Interdomain Movements in Metabotropic Glutamate Receptor Activation

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

aCentre 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 and cinacalcet, a positive allosteric modulator of the CaSR), and of several structural domains, some involved in ligand recognition, those in clinical trials (9–11). whereas others are responsible for signal transduction. In most The precise mechanisms of how agonist binding in the VFTs cases, the mechanism of how ligand interaction in the extracellu- leads to the 7TM conformational changes that are required for G lar domains leads to the activation of effector domains remains protein activation remain unknown. Structural and mutagenesis largely unknown. Here we examined how the extracellular ligand studies indicated that the closure of the VFT resulting from ag- fl binding to the venus ytrap (VFT) domains of the dimeric onist binding in the cleft represents a key step in receptor acti- metabotropic glutamate receptors activate the seven transmem- vation (12–16). It is assumed that this VFT conformational brane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). change is associated with a reorientation of the two VFTs in these We show that any of the four disulfide bridges of the CRD are dimeric receptors (15, 16), leading to a relative movement of the required for the allosteric coupling between the VFT and the 7TM two 7TMs and the activation of one of them (17). domains. More importantly, we show that a specific association of In most class C GPCRs, the VFT is linked to the 7TM through the two CRDs corresponds to the active state of the receptor. a 70-amino-acid-long cysteine-rich domain (CRD) containing nine Indeed, a specific crosslinking of the CRDs with intersubunit perfectly conserved Cys (18) (Fig. 1B). Although absent in the fi disul de bridges leads to fully constitutively active receptors, no GABAB receptor, several lines of evidence highlight the cru- longer activated by agonists nor by allosteric modulators. These cial role of the CRD in the allosteric connection between the data demonstrate that intersubunit movement at the level of the VFT and the 7TM in the other class C GPCRs. First, genetic CRDs represents a key step in metabotropic glutamate receptor mutations of cysteines in mGlu6 and CaSR CRDs have been activation. identified in human diseases, such as “night blindness” (19) and “hypocalciuric hypercalcemia” (20–22), respectively. Second, transmembrane signaling | G protein-coupled receptor | deletion of the CRD in mGluRs results in a loss of function (23). allosteric modulation Third, mutation of the disulfide bridge that interconnects the CRD to the VFT suppresses the allosteric coupling between the ost cell surface receptors are multimeric complexes of VFT and the 7TM (24). Fourth, sweet proteins such as brazzein which each subunit is produced through the association of M have been proposed to activate the sweet taste receptor by different domains throughout evolution (1–5). The activation interacting with the CRD (25). Recently, the crystal structures of of such receptor complexes is a result of coordinated confor- the dimeric VFT and CRD complex of mGlu3 bound to mational changes or movement of these different domains. Al- fi though an increasing amount of 3D crystal structures are becoming ve different agonists were solved (18). However, these struc- available, there is still limited information available on the structures did not lead to a clear role of the CRD in the activation tural basis of interdomain communication. process because all structures appeared to be in the expected “ ” Class C G protein-coupled receptors (GPCRs) represent key resting state. examples of such receptor complexes (6, 7). These receptors are In the present study, we further examined the role of the CRD fi fi obligatory dimers, either homo- or heterodimers, made by the in mGlu receptor activation. We rst con rm its critical role in association of two domains over evolutionary time; an extracel- the allosteric coupling between the VFT and the 7TM domains, lular bilobate venus flytrap (VFT) domain associated with a G but most importantly we show that a precise association of the protein activating 7 transmembrane (7TM) domain (Fig. 1A) (8). two CRDs within mGlu dimers leads to full receptor activation. The VFTs are evolved from certain types of bacterial periplas- Our study thus provides a clear demonstration that dimerization mic binding proteins, especially those of the leucine-isoleucine- and structural rearrangement at the CRD dimer interface is valine binding protein family involved in the transport of amino responsible for mGlu receptors activation. 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 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. (mGluRs), and the GABAB receptor; sugar (the sweet taste wrote the paper. receptors); or ions [the calcium-sensing receptor (CaSR)]. Ac- The authors declare no conflict of interest. cordingly, class C GPCRs represent exciting new targets for drug This article is a PNAS Direct Submission. development for both the pharmaceutical and food industries, 1To whom correspondence may be addressed. E-mail: [email protected], jean- as illustrated by the number of drugs targeting these receptors [email protected], or jfl[email protected]. already on the market (the GABAB receptor agonist baclofen, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. umami compounds, and various sweeteners such as aspartame, 1073/pnas.1107775108/-/DCSupplemental.

15480e15485 | PNAS | September 13, 2011 | vol. 108 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1107775108 Downloaded by guest on September 24, 2021 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 Each Cysteine of the CRD Is Required for Allosteric Coupling Between the VFT and the 7TM Domains. The crystal structure of the mGlu3 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 PHARMACOLOGY 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. 2. mGlu2 C500A mutant displays high constitutive activity. (A) Inositol- (Fig. S1C). In addition, the mutations did not prevent the 7TM phosphate (IP) accumulation in cells that express the wild-type mGlu2 and the indicated mutants without (basal) or after stimulation with gluta- domain from activating G proteins. The positive allosteric mate or LY487379, in conditions where the chimeric Gqi9 protein is coex- modulator (PAM), LY487379, which binds to the 7TM (26, 27), pressed. Data are expressed as the means ± SEM of at least three inde- activated all mutants except the C500A mutant (Fig. 1C); this is pendent experiments. (B) IP accumulation is proportional to the amount of in agreement with our previous reports that mGluR PAMs be- HA-tagged mGlu2 at the cell surface measured by ELISA for the wild-type after stimulation by glutamate and the constitutively active mutant come full agonists when the 7TM is disconnected from the VFT C500A. (C) IP accumulation of the mGlu2 C500A mutant is abolished by the (24, 28). Taken together, our results suggest that the structure of negative allosteric modulator MNI-137, but not by the competitive antago- the CRD in its entirety, stabilized by the four disulfide bridges, is nist LY341495.

Huang et al. PNAS | September 13, 2011 | vol. 108 | no. 37 | 15481 Downloaded by guest on September 24, 2021 Remarkably, the constitutive activity of mGlu2 C500A could be inhibited by the negative allosteric modulator (NAM), MNI- 137, 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 sta- bilizes 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 ex- amine whether a DTT sensitive link exists between these two Fig. 3. The constitutive activity of mGlu2 C500A mutant results from an domains, as this can be detected after thrombin cleavage (Fig. intersubunit disulfide bridge. (A) IP accumulation in cells that coexpress the S3B) (24), we showed that a disulfide bridge can indeed form indicated constructs resulting in a “heterodimeric” mGlu2 receptor, without between Cys234 and Cys519, when the two other Cys are mu- (basal) or after stimulation with glutamate, in the presence of the chimeric tated (Cys500 and Cys518) (Fig. S3C). However, this mutant Gqi9 protein. The pictogram displays the heterodimeric mGlu2 in which the subunits mGlu2-C1 and mGlu2-C2 are composed of HA-tagged mGlu2 where receptor did not display constitutive activity (Fig. S3D), sug- fi the C-terminal region was replaced by one of the GABAB1 and GABAB2 gesting that such a disul de bridge is unlikely responsible for the receptors, respectively, followed by the retention signal KKXX. (B) Western blot constitutive activity of the C500A mutant. in nonreducing or reducing conditions for wild-type and indicated mutants of Given that a Cys appears necessary at position 519 in the C500A mGlu2. The mutation C121A results in a receptor deleted of the intersubunit mutant for constitutive activity, and because mGlu receptors are disulfide bridge that could be resolved as monomer (see arrows) both in the well-established dimers, we speculated that the Cys519 may form absence and presence of DTT. Addition of the mutation C500A in the C121A an intersubunit disulfide bridge, then stabilizing an active form of mutant produces a receptor with a new intersubunit disulfide bridge and in this the mGlu dimer. To test this hypothesis, we took advantage of the double mutant the monomer could be resolved only in the presence of DTT. quality control system of the GABAB receptor to produce “het- ” erodimeric mGluR (13, 30), in which only one subunit carries the stabilize an active state of the receptor, we examined whether C500A mutation. As previously reported, the C-terminal tail of introducing additional Cys residues within the CRD could gen- mGlu2 was replaced either by that of the GABA subunit B1 erate constitutively active receptors. A series of 13 Cys mutants (mGlu2-C1) carrying the natural retention signal RSRR or by were produced on the basis of the known 3D structure of the a modified GABA C-terminal tail carrying a retention signal B2 mGlu3 CRD, at positions possibly corresponding to the dimer KKDL at its C terminus, just after the coiled coil domain (mGlu2- interface (15, 16, 18) (Fig. 4A). Among these, two mutants C2). We verified that only the mGlu2-C1–mGlu2-C2 heterodimer could indeed reach the cell surface, whereas the two homodimers (E516C and L521C) displayed a robust constitutive activity, and are retained in the endoplasmic reticulum (Fig. S4). This system L521C was fully active, i.e., displaying a constitutive activity as allowed us to show that only a receptor dimer carrying the C500A high as the glutamate-induced activity measured with the wild- type receptor (Fig. 4B). Western blot analysis of the C121A– mutation in both subunits displays a high constitutive activity, fi whereas any dimer carrying the mutation in a single subunit did not L521C double mutant con rmed that the additional Cys could fi C (Fig. 3A). This is consistent with the Cys519 being involved in an form an intersubunit disul de bridge (Fig. 4 ). intersubunit disulfide bridge. However, all of the mutations at the interface do not result in To firmly demonstrate this possibility, we examined whether a cross-linking between the subunits, for example the E502C C a DTT sensitive covalent link can be observed between both mutant (Fig. 4 ), indicating that the cross-links observed at the fi subunits of the C500A mutant in which the natural intersubunit interface of the two CRDs are speci c. Interestingly, the ap- disulfide bridge has been removed (by mutating Cys121 into pearance of a disulfide bridge between the two subunits does not Ala). As expected, monomers of the C121A mutant can be necessarily stabilize the active state of the receptor; indeed, cross- detected on a Western blot under nonreducing condition, whereas linking between the subunits can be observed with most mutants only the dimer can be detected in the wild-type and the double such as Q526C, P527C, E529C, and G541C, even though none of mutant C121A–C500A (Fig. 3B) [the latter being constitutively them display constitutive activity. active like the C500A mutant (Fig. S5)]. These data demonstrate that the mutation C500A leads to an intersubunit disulfide bridge Similar Constitutive Mutants in Other mGlu Receptors. We in- likely involving Cys519 and this is likely responsible for the con- vestigated whether similar mutations in the CRD of other mGlu stitutive activity of this mutant. receptors could also generate constitutively active mutants. This was examined on both mGlu5 and mGlu4 receptors, which belong Cysteine Scanning in the Putative CRD Interface Produces Consti- to group I and group III, respectively (Fig. 5), whereas the tutive Mutants. To firmly demonstrate that a covalent linkage aforementioned mGlu2 receptor belongs to group II mGlu between the CRDs within an mGlu dimer could be sufficient to receptors.

15482 | www.pnas.org/cgi/doi/10.1073/pnas.1107775108 Huang et al. Downloaded by guest on September 24, 2021 Fig. 4. Cysteine scanning in the putative CRD’sin- terface 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.

As observed with mGlu2, the mutation of the ﬁrst cysteine in the We further illustrate its important contribution for the allosteric CRD of mGlu4 and -5, C502 and C512, respectively, also result in coupling between the ligand binding VFT and the G protein constitutively active receptors that could not be further stimulated coupling 7TM domains, and most importantly, we show that a by the full agonists, L-AP4 and quisqualate, respectively (Fig. 5A). precise orientation of the two CRDs within an mGlu dimer is The weaker constitutive activity of mGlu4 C502A may be ex- associated with a full activity of the receptor. Because these key

plained by its lower expression level at the cell surface (Fig. 5B). data were reproduced with three distinct mGlu receptors, the PHARMACOLOGY Similar to mGlu2, the presence of the free partner Cys is required proposed mechanism is likely general to all class C GPCRs to stabilize the active state of these mGlu receptors, because the containing a CRD, then including the sweet and umami taste double mutants mGlu4 C502A–C521A and mGlu5 C512A– receptors (31), the CaSR (32), and the GPRC6a recently shown C531A did not display signiﬁcant constitutive activity (Fig. 5A) to be involved in the effect of osteocalcin (33). despite their appropriate cell surface expression (Fig. 5B). Many things were expected from the resolution of the crystal Also as observed with mGlu2, introduction of a Cys at the structure of the extracellular domain of mGlu receptors for the equivalent position of L251 (H523 and T533 in mGlu4 and -5, understanding of the activation mechanism of these complex respectively) also generated constitutively active mutants, whereas dimeric receptors (15, 16, 18). Although these studies revealed no such activity was measured when these residues were mutated that the VFT closure represents a ﬁrst step in the activation into Ala (Fig. 5A). process, how this conformational change is transmitted to the Taken together, these data illustrate how general our obser- 7TM to activate the G protein remains to be elucidated. The vation with mGlu2 is within the mGlu-like class C GPCRs. initial structures suggested that a relative movement from a resting (lobes 2 far apart) to an “active” (lobes 2 in closed Discussion contact) orientation of the VFTs could be involved in the acti- In the present study we provide important information on the vation process. However, recent structures are not consistent role of the CRD in the activation process of the mGlu receptors. with this proposal. Indeed active and resting orientations were

Huang et al. PNAS | September 13, 2011 | vol. 108 | no. 37 | 15483 Downloaded by guest on September 24, 2021 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 cross- link between the two subunits then providing a constitutively active re- Fig. 5. Similar constitutive mutants in mGlu4 and mGlu5 receptors. (A)IP ceptor, and the positions 529 (orange) that cross-link without producing accumulation in cells that express the wild-type mGlu4 or mGlu5 and the constitutive activity are highlighted. The distance between both Cα’softhe indicated mutants without (basal) or after stimulation with the indicated indicated residues are indicated. agonists, in the presence of the chimeric Gqi9 protein. (B) Amount of HA- tagged 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 mutant is not affected by the orthosteric antagonists known to least three independent experiments. prevent VFT closure. Such absence of inhibition is observed even though the antagonist binds to the receptor, then suggesting that observed with antagonist- and agonist-bound VFTs, respectively even when both VFTs are in the open state, the CRDs can still be (7, 18). In addition, a crystal structure of the dimeric complex in their active orientation (the Aoo conformation), as observed with both the VFT and the CRD did not clarify the role of the with the mGlu1 structure bound to a LY341495 (7). fl CRD in the activation process. Indeed, these structures were all Although some exibility exists in the mGlu receptor, some solved with five different bound agonists (18) and correspond to structural constraints are important between the CRD and the what was previously proposed to be the resting orientation with VFT. This is well illustrated by the requirement of the natural fi the two CRDs far apart, whereas both VFTs are closed. disul de bridge that links these two domains for the functional Our data show that a precise association of the two CRDs is coupling between the VFT and the 7TM (24). Such a constraint sufficient for full mGluR activation, indicating that the two CRDs is likely important for the VFTs to control the relative position of are likely contacting each other in the active state. Such a proposal the CRDs in the dimeric mGlu, thereby controlling receptor is consistent with a 3D model of the dimeric extracellular domain activity. Accordingly, it is likely that a change in the relative in which either one or both VFTs are in the closed state and their orientation of the VFTs resulting from agonist binding and VFT association in the active orientation (Acc or Aco; Fig. 6). In such closure can bring the CRDs in close contact, leading to the ac- models, all residues that, when mutated into Cys, lead to an tivation of one 7TM. However, the real amplitude of movement intersubunit disulfide bridge and a constitutively active receptor between the VFTs remains to be identified. Indeed, in the pro- are facing each other at a distance consistent with a disulfide bond posed resting orientation observed with the soluble VFTs, the formation. In particular, when comparing the conformation Aco CRDs are far apart, as will then be the 7TM domains. However, and Acc, the distance Cα-Cα between both Cys519 or Leu521 such proposed conformation of the dimer is not compatible with reduce from 24 to 16 Å, and 24 to 18 Å, respectively. These dis- FRET data obtained when CFP and YFP are fused to the in- tances are compatible with the disulfide bond formation due to tracellular loops of the receptor (17, 36). Indeed these data the thermal motion of proteins (34), but once the bond has revealed a close proximity between the 7TM domains, closer that formed, the distance between alpha-carbons in the two cysteines is what is expected on the basis of the resting orientation of the about 6 Å (35). It is interesting to note that even though inter- VFTs. Accordingly, either the CRD move relative to the VFT or subunit cross-linking was obtained with other residues (such as most likely the changes of the VFT relative position occurring Glu529), such mutant receptors did not display constitutive ac- during activation are not as important as those observed in the tivity, indicating that only a specific and precise association of the crystal structures. Consistent with this proposal, a precise asso- CRDs leads to G protein activation. ciation mode of the CRDs is required for receptor activation. The effects of competitive and noncompetitive antagonists In conclusion, due to their key role in the mechanism of acti- reveal that some flexibility exists in the mGlu receptor for the vation of the receptors, the CRDs may constitute the site of action interconnection between the VFT and 7TM domains. The con- of molecules that modulate the activity of the class C GPCRs. For stitutive activity of the cross-linked receptor was inhibited by instance, it was suggested that the mode of action of the sweet negative allosteric modulators acting in the 7TM domains, thereby proteins such as brazzein was through the binding to CRD of the indicating that the constitutive activity involves conformational T1R3 subunit of the heteromeric sweet taste receptor (25). As changes in the 7TM domain. This action of NAMs also indicates such, this study brings unique views on how the genetic association that the 7TM domains can be maintained in an inactive confor- during evolution of proteins able to bind amino acids, sugars, or mation even when the CRDs are linked together in their active ions, was successful in generating GPCRs activated by such association. In contrast, the constitutive activity of the cross-linked ligands, through the use of an intermediate CRD.

15484 | www.pnas.org/cgi/doi/10.1073/pnas.1107775108 Huang et al. Downloaded by guest on September 24, 2021 Materials and Methods Western Blotting, Cell Surface Quantification by ELISA, Ligand Binding Assay, Materials. LY341495 ((2S)-2-amino-2-[(1S,2S)-2-carboxy-cycloprop-1-yl]-3-(xanth- Intracellular Calcium Release, Inositol Phosphate, and Time-Resolved FRET 9-yl) propanoic acid) and LY487379 (2,2,2-trifluoro-N-[4-(2-methoxyphenoxy) Measurements. Detection of the HA-tagged constructs at the cell surface by phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) were purchased from Western blotting or ELISA with or without thrombin treatment were per- 3 Tocris Cookson. L-AP4 was obtained from Ascent Scientific. [ H]LY341495 formed as previously described (24, 38). Ligand binding assay in intact HEK- 3 was purchased from American Radiolabeled Chemicals. MNI-137 was a 293 cells using 2 nM [ H]LY341495, measurements of inositol phosphate gift from Gilles Tamagnan (Institute for Neurodegenerative Disorders, New accumulation, calcium signal in HEK-293 cells, and time-resolved FRET were Haven, CT). Human thrombin (CalBiochem) was obtained from Merck. performed as previously described (24).

Plasmids and Transfection. pRK5 plasmids encoding the HA-tagged wild-type Molecular Modeling. Homology models of the dimeric mGlu2 VFTs were mGlu2 from rat (24) was previously described. The construct HA-tagged generated using the crystal structures of mGlu1 VFT as templates: Protein mGlu2-C1-KKXX was obtained by introducing the restriction site NotI after Data Bank (PDB) accession no. 1EWT for Roo, 1EWK for Aco, and 1ISR for Acc. the codon encoding residue Leu846 in the C-terminal region of mGlu2, then Homology models of the mGlu2 CRD were generated using the crystal by replacing the last 26 residues in the C terminus of mGlu2 by the region 875– structure of mGlu3 CRD (PDB accession no. 2E4U). Models were manually 921 of the GABAB1 subunit C-terminal region followed by the retention refined with ViTO (39) using the sequence alignment of the mGlu VFTs. Final sequence KKTN. The construct HA-tagged mGlu2-C2-KKXX was obtained models were built using Modeler 7.0 (40) and evaluated using the dynamic using the same strategy except that the last 26 residues in the mGlu2 C ter- evolutionary trace as implemented in ViTO. minus were replaced by the region 772–820 of the GABAB2 subunit C-terminal region followed by the retention sequence KKTN. In these constructs, the C- ACKNOWLEDGMENTS. We thank Dr. Carsten Brock for the mGlu2-C1 and termini of mGlu2-C1-KKXX and mGlu2-C2-KKXX are SANLAAATGSSTNN- mGlu-C2 constructs, Pauline Scholler for her help in performing experiments, NEEEKSRLLEKENRELEKIIAEKEERVSELRHQLQSRQQLKKTN and SANLAAATST- Dr. Greg Stewart for English editing, and the ARPEGE (Pharmacology SVTSVNQASTSRLEGLQSENHRLRMKITELDKDLEEVTMQLQDTPEKKTN, respec- Screening-Interactome) platform facility at the Institut de Génomique Fonc- tively (the residue Leu846 of mGlu2 is underlined). tionnelle (Montpellier, France). P.R. and J-P.P. were supported by the Centre fi HEK-293 and COS-7 cells were cultured in DMEM supplemented with 10% National de la Recherche Scienti que (CNRS), the Institut National de la FBS and transfected by electroporation as described elsewhere (37). Ten Santé et de la Recherche Médicale (INSERM), and by Grants from the Agence Nationale de la Recherche [GPCR dimers (ANR-BLAN06-3_135092) and mGlu- millions cells were transfected with 2 μg of each plasmid of interest and μ Patho (ANR-08-NEUR-006-02) in the frame of ERA-Net NEURON] and by an completed to a total amount of 10 g with the plasmid encoding the pRK5 unrestricted grant from Senomyx. J.L. was supported by the National Basic fi empty vector. To allow ef cient coupling of the receptor to the phospho- Research Program of China (Grant 2007CB914200), the National Natural lipase C pathway, cells were also transfected with the chimeric G protein Science Foundation of China (Grant 30973514), and the Program of Intro- Gαqi9 (2 μg). ducing Talents of Discipline to Universities of Ministry of Education (B08029).

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