutation of the disulfide bridge that interconnects the CRD to the VFT suppresses the allosteric coupling between the VFT and the 7TM (24). Fourth, sweet proteins such as brazzein have been proposed to activate the sweet taste receptor by interacting with the CRD (25). Recently, the crystal structures of the dimeric VFT and CRD complex of mGlu3 bound to five different agonists were solved (18). However, these structures did not lead to a clear role of the CRD in the activation process because all structures appeared to be in the expected “resting” state. In the present study, we further examined the role of the CRD in mGlu receptor activation. We first confirm its critical role in the allosteric coupling between the VFT and the 7TM domains, but most importantly we show that a precise association of the two CRDs within mGlu dimers leads to full receptor activation. Our study thus provides a clear demonstration that dimerization and structural rearrangement at the CRD dimer interface is responsible for mGlu receptors activation.

Author contributions: S.H., J.L., J.-P.P., and P.R. designed research; S.H., J.C., M.J., and G.L. performed research; S.H., J.L., J.-P.P., and P.R. analyzed data; and S.H., J.L., J.-P.P., and P.R. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence may be addressed. E-mail: [email protected], [email protected], or jfl[email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1107775108/-/DCSupplemental.

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required for the transduction from agonist binding to G protein activation in the mGlu2 receptor. mGlu2 Cys-500 Ala Mutant Displays High Constitutive Activity. For three of the four disulfide bridges, mutation of either of the two Cys residues involved in disulfide bridge formation resulted in a similar phenotype (Fig. 1C). However, whereas the mutation of Cys519 produced a mutant that can be activated by LY487379, this PAM was incapable of activating the receptor in which the partner Cys (Cys500) was mutated. Additional experiments revealed that the C500A mutant displayed constitutive activity, as shown by measurement of inositol phosphate accumulation leading to a response similar to that obtained with the agonist activated wild-type receptor (Fig. 2 A and B). This explains why no ligand-induced calcium signals could be measured with this receptor (Fig. 1C). The full constitutive activity of the mutant C500A is further supported by the absence of potentiation of inositol accumulation by the agonist and the PAM (Fig. 2A).

Fig. 1. Cysteines of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. (A) Structural model of the mGlu receptor where the dimeric extracellular domain is from the crystal structure of mGlu3 (code PDB 2E4U) bound to two molecules of glutamate (green) and the 7TM domain is from the structure of the β2-adrenergic receptor (code PDB 2RH1). (B) Close-up view of the CRD in the model of mGlu2 obtained by molecular homology, in which the eight cysteines that form intradomain disulfide bridges are highlighted. (C) Effect of glutamate and of LY487379, an mGlu2 positive allosteric modulator, on Ca2+ release in cells expressing the wild-type mGlu2 or the indicated mutants, in the presence of the chimeric Gqi9 protein. Data are expressed as the means ± SEM of at least three independent experiments.

Results

extracellular domain revealed that the CRD is stabilized by four intradomain disulfide bridges (Fig. 1B), and is linked to the VFT by an additional disulfide bridge involving Cys234 (18, 24). In the present study, we have analyzed the importance of each of these disulfide bridges by changing the conserved cysteines to alanines in the rat mGlu2 CRD. We found that all these mutations abolished agonist-induced responses (Fig. 1C), even though all mutants were correctly expressed at the cell surface (Fig. S1A), bound agonist with a wild-type affinity (Fig. S1B), and are still able to form dimers as revealed by cell surface time-resolved fluorescence resonance energy transfer (FRET) measurements (Fig. S1C). In addition, the mutations did not prevent the 7TM domain from activating G proteins. The positive allosteric modulator (PAM), LY487379, which binds to the 7TM (26, 27), activated all mutants except the C500A mutant (Fig. 1C); this is in agreement with our previous reports that mGluR PAMs become full agonists when the 7TM is disconnected from the VFT (24, 28). Taken together, our results suggest that the structure of the CRD in its entirety, stabilized by the four disulfide bridges, is Huang et al.

PHARMACOLOGY

Each Cysteine of the CRD Is Required for Allosteric Coupling Between the VFT and the 7TM Domains. The crystal structure of the mGlu3

Fig. 2. mGlu2 C500A mutant displays high constitutive activity. (A) Inositolphosphate (IP) accumulation in cells that express the wild-type mGlu2 and the indicated mutants without (basal) or after stimulation with glutamate or LY487379, in conditions where the chimeric Gqi9 protein is coexpressed. Data are expressed as the means ± SEM of at least three independent experiments. (B) IP accumulation is proportional to the amount of HA-tagged mGlu2 at the cell surface measured by ELISA for the wild-type after stimulation by glutamate and the constitutively active mutant C500A. (C) IP accumulation of the mGlu2 C500A mutant is abolished by the negative allosteric modulator MNI-137, but not by the competitive antagonist LY341495.

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Remarkably, the constitutive activity of mGlu2 C500A could be inhibited by the negative allosteric modulator (NAM), MNI137, which is known to act in the 7TM (Fig. 2C) (29). However, it was not inhibited by the competitive antagonist, LY341495, which prevents VFT closure, even though this compound binds to the receptor with a normal affinity, suggesting that the VFT can still adopt an open conformation, without preventing G protein activation by the 7TM. However, the presence of the VFT and of the interdomain disulfide bridge between the VFT and CRD are necessary, because their removal abolished constitutive activity induced by the C500A mutation (Fig. S2). Altogether, these data revealed that the C500A mutation stabilizes the 7TM in an active state, even though the VFT can remain open, suggesting that the mutation likely affects a key element involved in the signal transfer from the VFT to the 7TM. Constitutive Activity of C500A Results from an Intersubunit Disulfide Bridge. We investigated whether the constitutive activity could

result from an aberrant intrasubunit disulfide bridge between the VFT and the CRD. In the mGlu3 VFT–CRD crystal structures, Cys500 is in close proximity to three other Cys residues: Cys234 from the VFT linked to Cys518 of the CRD, and of course Cys519, to which it is naturally linked (Fig. S3A). By inserting a thrombin cleavage site between the VFT and the CRD to examine whether a DTT sensitive link exists between these two domains, as this can be detected after thrombin cleavage (Fig. S3B) (24), we showed that a disulfide bridge can indeed form between Cys234 and Cys519, when the two other Cys are mutated (Cys500 and Cys518) (Fig. S3C). However, this mutant receptor did not display constitutive activity (Fig. S3D), suggesting that such a disulfide bridge is unlikely responsible for the constitutive activity of the C500A mutant. Given that a Cys appears necessary at position 519 in the C500A mutant for constitutive activity, and because mGlu receptors are well-established dimers, we speculated that the Cys519 may form an intersubunit disulfide bridge, then stabilizing an active form of the mGlu dimer. To test this hypothesis, we took advantage of the quality control system of the GABAB receptor to produce “heterodimeric” mGluR (13, 30), in which only one subunit carries the C500A mutation. As previously reported, the C-terminal tail of mGlu2 was replaced either by that of the GABAB1 subunit (mGlu2-C1) carrying the natural retention signal RSRR or by a modified GABAB2 C-terminal tail carrying a retention signal KKDL at its C terminus, just after the coiled coil domain (mGlu2C2). We verified that only the mGlu2-C1–mGlu2-C2 heterodimer could indeed reach the cell surface, whereas the two homodimers are retained in the endoplasmic reticulum (Fig. S4). This system allowed us to show that only a receptor dimer carrying the C500A mutation in both subunits displays a high constitutive activity, whereas any dimer carrying the mutation in a single subunit did not (Fig. 3A). This is consistent with the Cys519 being involved in an intersubunit disulfide bridge. To firmly demonstrate this possibility, we examined whether a DTT sensitive covalent link can be observed between both subunits of the C500A mutant in which the natural intersubunit disulfide bridge has been removed (by mutating Cys121 into Ala). As expected, monomers of the C121A mutant can be detected on a Western blot under nonreducing condition, whereas only the dimer can be detected in the wild-type and the double mutant C121A–C500A (Fig. 3B) [the latter being constitutively active like the C500A mutant (Fig. S5)]. These data demonstrate that the mutation C500A leads to an intersubunit disulfide bridge likely involving Cys519 and this is likely responsible for the constitutive activity of this mutant. Cysteine Scanning in the Putative CRD Interface Produces Constitutive Mutants. To firmly demonstrate that a covalent linkage

between the CRDs within an mGlu dimer could be sufficient to 15482 | www.pnas.org/cgi/doi/10.1073/pnas.1107775108

Fig. 3. The constitutive activity of mGlu2 C500A mutant results from an intersubunit disulfide bridge. (A) IP accumulation in cells that coexpress the indicated constructs resulting in a “heterodimeric” mGlu2 receptor, without (basal) or after stimulation with glutamate, in the presence of the chimeric Gqi9 protein. The pictogram displays the heterodimeric mGlu2 in which the subunits mGlu2-C1 and mGlu2-C2 are composed of HA-tagged mGlu2 where the C-terminal region was replaced by one of the GABAB1 and GABAB2 receptors, respectively, followed by the retention signal KKXX. (B) Western blot in nonreducing or reducing conditions for wild-type and indicated mutants of mGlu2. The mutation C121A results in a receptor deleted of the intersubunit disulfide bridge that could be resolved as monomer (see arrows) both in the absence and presence of DTT. Addition of the mutation C500A in the C121A mutant produces a receptor with a new intersubunit disulfide bridge and in this double mutant the monomer could be resolved only in the presence of DTT.

stabilize an active state of the receptor, we examined whether introducing additional Cys residues within the CRD could generate constitutively active receptors. A series of 13 Cys mutants were produced on the basis of the known 3D structure of the mGlu3 CRD, at positions possibly corresponding to the dimer interface (15, 16, 18) (Fig. 4A). Among these, two mutants (E516C and L521C) displayed a robust constitutive activity, and L521C was fully active, i.e., displaying a constitutive activity as high as the glutamate-induced activity measured with the wildtype receptor (Fig. 4B). Western blot analysis of the C121A– L521C double mutant confirmed that the additional Cys could form an intersubunit disulfide bridge (Fig. 4C). However, all of the mutations at the interface do not result in a cross-linking between the subunits, for example the E502C mutant (Fig. 4C), indicating that the cross-links observed at the interface of the two CRDs are specific. Interestingly, the appearance of a disulfide bridge between the two subunits does not necessarily stabilize the active state of the receptor; indeed, crosslinking between the subunits can be observed with most mutants such as Q526C, P527C, E529C, and G541C, even though none of them display constitutive activity. Similar Constitutive Mutants in Other mGlu Receptors. We investigated whether similar mutations in the CRD of other mGlu receptors could also generate constitutively active mutants. This was examined on both mGlu5 and mGlu4 receptors, which belong to group I and group III, respectively (Fig. 5), whereas the aforementioned mGlu2 receptor belongs to group II mGlu receptors. Huang et al.

As observed with mGlu2, the mutation of the first cysteine in the CRD of mGlu4 and -5, C502 and C512, respectively, also result in constitutively active receptors that could not be further stimulated by the full agonists, L-AP4 and quisqualate, respectively (Fig. 5A). The weaker constitutive activity of mGlu4 C502A may be explained by its lower expression level at the cell surface (Fig. 5B). Similar to mGlu2, the presence of the free partner Cys is required to stabilize the active state of these mGlu receptors, because the double mutants mGlu4 C502A–C521A and mGlu5 C512A– C531A did not display significant constitutive activity (Fig. 5A) despite their appropriate cell surface expression (Fig. 5B). Also as observed with mGlu2, introduction of a Cys at the equivalent position of L251 (H523 and T533 in mGlu4 and -5, respectively) also generated constitutively active mutants, whereas no such activity was measured when these residues were mutated into Ala (Fig. 5A). Taken together, these data illustrate how general our observation with mGlu2 is within the mGlu-like class C GPCRs. Discussion In the present study we provide important information on the role of the CRD in the activation process of the mGlu receptors. Huang et al.

We further illustrate its important contribution for the allosteric coupling between the ligand binding VFT and the G protein coupling 7TM domains, and most importantly, we show that a precise orientation of the two CRDs within an mGlu dimer is associated with a full activity of the receptor. Because these key data were reproduced with three distinct mGlu receptors, the proposed mechanism is likely general to all class C GPCRs containing a CRD, then including the sweet and umami taste receptors (31), the CaSR (32), and the GPRC6a recently shown to be involved in the effect of osteocalcin (33). Many things were expected from the resolution of the crystal structure of the extracellular domain of mGlu receptors for the understanding of the activation mechanism of these complex dimeric receptors (15, 16, 18). Although these studies revealed that the VFT closure represents a first step in the activation process, how this conformational change is transmitted to the 7TM to activate the G protein remains to be elucidated. The initial structures suggested that a relative movement from a resting (lobes 2 far apart) to an “active” (lobes 2 in closed contact) orientation of the VFTs could be involved in the activation process. However, recent structures are not consistent with this proposal. Indeed active and resting orientations were PNAS | September 13, 2011 | vol. 108 | no. 37 | 15483

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Fig. 4. Cysteine scanning in the putative CRD’s interface produces mGlu2 with constitutive activity. (A) Molecular model of mGlu2 where the residues mutated into Cys are in yellow or orange. The positions where the presence of a Cys leads to constitutive activity are highlighted in orange. (B) IP accumulation in cells that express the wild-type mGlu2 and the indicated mutants without (basal) or after stimulation with glutamate, in the presence of the chimeric Gqi9 protein. (C) Western blot analysis of HA-tagged mGlu2 that carry the mutations described in B in addition to the C121A mutation. No monomers in denaturating conditions in the absence of DTT were observed for the fully constitutively active mutant L521C that could not be further activated by glutamate. In denaturating conditions in the presence of 10 mM DTT, the wild-type mGlu2 and all of the indicated mutants could be solved as monomers (arrows), indicating the presence of a disulfide bridge between the two subunits of the wild-type mGlu2 and of the indicated mutants.

Fig. 5. Similar constitutive mutants in mGlu4 and mGlu5 receptors. (A) IP accumulation in cells that express the wild-type mGlu4 or mGlu5 and the indicated mutants without (basal) or after stimulation with the indicated agonists, in the presence of the chimeric Gqi9 protein. (B) Amount of HAtagged mGlu4 or mGlu5 at the cell surface measured by ELISA for wild-type and the indicated mutants. Data are expressed as the means ± SEM of at least three independent experiments.

observed with antagonist- and agonist-bound VFTs, respectively (7, 18). In addition, a crystal structure of the dimeric complex with both the VFT and the CRD did not clarify the role of the CRD in the activation process. Indeed, these structures were all solved with five different bound agonists (18) and correspond to what was previously proposed to be the resting orientation with the two CRDs far apart, whereas both VFTs are closed. Our data show that a precise association of the two CRDs is sufficient for full mGluR activation, indicating that the two CRDs are likely contacting each other in the active state. Such a proposal is consistent with a 3D model of the dimeric extracellular domain in which either one or both VFTs are in the closed state and their association in the active orientation (Acc or Aco; Fig. 6). In such models, all residues that, when mutated into Cys, lead to an intersubunit disulfide bridge and a constitutively active receptor are facing each other at a distance consistent with a disulfide bond formation. In particular, when comparing the conformation Aco and Acc, the distance Cα-Cα between both Cys519 or Leu521 reduce from 24 to 16 Å, and 24 to 18 Å, respectively. These distances are compatible with the disulfide bond formation due to the thermal motion of proteins (34), but once the bond has formed, the distance between alpha-carbons in the two cysteines is about 6 Å (35). It is interesting to note that even though intersubunit cross-linking was obtained with other residues (such as Glu529), such mutant receptors did not display constitutive activity, indicating that only a specific and precise association of the CRDs leads to G protein activation. The effects of competitive and noncompetitive antagonists reveal that some flexibility exists in the mGlu receptor for the interconnection between the VFT and 7TM domains. The constitutive activity of the cross-linked receptor was inhibited by negative allosteric modulators acting in the 7TM domains, thereby indicating that the constitutive activity involves conformational changes in the 7TM domain. This action of NAMs also indicates that the 7TM domains can be maintained in an inactive conformation even when the CRDs are linked together in their active association. In contrast, the constitutive activity of the cross-linked 15484 | www.pnas.org/cgi/doi/10.1073/pnas.1107775108

Fig. 6. Model for the mechanism of mGlu receptor activation. Models of mGlu2 where the dimeric VFT domains are in the resting (R) or active (A) conformations according to the three states observed in the mGlu1 crystal structures (15, 16). Each VFT is adopting an open (o) or closed (c) conformation, and the CRD model was obtained by homology modeling using the crystal structure of the whole extracellular domain of mGlu3 (18). The C termini of the CRD (arrowheads), the positions 519 and 521 (red) that crosslink between the two subunits then providing a constitutively active receptor, and the positions 529 (orange) that cross-link without producing constitutive activity are highlighted. The distance between both Cα’s of the indicated residues are indicated.

mutant is not affected by the orthosteric antagonists known to prevent VFT closure. Such absence of inhibition is observed even though the antagonist binds to the receptor, then suggesting that even when both VFTs are in the open state, the CRDs can still be in their active orientation (the Aoo conformation), as observed with the mGlu1 structure bound to a LY341495 (7). Although some flexibility exists in the mGlu receptor, some structural constraints are important between the CRD and the VFT. This is well illustrated by the requirement of the natural disulfide bridge that links these two domains for the functional coupling between the VFT and the 7TM (24). Such a constraint is likely important for the VFTs to control the relative position of the CRDs in the dimeric mGlu, thereby controlling receptor activity. Accordingly, it is likely that a change in the relative orientation of the VFTs resulting from agonist binding and VFT closure can bring the CRDs in close contact, leading to the activation of one 7TM. However, the real amplitude of movement between the VFTs remains to be identified. Indeed, in the proposed resting orientation observed with the soluble VFTs, the CRDs are far apart, as will then be the 7TM domains. However, such proposed conformation of the dimer is not compatible with FRET data obtained when CFP and YFP are fused to the intracellular loops of the receptor (17, 36). Indeed these data revealed a close proximity between the 7TM domains, closer that what is expected on the basis of the resting orientation of the VFTs. Accordingly, either the CRD move relative to the VFT or most likely the changes of the VFT relative position occurring during activation are not as important as those observed in the crystal structures. Consistent with this proposal, a precise association mode of the CRDs is required for receptor activation. In conclusion, due to their key role in the mechanism of activation of the receptors, the CRDs may constitute the site of action of molecules that modulate the activity of the class C GPCRs. For instance, it was suggested that the mode of action of the sweet proteins such as brazzein was through the binding to CRD of the T1R3 subunit of the heteromeric sweet taste receptor (25). As such, this study brings unique views on how the genetic association during evolution of proteins able to bind amino acids, sugars, or ions, was successful in generating GPCRs activated by such ligands, through the use of an intermediate CRD. Huang et al.

Materials. LY341495 ((2S)-2-amino-2-[(1S,2S)-2-carboxy-cycloprop-1-yl]-3-(xanth9-yl) propanoic acid) and LY487379 (2,2,2-trifluoro-N-[4-(2-methoxyphenoxy) phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) were purchased from Tocris Cookson. L-AP4 was obtained from Ascent Scientific. [3H]LY341495 was purchased from American Radiolabeled Chemicals. MNI-137 was a gift from Gilles Tamagnan (Institute for Neurodegenerative Disorders, New Haven, CT). Human thrombin (CalBiochem) was obtained from Merck.

Western Blotting, Cell Surface Quantification by ELISA, Ligand Binding Assay, Intracellular Calcium Release, Inositol Phosphate, and Time-Resolved FRET Measurements. Detection of the HA-tagged constructs at the cell surface by Western blotting or ELISA with or without thrombin treatment were performed as previously described (24, 38). Ligand binding assay in intact HEK293 cells using 2 nM [3H]LY341495, measurements of inositol phosphate accumulation, calcium signal in HEK-293 cells, and time-resolved FRET were performed as previously described (24). Molecular Modeling. Homology models of the dimeric mGlu2 VFTs were generated using the crystal structures of mGlu1 VFT as templates: Protein Data Bank (PDB) accession no. 1EWT for Roo, 1EWK for Aco, and 1ISR for Acc. Homology models of the mGlu2 CRD were generated using the crystal structure of mGlu3 CRD (PDB accession no. 2E4U). Models were manually refined with ViTO (39) using the sequence alignment of the mGlu VFTs. Final models were built using Modeler 7.0 (40) and evaluated using the dynamic evolutionary trace as implemented in ViTO.

Plasmids and Transfection. pRK5 plasmids encoding the HA-tagged wild-type mGlu2 from rat (24) was previously described. The construct HA-tagged mGlu2-C1-KKXX was obtained by introducing the restriction site NotI after the codon encoding residue Leu846 in the C-terminal region of mGlu2, then by replacing the last 26 residues in the C terminus of mGlu2 by the region 875– 921 of the GABAB1 subunit C-terminal region followed by the retention sequence KKTN. The construct HA-tagged mGlu2-C2-KKXX was obtained using the same strategy except that the last 26 residues in the mGlu2 C terminus were replaced by the region 772–820 of the GABAB2 subunit C-terminal region followed by the retention sequence KKTN. In these constructs, the Ctermini of mGlu2-C1-KKXX and mGlu2-C2-KKXX are SANLAAATGSSTNNNEEEKSRLLEKENRELEKIIAEKEERVSELRHQLQSRQQLKKTN and SANLAAATSTSVTSVNQASTSRLEGLQSENHRLRMKITELDKDLEEVTMQLQDTPEKKTN, respectively (the residue Leu846 of mGlu2 is underlined). HEK-293 and COS-7 cells were cultured in DMEM supplemented with 10% FBS and transfected by electroporation as described elsewhere (37). Ten millions cells were transfected with 2 μg of each plasmid of interest and completed to a total amount of 10 μg with the plasmid encoding the pRK5 empty vector. To allow efficient coupling of the receptor to the phospholipase C pathway, cells were also transfected with the chimeric G protein Gαqi9 (2 μg).

ACKNOWLEDGMENTS. We thank Dr. Carsten Brock for the mGlu2-C1 and mGlu-C2 constructs, Pauline Scholler for her help in performing experiments, Dr. Greg Stewart for English editing, and the ARPEGE (Pharmacology Screening-Interactome) platform facility at the Institut de Génomique Fonctionnelle (Montpellier, France). P.R. and J-P.P. were supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), and by Grants from the Agence Nationale de la Recherche [GPCR dimers (ANR-BLAN06-3_135092) and mGluPatho (ANR-08-NEUR-006-02) in the frame of ERA-Net NEURON] and by an unrestricted grant from Senomyx. J.L. was supported by the National Basic Research Program of China (Grant 2007CB914200), the National Natural Science Foundation of China (Grant 30973514), and the Program of Introducing Talents of Discipline to Universities of Ministry of Education (B08029).

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PHARMACOLOGY

Materials and Methods