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Illuminating the activation mechanisms and allostericproperties of metabotropic glutamate receptorsEtienne Doumazanea,b,c,1, Pauline Schollera,b,c,d,1, Ludovic Fabrea,b,c,d, Jurriaan M. Zwierd, Eric Trinquetd,Jean-Philippe Pina,b,c,2, and Philippe Rondarda,b,c,2

aCentre National de la Recherche Scientifique, Unit Mixte de Recherche 5203, Institut de Gnomique Fonctionnelle, F-34000 Montpellier, France; bInstitut

National de la Sant et de la Recherche Mdicale U661, F-34000 Montpellier, France; cUniversits de Montpellier 1 and 2, F-34000 Montpellier, France;and dCisbio Bioassays, F-30200 Codolet, France

Edited* by Jean-Pierre Changeux, Institut Pasteur, Paris Cedex 15, France, and approved February 5, 2013 (received for review September 19, 2012)

In multimeric cell-surface receptors, the conformational changes of

the extracellular ligand-binding domains (ECDs) associated with

receptor activation remain largely unknown. This is the case for

the dimeric metabotropic glutamate receptors even though a num-

ber of ECD structures have been solved. Here, using an innovativeapproach based on cell-surface labeling and FRET, we demonstrate

that a reorientation of the ECDs is associated with receptor and

G-protein activation. Our approach helps identify partial agonists

and highlights allosteric interactions between the effector and

binding domains. Any approach expected to stabilize the active

conformation of the effector domain increased the agonist potency

in stabilizing the active ECDs conformation. These data provide keyinformation on the structural dynamics and drug action at metab-

otropic glutamate receptors and validate an approach for tackling

such analysis on other receptors.

allostery | biosensor | snap-tag | time-resolved FRET

Many cell-surface receptors are multimers of proteins com-posed of several domains (13), including extracellulardomains (ECDs) involved in endogenous ligand recognition andtransmembrane domains (TMDs) responsible for intracellularsignal transduction. Analysis of the conformational changes of theECDs associated with receptor activation is crucial to understandthe detailed mechanism involved in receptor activation and for

the development of new innovative drugs. However, limited in-formation is available on how the conformational changes inthese proteins lead to receptor activation, especially in living cells.

The eight glutamate-activated G-proteincoupled receptors(GPCRs), called metabotropic glutamate receptors (mGluRs),are key examples of multidomain and multimeric receptors (Fig.1A). These receptors are strict dimers (46), and each subunitis made of a large ECD associated with a seven-helix TMD re-sponsible for G-protein activation and downstream signaling (7).The mGluRs are key elements involved in the regulation ofsynaptic activity (8), and therefore they represent promisingtargets in drug development for the treatment of multiple neu-rologic and psychiatric diseases (9). More generally, the mGluRsare part of the class C GPCR family that contains structurally

related receptors such as the receptors for sweet and umami taste,calcium, basic amino acids, and the inhibitory neurotransmitterGABA (10, 11).

Crystallographic studies of the isolated dimeric ECDs andmutagenesis analyses have provided a clear view of the structureof the dimeric ECD and of the initial steps of mGluR activation.The ECD is composed of a Venus flytrap (VFT) bilobate domaincontaining the agonist binding site (1215) and a cysteine-richdomain (CRD) that connects the VFT to the TMD (16). TheVFTs exist in two major states: an open state (o) in absence ofligand and stabilized by antagonists, and a closed state (c) sta-bilized by agonists and required for receptor activation (1215).The VFT dimer is in equilibrium between various conformations,depending on whether one or two VFTs are open or closed. Inthe absence of ligand or in the presence of antagonists, both

VFTs are opened (oo). The binding of one or two agoniststriggers the closure of one (co) or two VFTs (cc) in the dimer,resulting in receptor activation (13).

However, how the closure of at least one VFT affects theconformational state of the TMD during receptor activationremains controversial. Based on the initial X-ray structures (7,1416), the predominant model proposes that the closure of theVFTs in the dimer is associated with a large change in the rel-ative orientation of the two VFTs, from a resting (R) to an active(A) orientation defining three major states: Roo, Aco, and Acc(Fig. 1C). The major consequence of this reorientation is that thesecond lobes of the VFTs, which are distant in the R state, be-come closer in the A conformation, stabilizing the active state ofthe TMDs. However, as shown in Fig. 1C, if we consider allstructures deposited in the Protein Data Bank (PDB) (SI Ap-pendix, Table 1), no correlation emerges between the specificconformations of the dimeric VFTs and the nature (i.e., agonistor antagonist) of the cocrystallized ligand. Indeed, agonist-boundVFTs can be found in the R orientation, and the A orientationalso has been observed with bound antagonists (Fig. 1C).

To understand mGluR dynamics better and to resolve thisapparent paradox, we examine here the conformational changesof the ECDs associated with the activation of mGluRs at thesurface of living cells. To do so, we used a combination of in-novative technologies enabling the exclusive labeling of cell-sur-

face receptors withfl

uorophores compatible with time-resolvedFrster (fluorescence-based) resonance energy transfer (trFRET)measurements. With this approach, we clearly demonstrate thata large movement between the ECDs is associated with receptorand G-protein activation. Our approach also allows the detectionof partial agonists and reveals precise allosteric interactions be-tween the ECD and TMD of the mGluRs. Finally, it supplies im-portant information on the mode of action of allosteric regulators,either small molecules or associated proteins such as G proteins.

Results

Engineering of mGluRs to Analyze Conformational Changes of the

Dimeric ECDs. To study the conformational changes of theECDs in the context of the full-length mGluRs, we developeda trFRET-based strategy. We fused the 20-kDa SNAP-tag en-

zyme which can be covalently labeled with fluorophores (17) to

Author contributions: E.D., P.S., L.F., J.-P.P., and P.R. designed research; E.D., P.S., and L.F.

performed research; E.D., P.S., L.F., J.M.Z., E.T., J.-P.P., and P.R. analyzed data; and E.D.,

P.S., J.-P.P., and P.R. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

See Commentary on page 5742.

1E.D. and P.S. contributed equally to this work.

2To whom correspondence may be addressed. E-mail: [email protected] or

[email protected].

See Author Summary on page 5754 (volume 110, number 15).

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

1073/pnas.1215615110/-/DCSupplemental.

E1416E1425 | PNAS | Published online March 4, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1215615110
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the N-terminal end of the receptor. This strategy has a number of

advantages. First, because mGluRs are strict homodimers (46),the FRET signal results exclusively from the energy transferbetween the two protomers (5). Second, according to the avail-able crystal structures of mGlu ECDs (14, 15), the distance be-tween the N termini varies from 2.8 nm in the R conformationto 3.3 nm in the A conformation, a variation compatible withchanges in FRET efficiency (Fig. 1C). Thus, we hypothesizedthat if the N termini are labeled with FRET-compatible probes,the R-to-A conformation switch may result in a decrease in theFRET efficiency. Third, because of its accessibility, the labelingof the N termini of the mGluR does not interfere with the re-ceptor function as measured by G-protein coupling (5). Fourth,and most importantly, the SNAP-tag enzyme can be labeled se-lectively and irreversibly using cell-impermeant benzylguanine-

derived fluorescent substrates allowing a specific labeling of cell-surface receptors only (5, 6, 17). This latter approach preventsany FRET signal originating from intracellular receptors thatcannot be activated by agonists.

We expressed SNAP-mGlu2 in HEK293 cells and labeled themwith a trFRET donor, SNAP-Lumi4-Tb, and a green fluorescentacceptor, SNAP-Green (Fig. 2A). Concentrations were chosen tolabel surface subunits with both fluorophores equivalently. Con-sequently, half of all mGlu2 homodimers at the surface carry one

donor and one acceptor molecule (5). We then recorded thetrFRET signal as the Green-labeled acceptor delayed emission at520 nm following Lumi4-Tb excitation at 337 nm.

Glutamate Induces a Relative Movement of the ECDs. We observedthat glutamate application resulted in a large decrease in theFRET signal (Fig. 2B), a variation that was independent of thenumber of cell-surface receptors (SI Appendix, Fig. S1). Impor-tantly, a competitive antagonist, LY341495, completely restoredthe emission signal, ruling out any nonspecific effect of glutamateon the FRET efficiency. Of note, the FRET signal recorded afterantagonist application is higher than that recorded in the controlcondition, suggesting either a slight constitutive activity of thereceptor or ambient glutamate in the assay medium. Eventually,no such agonist-mediated FRET change was observed with a mu-tant mGlu2 insensitive to glutamate, mGlu2-YADA (Fig. 2C)(18). Finally, agonist application is associated with an increasein lifetime of the donor fluorophore, consistent with a decrease inFRET efficiency (SI Appendix, Fig. S2).

Three main points are consistent with the decrease in FRETefficiency originating from a relative movement of the ECDs(Fig. 2B). First, the kinetics of the change is very rapid (t1/2 lessthan 1 s), a characteristic that suggests a conformational changerather than the recruitment or the dissociation of a partnersubunit or even the internalization of the receptor (19). Second,

with the rare earth cryptate Lumi4-Tb, FRET efficiency dependsalmost exclusively on the distance between the two fluorophoresand not on the orientation (20). Third, as expected from an energytransfer, the lifetime of the donor is decreased significantly in the

presence of the acceptor under the control condition but to a lesserextent in the presence of agonist, consistent with a lower FRETefficiency when the receptor is activated (SI Appendix, Fig. S2).

Taken together, our data show that the decrease in FRETefficiency induced by glutamate and reversed by a competitiveantagonist likely reflects a reorientation of the dimeric ECDs.

Intramolecular FRET Is Linked to Receptor Activation. To prove thatthe change in FRET is related to the activation process ofmGluRs, we tested four different selective and nonselective mGlu2agonists on our FRET sensor (Fig. 3A). They all consistently in-duced a decrease in the FRET efficiency in a dose-dependentmanner with potencies (fitted EC50) in the range of those mea-sured using cellular functional assays based on the coupling ofmGlu2 to the phospholipase C signaling pathway through the use

of the chimeric G protein Gqi9 (Fig. 3B). The EC50 measured inthe inositol phosphate (IP) accumulation assay and in the calciummobilization assay correlated strongly with those determined inthe FRET assay (Fig. 3B). Of note, the EC50 values for IP accu-mulation and calcium assays are lower (reflecting higher potency)than those obtained using the FRET sensor, showing that higherconcentrationsof agonist areneeded to elicita full FRET response.However, theKivalues for the FRET assay perfectly matched thoseobtained in radioactive ligand-binding experiments (Fig. 3C).Eventually, the agonist-mediated change in FRET is fully reversedby a series of antagonists with Ki values consistent with thosereported in the literature (SI Appendix, Fig. S3) (21).

Interestingly, (2S,2R,3R)-2-(2,3-Dicarboxycyclopropyl)glycine(DCG-IV) and(2S,1S,2S)-2-(Carboxycyclopropyl)glycine(LCCG-I)lead to a partial reduction in FRET efficiency (Fig. 3A), suggesting

Fig. 1. Structural organization of the mGluRs and crystal structures of the

dimeric ECDs. (A) Cartoon illustrating the full-length dimeric mGluRs in

which each subunit is composed of an ECD made of a bilobate VFT and a CRD

that is linked directly to the TMD. The glutamate-binding pocket is between

the first and second lobes of the VFTs. (B) Crystal structures of the ECD of

mGlu1, 3, 5, and 7 (see PDB numbers in C) obtained in presence or absence

of the CRD and in complex with agonists and competitive antagonists. ( C) In

the different structures (7, 1416), the dimeric ECDs were observed in resting(R) and active (A) orientation whether they were bound to agonist (green) or

to antagonist (red), raising the question of the true inactive and active ori-

entations of the ECD. In these structures, the VFTs are in open (o) or closed

(c) state, thus defining the major states of the ECDs: Roo, Rcc, Aoo, Aco, and

Acc. In the dimer, the VFT in front is represented in dark blue, and the

second one in the back is shown in purple. The main difference between the

inactive and active states is that the second lobes are distant in the inactive

state and closed in the active state. Only one structure representative of

each conformation and corresponding to the underlined PDB number is

shown with two molecules of agonists or antagonists. Double-headed

arrows indicate the distances between the two N termini. The C of the first

N terminus residue in the crystal structures is highlighted in orange.

Doumazane et al. PNAS | Published online March 4, 2013 | E1417

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partial agonist activities. This reduction was confirmed in a func-tional calcium assay by testing the effects of maximal doses ofDCG-IV and LCCG-I on cells transfected with low amounts ofreceptor plasmid DNA to avoid saturation of the response andreceptor reserve (Fig. 3D). In addition, as expected for a partialagonist, DCG-IV partly inhibited the maximal effect of glutamate(Fig. 3E).

FRET Change Is Associated with ECDs Reorientation and Receptor

Activation. Two sets of data demonstrate that the FRETchange is correlated with a reorientation of the ECDs and theactivation of the receptor. First, we locked the R conformationby introducing a glycan wedge expected to prevent the associa-tion of the second lobes (Fig. 4A), a strategy that has been used

previously to block the GABAB receptor in its inactive confor-mation by steric hindrance generated by the engineered glyco-sylation (22). We introduced an NXS motif, which is recognizedby the endoplasmic reticulum machinery as being modified withN-linked glycosylation, at residue 515, at the level of the CRDdimer interface (23). In the absence of ligand, we found that theFRET efficiency measured with this mutant is similar to thatmeasured on the wild-type receptor but that the effects of gluta-mate or the most potent agonist LY379268 on the FRET effi-ciency are strongly impaired (Fig. 4A). Accordingly, the activationof this mutant measured in the IP assay is strongly impaired (Fig.4A). Second, we used a mutant (L521C) that is locked in anactive conformation by an engineered disulfide bond that cross-links the two CRDs (23). We found that the FRET signal in-tensity of the mutant L521C is similar to that measured on the

wild-type receptor in the presence of saturating concentrationsof agonist (Fig. 4B). Interestingly, the competitive antagonistLY341495 significantly increased the FRET efficiency of theconstitutively active mutant L521C (Fig. 4B) without reachingthe level of the inactive receptor and without specificallychanging the basal activity of this mutant receptor.

In conclusion, these data indicate that a relative movement ofthe ECDs, especially at the level of the second lobes, is bothnecessary and sufficient to control the activation of the TMDs byorthosteric ligands. They also are consistent with the initialstructural observation of an R-to-A conformational change ofthe dimeric VFT being associated with receptor activation.

ECDs Movement in Other mGlu Receptors. As shown in Fig. 1, the

structures that do not fit with the R-to-A model for receptoractivation are those of the mGlu1 VFTs bound to the antagonistLY341495 (PDB 3KS9), which are found in a relative A orien-tation, and those of the mGlu3 VFTs occupied by five differentagonists, including glutamate and DCG-IV, which are all in theR orientation. Thus it is possible that our observations withmGlu2, may not apply to either mGlu1 or mGlu3 receptors.However, all three SNAP-tagged mGlu1, mGlu2, and mGlu3receptors show a high FRET efficiency in their inactive confor-mation when bound to the antagonist LY341495 and a lowFRET efficiency in the presence of agonist (Fig. 2C and SI Ap-pendix, Fig. S4). For mGlu1, the low FRET efficiency measuredin the presence of glutamate is dose-dependently reversed inhigh values by increasing LY341495 concentrations (Fig. 2D).For mGlu3, all four agonists observed in the crystal structure in

Fig. 2. Glutamate induces a relative movement of the ECDs in the full-length mGluRs. (A) Cartoon illustrating the full-length SNAP-mGlu2 (ST-mGlu2) labeled

with the Lumi4-Tb donor and the Green fluorescent acceptor, with a high FRET signal in the absence of ligand or in presence of the antagonist LY341495

(LY34) and a low FRET signal in the presence of glutamate or the agonist LY379268 (LY37). ( B) trFRET signal between the two ECDs after cell-surface labeling

of SNAP-mGlu2expressing cells with SNAP-Lumi4-Tb and SNAP-Green in the presence of a saturating concentration of glutamate (injection at t = 30 s) and

then in the presence of the competitive antagonist LY341495 (injection at t = 60 s). The trFRET signal is represented as the ratio of the acceptor-sensitized

emissions measured in two time windows (Materials and Methods) and is normalized to the maximum signal obtained in the presence of a saturating

concentration of antagonist. (C) trFRET signal of wild-type SNAP-mGlu2, the ligand-bindingdeficient SNAP-mGlu2-YADA mutant in which the two residues

Y216 and D295 important for agonist binding in the VFT are mutated to alanine, and wild-type SNAP-mGlu1 and SNAP-mGlu3. All receptors were labeled

with Lumi4-Tb and Green in the presence of a saturating concentration of competitive antagonist (LY34) or agonist (LY37). (D and E) Intramolecular trFRET

signal measured on donor- and acceptor-labeled SNAP-mGlu1 and SNAP-mGlu3 in the presence of the indicated antagonists or agonist found in the crystal

structures of mGlu1 and mGlu3.

E1418 | www.pnas.org/cgi/doi/10.1073/pnas.1215615110 Doumazane et al.
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the resting state glutamate; 1S,3R-ACPD; 2R,4R-APDC; and

DCG-IVdecrease the FRET efficiency, as does glutamate onmGlu2 and mGlu1 (Fig. 2E), with potencies similar to thosemeasured with other assays on this receptor (21). Of interest, andas observed with mGlu2, DCG-IV displays a partial effect. Takentogether, our data are consistent with a similar reorientation ofthe ECDs of all three mGluRs tested (mGlu1, mGlu2, andmGlu3) during receptor activation, from the R orientation in theinactive state to the A orientation in the active state.

Allosteric Control of the Relative Movement of the ECDs by the TMD.

If the movement of the ECDs is critical for the allosteric controlof the conformational state of the TMD, then such coupling alsoshould occur in the other direction, from the TMD to the ECD.In other words, stabilizing the active or inactive state of the TMD

should affect the dynamics of the ECD movement. In mGluRs,synthetic negative and positive allosteric modulators (NAMs andPAMs, respectively) interacting in the TMD have been identified(Fig. 5A). NAMs act as noncompetitive inhibitors and often dis-play inverse agonist activity (24, 25), whereas PAMs enhance thepotency or/and efficacy of agonists (26) but have no or weak directagonist activity when applied alone in the absence of agonist.

We examined the effect of both the mGlu2 NAM Ro64-5229and the mGlu2 PAM LY487379 on the agonist-induced FRETchange (Fig. 5A). As illustrated in Fig. 5 B and C, the NAMslightly decreased the agonist efficacies and slightly reduced ag-onist potencies; meanwhile the PAM enhanced both agonistefficacies and potencies. Of note, the partial agonists DCG-IVand LCCG-I become full agonists in the presence of PAM.Consistent with the absence of agonist activity of the PAM in the

concentration range tested, no significant effect of the PAM applied

alone was observed in the FRET assay (SI Appendix, Fig. S5).This finding further supports the proposal that, in the absenceof agonist, the inactive ECD dimer prevents this mGlu2 PAMfrom activating the receptor.

We also examined whether the TMD per se had some in-fluence on the conformational dynamics of the ECD dimer byexpressing the mGlu2 ECD or the mGlu2 VFT attached to theplasma membrane through a glycosyl phosphatidylinositol (GPI)anchor (Fig. 5D) (27). Interestingly, such constructs behave likethe full-length mGlu2 with almost the same basal and agonist-induced FRET efficiency and with the same agonist potencies.However, as expected, neither the PAM nor the NAM had anyinfluence on the effect of agonists on these truncated receptorslacking the TMD.

These data reveal that theactive conformationof theECD dimeris stabilized further by compounds stabilizing the active form of theTMD and that the TMD per se is not required for the agonist toinduce or stabilize the A conformation of the ECD dimer.

Mutations in the TMD Control the Conformation of the Dimeric ECD.

A number of mutations in the TMD of class C GPCRs whichresult in either a gain or a loss of function have been reported,either through site-directed mutagenesis (28) or through theanalysis of human gene sequences (2932). For example, bothgain- and loss-of-function mutations identified in the calcium-sensing receptor gene are responsible for autosomal dominanthypocalcemia and familial hypocalciuric hypercalcemia, respec-tively (33). We examined whether similar mutations introducedin the mGlu2 TMD can affect the dynamics of the ECD dimer.

Fig. 3. The relative movement of the ECDs is correlated with receptor function. (A) Intramolecular trFRET signal measured on donor- and acceptor-labeled

SNAP-mGlu2 in the presence of increasing concentrations of the indicated selective (LY35 and LY37) and nonselective group II mGluRs agonists (glutamate,

LCCG-I, and DCG-IV). (B) Correlation between the potency of the agonists determined by IP accumulation and their potency in intramolecular trFRET and in

calcium mobilization assays. (C) Correlation between the Ki of the agonists determined in intramolecular trFRET in the presence of 3 nM LY34 and the Kidetermined in a competitive radioligand-binding study using 3 nM of the tritiated LY34. (D) Response in the calcium mobilization assay with the indicated

agonists (10 M LY37 and 100 M LY35, DCG-IV, and LCCG-I) for the quantities of SNAP-mGlu2 at the cell surface measured by the fluorescence emission of

the Lumi4-Tb bound to the receptor. (E) Intramolecular trFRET signal for labeled SNAP-mGlu2 with increasing concentrations of the partial agonist DCG-IV in

the presence or absence of 100 M glutamate.


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We first examined the effect of two mutations in the mGlu2TMD (Q679V and C770A), equivalent to activating mutationsidentified in the mGlu8 receptor (Fig. 6A) (34, 35). Both mutantsdisplay a small but significant basal activity and higher agonistpotency in the IP assay (Fig. 6B, Upper). In the ECD FRET assay(Fig. 6B, Middle) neither mutant displays strong basal activity,but both display higher agonist potency. Consistent with thiseffect (possibly resulting from a better stabilization of the activeconformation of the TMD by the mutations), the addition ofa NAM restores a wild-type potency for agonists (Fig. 6B, Mid-dle). In addition, and as observed with the PAM on the wild-typereceptor, both mutations converted the partial agonist LCCG-I

into a full agonist (Fig. 6B

,Lower

), again as is consistent with theproposal that these mutations further stabilize an active con-formation of the TMD upon agonist activation.

We also examined the effect of two loss-of-function mutationsof the highly conserved residue Phe756 located in the third in-tracellular loop of class C GPCRs (Fig. 6B) (36, 37). The F756Dmutant behaves like the wild type in the ECD FRET assay foragonist potency and efficacy measured in the absence and in thepresence of either a PAM or a NAM, even though a complete lossof coupling to the G protein was observed (Fig. 6D, Upper).Surprisingly, the F756P mutant, which also prevents G-proteincoupling, displays increased agonist potency in affecting the ECDFRET sensor (Fig. 6D, Middle), an effect not significantly po-tentiated by a PAM (SI Appendix, Fig. S6) but largely inhibited bya NAM (Fig. 6D, Middle). Moreover, as observed with the wild-

type receptor in the presence of a PAM, the efficacy of the partialagonist LCCG-I is increased greatly to reach that of a full agonist(Fig. 6D, Lower). These data are consistent with the constraintsbrought by the Pro residue stabilizing the overall TMD in anactive-like conformation but at the same time preventingG-protein interaction and activation.

G Protein Controls the Conformation of the Dimeric ECD. Manyproteins are known to interact directly with mGluRs for cellsignaling or for modulating receptor activity (38). We analyzedthe influence of the Go protein coupling on the conformationof the ECDs of mGlu2 (Fig. 7A). To improve the couplingbetween Go and mGlu2, we used the Go mutant G203T known

to have a low affinity for both GDP and GTP and resulting in astabilization of the G empty state that displays dominant neg-ative properties (39). The coexpression of this G-protein mu-tant alone (SI Appendix, Fig. S7) or in combination with thesubunit G22 (Fig. 7B) enhanced the potency of agonists forSNAP-mGlu2, showing that the G-protein coupling increasesagonist potency.

Consistent with the mutation F756P preventing G-proteincoupling when already in an active-like conformation, the agonistpotency on the ECD FRET-based assay is similar to that ob-served with the wild-type receptor coexpressed with the activeform of the G protein (Go G203T), whereas the coexpression ofthis mutant G protein had no effect on agonist potency on theF756P mutant (Fig. 7C). In contrast, the agonist potency of theother loss-of-function mutant, F756D, in the ECD FRET-based

Fig. 4. The relative movement of the ECDs is necessary and sufficient for mGlu2 activation. Intramolecular trFRET signal (Upper) and IP accumulation (Lower)

measured for the mGlu2 receptor mutant 515NGlyc blocked in the resting conformation (A) and the mutant L521C locked in the constitutively active state ( B)

in transfected cells incubated with the agonist LY37 or the competitive antagonist LY34 and in the presence of the chimeric Gqi9 protein.

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assay is similar to that of the wild type. As is consistent with itsinability to couple to G protein, the addition of Go G203T hadno effect (Fig. 7C).

Discussion

The present study reports important information on the activa-tion process of the dimeric mGluRs, and solves an issue not yetclarified through X-ray crystallography studies. Our approachthat allowed the efficient analysis of cell-surface conformationalchanges by FRET showed that a large change in conformation atthe level of the ECD dimer is intimately linked with mGluRs andG-protein activation, led to the identification of partial agonist,and revealed the allosteric control of the ECD dynamics by the

TMD. This finding clarifies our view on the activation mecha-nism of mGluRs and its regulation by G proteins and helps inunderstanding the allosteric communication among the differentdomains of these dimeric receptors.

The agonist-induced decrease in intramolecular FRET effi-ciency is caused by a large change in the distance that separatesboth fluorophores because trFRET is barely sensitive to therelative fluorophore dipole orientation (20). Such movement is

well related to receptor activation because (i) agonist potenciescorrelate with those measured using cell-based functional assays;(ii) mutations expected to prevent ECD movement preventedboth agonist-induced FRET change and receptor activation; and(iii) locking the receptor in its active state resulted in agonist-like

Fig. 5. The relative movement of the ECDs is controlled allosterically by the TMD. (A) Cartoon illustrating that the PAM stabilizes the active conformation of

mGlu2 in presence of the agonist (Left), and the NAM stabilizes the resting conformation (Right). (B) Intramolecular trFRET signal measured for the indicated

agonists in the presence of either 1 M of the PAM LY487379 or 1 M of the NAM Ro64-5229. (C) Potency (Left) and efficacy (Right) of the indicated agonists

in absence or presence of the NAM or PAM in the conditions described in A. Data in ACare mean SEM of three individual experiments, each performed in

triplicate. *P< 0.05, one-way ANOVA followed by a Bonferronis multiple comparison test. (D) Intramolecular trFRET signal measured for the isolated mGlu2

ECD (SNAP-mGlu2-499GPI) at the surface of living cells.


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trFRET efficiency. These data are consistent with the confor-mational change from resting R to active A orientation resultingfrom glutamate binding, as initially proposed based on the firstmGlu1 VFT dimer structures (40). Our finding that similarchanges in trFRET are observed with the full-length mGlu1,mGlu2, and mGlu3 receptors upon agonist binding indicate

that a similar conformational change occurs in all mGluRs uponactivation. Of note, the same change is observed with VFTsinserted in the plasma membrane with a GPI anchor. Two pos-sibilities could explain the two nonpreferential conformationsobserved in crystals, i.e., the Rcc stabilized by an agonist andthe Aoo in presence of antagonist for mGlu3 and mGlu1, re-spectively (Fig. 1C). These conformations could result fromconstraints in the packing mode of the proteins in the crystal thatstabilize a less stable conformation of the mGluRs. Alternately,they could result from a lack of constraints in the absence of theplasma membrane attachment of the VFTs.

Our FRET data also provide important information on howthe ECDs reorientation is related to the efficacy of the differentagonists. We found that the well-known group II mGluR ago-nists DCG-IV and LCCG-I display a lower efficacy than the

other agonists tested in the intramolecular FRET assay. Ouradditional functional studies confirmed that these drugs indeedare partial agonists. Partial activity usually is difficult to detectfor drugs that still have a high efficacy close to that of a fullagonist, as observed with DCG-IV and LCCG-I, especially withreceptors overexpressed in cell lines and when measuring am-

plified GPCR-mediated responses. This difficulty highlights thepower of our trFRET-based sensor for ligand characterization.Such a partial agonist action may be explained by a partial VFTclosure and then a partial reorientation of the ECDs. Alterna-tively, partial agonism may be caused by a lower stability of theclosed state of the VFTs so that only a fraction of the agonist-occupied receptors reach the A orientation. Because the mGlu3VFT structure bound to the partial agonist DCG-IV (Fig. 2E)

was found in a fully closed state, as observed with the full ago-nists, it is likely that the highly homologous mGlu2 VFT also canreach a fully closed state upon DCG-IV binding. Accordingly, thepartial DCG-IV and LCCG-I activities likely result from a lowerstability of the fully closed state. Further experiments analyzingsingle molecules may be needed to confirm this proposal.

Fig. 6. Mutations in TMD can influence the relative movement of the ECDs. (A) Cartoon illustrating the TMD punctual mutations equivalent to activating

mutations in mGlu8. (B) IP accumulation (Upper) and intramolecular trFRET for the wild type and the indicated SNAP-mGlu2 constructs for the full agonist in

presence or absence of 10 M of the NAM Ro64-5229 (Middle) or for the partial agonist (Lower). (C) Cartoon to illustrate the mutations in the third in-

tracellular loop at the residue F756 of mGlu2. (D) IP accumulation (Upper) and intramolecular trFRET for the wild type and the indicated SNAP-mGlu2

constructs for the full agonist in presence or absence of 10 M Ro64-5229 (Middle) or for the partial agonist (Lower).

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Our data also bring information on the structural dynamics ofthe ECD when the receptor is blocked in the active conforma-tion. Interestingly, the antagonist LY341495 does increase theintramolecular FRET efficiency in the receptor locked in an

active state through an engineered intersubunit disulfide bridge.Such a change in FRET is not associated with a decrease inreceptor activity, indicating that even though the receptor islocked in the active state at the level of the CRD and TMD,some flexibility still remains at the level of the VFTs. BecauseLY341495 is not accepted in a closed VFT, it is likely that eventhough the VFT dimer is maintained in the A orientation,binding of LY341495 forces both VFTs to reach an open con-formation, as observed in the mGlu1 ECD structure with boundLY341495 (PDB 3KS9; Fig. 1C). Accordingly, the increase inFRET efficiency measured with LY341495 may result froma change from the Acc state to an Aoo state and suggests thatboth VFTs are in the closed form (Acc) in this constitutivelyactive mutant. This proposal is supported by the low potency of

LY341495 in inducing a FRET change, 200 times lower than itspotency as an antagonist on the wild-type receptor.

Our results also reveal a clear allosteric communication be-tween the TMD and the ECD, a molecular mechanism crucial

for the functioning of the mGluRs. Indeed, stabilization of theTMD in an active conformation, using either a PAM or in-troducing mutations, influences the ECD dimer dynamics, as il-lustrated by the increased potency of agonists in the intramolecularFRET assay. However, in the absence of agonist neither thesemutations nor PAM application leads to activation of the re-ceptor as measured through the intracellular functional assayand does not induce a change in the VFT dimer conformation asrevealed by the absence of FRET change. This result confirmsthat the receptor activation is dependent on the conformationalchange of the ECD dimer and highlights the important energybarrier brought by the ECD that limits the ability of the dimericTMD to be fully activated (41, 42).

NAMs that are known to stabilize the TMD in the inactivestate only slightly decrease agonist potency in changing the ECD

Fig. 7. G protein controls the conformation of the ECDs. (A) Cartoon illustrating the key intermediate of the GDP-to-GTP exchange, the agonist-receptor-

Gempty complex, in which the agonist displays a reduced dissociation rate and therefore a higher apparent affinity. This intermediate state is mimicked by a G

mutant, Go (G203T) that has a low affinity for GDP and GTP. (B) Intramolecular trFRET signal on donor- and acceptor-labeled SNAP-mGlu2 in the presence of

the Go mutant cotransfected with G22. (C) Intramolecular trFRET signal for the constructs SNAP-mGlu2 mutated in the third intracellular loop in the

presence of the wild type or the mutated Go(G203T) protein cotransfected with G22.


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dimer orientation. Thus the A orientation of the VFT dimer canbe obtained even when both TMDs are in the inactive state. Thisfinding is consistent with our recent observation on the mecha-nism of the TMDs activation in which a relative movement ofthe TMDs occurs first, followed by a change in conformation inthe TMD (43).

Our study also revealed that loss-of-function mutations can havedifferent phenotypes, as illustrated by the F756P and F756Dmutations in the third intracellular loop. Although both mutants

are unable to activate G proteins, the F756P mutant appears tolower the energy barrier to reach an active-like state, increasingagonist potency in the ECD FRET-based assay, increasing partialagonist efficacy and being insensitive to the PAM while returning toan inactive-like behavior in the presence of a NAM.

Our experimental system also is able to detect the conforma-tional change of the ECDs induced by interacting proteins suchas G proteins. Indeed, the G protein in its empty form is wellknown to stabilize the high agonist affinity state of GPCRs andthen the active state of the receptor (44). Consistent with thismodel, we found that overexpressing the Go dominant negativemutant that displays a low affinity for both GDP and GTP hasa direct effect on the ECD dimer dynamics and, indeed, increasesagonist potency. The effect observed is consistent with the smalldecrease in agonist-binding affinity upon GTPS treatmentreported for many mGlu receptors (45).

When comparing agonist potencies in the intramolecularFRET assay and in the IP accumulation and calcium releasefunctional assays, we found that potencies are 310 times higher(lower EC50s) in the functional assays based on signaling. Oneshould note that, because the FRET-based assay is intimatelylinked to the conformational change of the receptor, potenciesmeasured in such an assay are very close to the binding affinity ofthe agonist. In contrast, signal amplification resulting from themeasurement of downstream signaling events likely increasesagonist potencies. One also may consider another, not exclusivepossibility. Because of the increase in agonist affinity resultingfrom the formation of the receptorG protein (RG) complexthat likely corresponds to the real active form of the complex, it

is likely that the agonist potency measured in any functionalassay is related to the agonist affinity on this RG complex. In-stead, the intramolecular FRET assay measured agonist affinityon all cell-surface receptors, only a fraction of which are in theactive RG state, and found a lower apparent affinity than on anyother functional assays.

Finally, our experimental approach demonstrates an efficientand robust tool to analyze the effect of ligands on the mGluRs atthe surface of living cells. Both full-length receptors and isolatedVFTs can be analyzed to define whether the ligand binds to theECD or the TMD of the mGluRs. Our approach, which is easy touse and allows intramolecular FRET measurements in microtiterplates, likely will be useful in studying the activation process ofother cell-surface multimeric complexes. It may be developed asa reliable assay for drugs screening.

Taken together, our data bring clear evidence that a change inthe relative orientation of the mGlu ECDs is a key step in re-ceptor activation. Consistent with such a movement controllingthe active state of the receptor, we found that stabilizing theTMD in an active state further stabilizes the ECD dimer in itsactive orientation, as illustrated by the large increase in agonistpotency. These data provide key information on the structuraldynamics and drug action at mGluRs and validate using such anapproach to perform such analysis on other receptors.

Materials and MethodsMaterials. Glutamate was purchased from Sigma-Aldrich. LY379268, LY354740,

and LY341495 were from Abcam Biochemicals; DCG-IV, LY487379, Ro64-5229,

and (2S)--Ethylglutamic acid (EGLU) were from Tocris Bioscience; and [ 3H]-

LY341495 was from the American Radiolabeled Chemicals. Lipofectamine

2000 and Fluo4-AM were from Life Technologies. SNAP-Lumi4-Tb, SNAP-

Green, and Tag-Lite buffer were from Cisbio Bioassays.

Plasmids and Transfection. pRK5 plasmids encoding the HA-SNAPtagged

wild-type mGlu1, 2, and 3 from rat and L521C mutant mGlu2 were de-

scribed previously (5, 23). The single mutations in the pRK5 plasmid

encoding rat Go protein and HA-SNAPtagged mGlu2 and the 499GPI

construct were generated using QuikChange mutagenesis protocol (Agi-

lent Technologies) or were obtained from synthetic cDNA from Genecust.

HEK293 cells were cultured in DMEM (Life Technologies) supplemented

with 10% (vol/vol) fetal calf serum (Lonza). Cells were transfected byelectroporation or by the reverse Lipofectamine 2000 protocol as pre-

viously described (5). To maintain a low concentration of ambient gluta-

mate, cells were cotransfected with the cDNA of the glutamate

transporter EAAC1 and incubated in DMEM Glutamax medium (Life

Technologies) at least 2 h before the different assays were performed.

IP and Intracellular Calcium Measurements. Measurement of IP accumulation

in HEK293 cells was determined using the IP-One HTRF kit (CisBio Bioassays)

according to the manufacturers recommendations, and the calcium mobi-

lization signal was determined in HEK293 cells with a calcium-sensitive

fluorescent dye (Fluo-4, Life Technologies), as previously described (46).

Cell-Surface Quantification and Ligand-Binding Assay. Amounts of SNAP-

tagged constructs at the cell surface were quantified as previously described

(5). HEK293 cells were incubated at 37 C for 1 h with a solution of 300 nM

SNAP-Lumi4-Tb and were washed three times with tag-Lite buffer. Afterexcitation with a laser at 337 nm, the fluorescence of the Lumi4-Tb was

collected at 620 nm for 450 s after a 50-s delay on a PHERAstar FS

(BMG Labtech).

For the ligand-binding assay, intact HEK293 cells in 10-cm dishes were

collected 24 h after transfection and incubated with 3 nM [ 3H]-LY341495

(corresponding to the Kd of this radioligand) and various concentrations of

cold ligands for 1 h at room temperature. Cells then were collected on

filters and washed three times with a solution of 0.9% NaCl. Filters were

placed in tubes containing a liquid scintillation mixture solution, and ra-

dioactivity was measured with a Packard beta counter (Perkin Elmer).

Unspecific binding was determined in presence of 10 M LY379268. Kivalues were calculated according to the following equation: Ki = IC50/(1 +

[RL]/Kd), where [RL] and Kd are the concentration and dissociation constant

of the radioligand, respectively.

FRET Measurements. For labeling, 24 h after transfection, HEK293 cells wereincubated at 37 C for 1 h with a solution of 100 nM of SNAP-Lumi4-Tb and

60 nM of SNAP-Green in Tag-Lite buffer (5). After being labeled, cells were

washed three times with Tag-Lite, and drugs were added. The trFRET meas-

urements were performed in Greiner black 96-well plates on a PHERAstar FS

microplate reader with the following setup: after excitation with a laser at

337 nm (40 flashes per well), the fluorescence was collected at 520 nm for

a 50-s reading after a 50-s delay after excitation (window 1) or for a 400-s

reading after a 1,200-s delay (window 2) (SI Appendix, Fig. S2). The acceptor

ratio was determined by dividing the signal measured in window 1 by the

signal measured in window 2 and then was plotted on logarithmic scale. The

intensity level of window 2 was above noise level by at least a factor of 5 to

avoid erroneous divisions. We chose this representation because, in the

range of acceptor ratio measured, the log of the ratio is correlated linearly

with the distance between Lumi4Tb and the Green acceptor used (Frster

radius of pair is 4.6 nm). Kinetic trFRET measurements were performed with

the same setup, with signal captured every 1 s during 90 s.

Statistical Analysis. When indicated, statistical analyses of the data were

performed on results from three individual experiments, each performed in

triplicate. Mean SEM were plotted, and a one-way ANOVA test was per-

formed followed by a Bonferronis multiple comparison test using the

analysis software GraphPad Prism. P< 0.05 was considered significant.

ACKNOWLEDGMENTS. We thank Drs. Guillaume Lebon, Pierre Paoletti, andGreg Stewart for constructive discussions. The intracellular calcium release,inositol phosphate assays, and homogeneous time-resolved fluorescenceexperiments were performed on the ARPEGE (Pharmacology Screening-Interactome) platform facility at the Institut de Gnomique Fonctionnelle.E.D. was supported by a fellowship from the Fondation pour la RechercheMdicale and from the Association Schizo Oui. P.R. and J.-P.P. were sup-ported by the Centre National de la Recherche Scientifique, the InstitutNational de la Sant et de la Recherche Mdicale, by grants from the Agence

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Nationale de la Recherche [G-proteincoupled receptor dimers ANR-BLAN06-3_135092, mGluPatho (ANR-08-NEUR-006-02 in the frame of ERA-

Net NEURON), and Glusense (ANR-09-BIOT-018)] and the Fondation Betten-court Schueller, and by an unrestricted grant from Senomyx.

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