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J. Biol. Chem., Vol. 280, Issue 26, 24380-24385, July 1, 2005
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¶¶
From the
CNRS UMR5203, INSERM, U661, University Montpellier I, University Montpellier II, Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, 141 Rue de la Cardonille, Montpellier F-34094 Cedex 5, France, ||Department of Molecular Pharmacology, Institute of Experimental Medicine, Academy of Science of Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic, **Cis Bio International, 30204 Bagnols-sur-Cèze, France, Laboratory of Pharmacological and Toxicological Chemistry and Biochemistry, CNRS UMR8601, University Paris-V, 75270 Paris Cedex 6, France
Received for publication, March 10, 2005 , and in revised form, April 25, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Within the large family of GPCRs, those of the class C constitute a good model to study the relevance of GPCR dimerization in G-protein activation, because all are constitutive dimers, either homodimers linked by a disulfide bridge (4, 5) or obligatory heterodimers (6–10). This class of GPCRs includes the receptors for the main neurotransmitters, glutamate and GABA, the calcium-sensing receptor, the receptors for sweet and umami taste, plus several pheromone receptors (11). Like any other GPCRs, these receptors possess a heptahelical domain (HD) involved in G-protein activation. However, they possess a large extracellular domain (ECD) where agonists bind. Dimerization of class C GPCRs is a prerequisite for their function (12). Indeed agonist binding in the ECD stabilizes its closed state (13–16) and leads to a change in the relative orientation of the two ECDs within the dimer. This major change in conformation of the dimer of ECDs is assumed to be associated with a conformational change in the dimer of HDs, leading to its activation (12, 17).
Positive allosteric modulators (PAMs) constitute a new class of synthetic ligands acting on GPCRs, which have been discovered within the last years. In contrast to full agonists, PAMs are either devoid of agonist activity or act as very partial agonists but significantly enhance the potency and/or efficacy of agonists. Such molecules offer an excellent alternative to agonists in therapeutics (18–21) as illustrated with cinacalcet, the first GPCR PAM now on the market (22). A number of such compounds have been identified for class C GPCRs and have been shown to bind in a site equivalent to the retinal binding pocket of opsins (23–25). Recently, we reported that PAMs act as full agonists on either mGlu or GABAB receptors deleted of their large ECD (26, 27). This indicates that PAMs can stabilize the active state of the HD, an effect inhibited by the ECD in the absence of agonist.
In the present study, we have examined whether one or two PAM(s) is/are required per mGlu dimer to enhance the effect of agonists. Using a system that allows us to control the subunit composition within a defined dimer (28, 29), our results demonstrate that only one PAM is sufficient. Moreover, our work brings new data supporting the proposal that only one HD within these homodimeric receptors can reach an active conformation, further demonstrating the asymmetric functioning of the dimer of HDs. This represents an additional evidence for dimeric GPCRs activating a single heterotrimeric G-protein at a time.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmids, Mutagenesis, and Construction of Chimeric mGlu Receptors—To perform ELISA or fluorescence resonance energy transfer (FRET) experiments, a hemagglutinin (HA) or a Myc tag were introduced in the N-terminal end, right after the signal peptide of the different receptors. The construction of the N-terminal HA- or Myc-tagged rat mGlu1 or mGlu5a, pRKG5a-NHA, have been described already (6, 31). As previously reported for different mGlu (32, 33) or GABAB receptors (6), the presence of these tags did not modify the expression or the pharmacological properties of the receptor. The mGlu1C1, mGlu5C1, mGlu1C2, and mGlu5C2 chimera have been obtained by replacing the C terminus of HA- or Myc-mGlu1 and HA- or Myc-mGlu5a by that of GABAB1a and GABAB2, respectively (28, 29). The mGlu5 mutant sensitive to Ro01-6128, mGlu5Ro, has been obtained by introducing three point mutations in mGlu5, P654S and S657V in the third transmembrane segment and L743V in the fifth transmembrane segment (24). Mutations were obtained using the QuikChange® strategy (Stratagene), and all constructions were verified by DNA sequencing.
Cell Culture and Transfection—HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (penicillin and streptomycin, 100 units/ml final concentration). Cells were transiently transfected by electroporation as described elsewhere (34). To avoid any influence of glutamate in the assay medium released by the cells, the high affinity glutamate transporter EAAC1 was also co-transfected with the receptor.
Cell Surface Quantification by ELISA—Cell surface and total cell expression of N-terminal HA-tagged receptors was quantified by ELISA as described elsewhere (27). Transfected cells were first fixed with paraformaldehyde, then permeabilized or not (to measure total or cell surface expression, respectively) using Triton X-100 and incubated for 30 min with rat monoclonal anti-HA antibody (clone 3F10, Roche Diagnostics). Cells were then incubated with a secondary goat antibody conjugated to peroxidase (Jackson ImmunoResearch, West Grove, PA), which was further detected and quantified by chemiluminescence using Supersignal West Femto (Pierce) and a Wallac Victor2 luminescence counter (Molecular Devices, St Gregoire, France).
Time-resolved (TR)-FRET Analysis—The protein-protein interaction at the surface of living cells was analyzed by TR-FRET. These experiments are based on the use of specific antibodies against each partner and labeled either with Eu3+-cryptate or with AlexaFluor 647, which allows the measurement of a FRET signal in a time-resolved manner if both fluorophores are in close proximity. The TR-FRET experiments were performed as previously described (35). Briefly, HEK293 cells expressing the indicated HA- or Myc-tagged receptor subunits were labeled with monoclonal anti-HA (12CA5) and/or anti-Myc (9E10, American Type Culture Collection number CRL-1729) carrying either Eu3+-cryptate pyridine bipyridine or AlexaFluor 647 (provided by Cis Bio International Research). Fluorescence of Eu3+-cryptate and TR-FRET signals were measured at 620 and 665 nm, respectively, 50 µs after excitation at 337 nm, using a RubyStar fluorometer (BMG Labtechnologies, Champigny-sur-Marne, France). In parallel, the total fluorescence emitted at 682 nm by the bound Alexa647 antibodies was quantified after excitation at 640 nm using an AnalystTM reader (Molecular Devices). The FRET signal was measured as 
R using the following equation, r = [(R665/620)POS – (R665/620)NEG], where (R665/620)POS is the ratio of the 665 nm signal over that at 620 nm measured in the presence of both antibodies, and (R665/620)NEG is the same ratio measured in the absence of the acceptor-labeled antibody.
Functionnal Assays—Inositol phosphate (IP) accumulation and measurement of intracellular Ca2+ mobilization in transfected cells were performed in 96-well microplates as already described (27). The dose-response curves were fitted using the GraphPad Prism program and the following equation, y = [(ymax – ymin)/(1 + (x/EC50)n)] + ymin, where EC50 is the concentration of the compound necessary to obtain the half-maximal effect and n is the Hill coefficient.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Construction of a mGlu5 Triple Mutant (mGlu5Ro) Sensitive to Ro01-6128 —Ro01-6128 and DFB are known specific PAMs of mGlu1 and mGlu5, respectively (24, 27, 36). Accordingly, the EC50 value of quisqualate (a potent mGlu1 and mGlu5 agonist) on mGlu1 was significantly decreased in the presence of Ro01-6128 but not in the presence of DFB (Fig. 1A). Conversely, DFB significantly decreased the EC50 value of quisqualate on mGlu5, whereas Ro01-6128 had no effect (Fig. 1B). To have access to a mGlu5 receptor sensitive to the mGlu1 PAM, three point mutations were introduced into the mGlu5 HD according to Knoflach et al. (24). As described previously, this mGlu5Ro mutant was sensitive to Ro01-6128, although the increase in agonist potency is not as large as that measured on mGlu1 but retained its sensitivity to DFB (Fig. 1C).
Generation of mGlu5Ro:mGlu5 Heterodimers—By co-expressing mGlu5Ro and mGlu5 in the same cells, one is expecting that 
50% of the surface receptors correspond to the mGlu5Ro:mGlu5 heterodimer. To largely increase the proportion of this receptor combination, we took advantage of the quality control system of the GABAB receptor. Indeed in this heterodimeric receptor the GABAB1 subunit is retained in the endoplasmic reticulum unless its cytoplasmic endoplasmic reticulum retention signal is masked by the C-terminal tail of the associated GABAB2 subunit (7). We previously reported that this quality control system can be transferred to mGlu receptors by swapping their C-terminal tail by that of GABAB1 (C1) or GABAB2 (C2) (28, 29). Moreover, our previous data demonstrated that the G-protein-coupling efficacies of mGlu receptors bearing either the C1 or C2 tail were equivalent.
Here, we swapped the C-terminal tail of mGlu5Ro for that of GABAB1. This mGlu5RoC1 subunit does not reach the cell surface, unless co-expressed with mGlu5C2 that possesses the C-terminal tail of GABAB2, as shown by ELISA performed on intact cells (Fig. 2, A and B) and functional analysis (data not shown) (28, 29). The presence of the HA-mGlu5RoC1:myc-mGlu5C2 dimers at the cell surface was verified by a HTRF approach (35), using anti-HA and anti-myc antibodies labeled with europium-cryptate and AlexaFluor 647, respectively (Fig. 2C). No such a HTRF signal was measured in cells expressing HA-mGlu5 and myc-V2 receptors despite a similar expression level of these two receptors, demonstrating the specificity of this assay (data not shown) (28). Moreover, although a clear HTRF signal was obtained in cells expressing HA-mGlu5 using anti-HA antibodies labeled with either the donor or acceptor fluorophores, no such signal could be detected in cells co-expressing HA-mGlu5RoC1 and myc-mGlu5C2 (Fig. 2C). This demonstrates the absence of HA-mGlu5RoC1 homodimers at the cell surface. Although mGlu5C2 dimers are expected to reach the cell surface in cells expressing both this subunit and mGlu5RoC1, the proportion of mGlu5C2 homodimers can be maintained to a minimum by transfecting the cells with a higher proportion of mGlu5RoC1 plasmid (29).
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Generation of Heterodimeric mGlu1:mGlu5 Receptors—To further confirm that a single PAM per dimer can potentiate the activity of these receptors, we aimed at generating a receptor dimer in which each subunit can be targeted by a specific PAM. To that aim, we examined whether a mGlu1:mGlu5 heterodimer could be generated in cells co-transfected with HA-mGlu1C1 and Myc-mGlu5C2 or with HA-mGlu5C1 and Myc-mGlu1C2.
As shown in Fig. 3A, although mGlu1C1 and mGlu5C1 expressed alone are retained in the endoplasmic reticulum, mGlu5C2 and mGlu1C2 allow the surface targeting of mGlu1C1 and mGlu5C1, respectively. As described above, the presence of HA-mGlu1C1:myc-mGlu5C2 and HA-mGlu5C1: myc-mGlu1C2 heterodimers at the cell surface was clearly demonstrated with the HTRF approach using anti-HA and anti-Myc antibodies labeled with donor or acceptor fluorophores (Fig. 3B). In addition, mGlu1C1 or mGlu5C1 homodimers could not be detected with this technique in these same cells. Because no mGlu1:mGlu5 heterodimers have been reported with the wild-type subunits (4), the replacement of the C-terminal tails of these receptors by those of GABAB1 and GABAB2 is sufficient to allow the formation of such heterodimers.
Heterodimeric mGlu1:mGlu5 Receptors Are Positively Modulated by Both Ro01-6128 and DFB—As shown in Fig. 3, both the mGlu1 and mGlu5 PAMs potentiated the effect of quisqualate in cells co-expressing mGlu1C1 and mGlu5C2 (Fig. 3C) and in cells co-expressing mGlu5C1 and mGlu1C2 (Fig. 3D), as indicated by the increase in agonist potency (decrease of the EC50). In cells expressing mGlu1C1 and mGlu5C2, the potentiation by Ro01-6128 can only result from the effect of this PAM on the mGlu1C1:mGlu5C2 heterodimer, because this receptor combination is the only one at the cell surface containing a Ro01-6128 binding site. Similarly, the effect of DFB on cells expressing mGlu5C1 and mGlu1C2 can only result from the enhancement of the agonist potency at the mGlu5C1:mGlu1C2. This further demonstrates that a single PAM per dimer is sufficient to enhance agonist potency at these receptors. Of interest, when both PAMs were added simultaneously, no significant further potentiation was observed, suggesting that a single PAM per dimer is sufficient for the full enhancement of the receptor dimers activity (Fig. 3, C and D).
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PAMs Act as Non-competitive Antagonists when Interacting in the Subunit Unable to Activate G-proteins—As shown in Fig. 4, the mGlu5 PAM DFB enhanced agonist activity at the mGlu5C1:mGlu1XC2 combination, indicating that potentiation can be observed when PAMs bind in the functional HD. Of interest, when the PAM binds in the HD unable to activate G-proteins, then it acts as a non-competitive antagonist rather than a positive modulator. Indeed, the mGlu1 PAM Ro01-6128 decreases the maximal agonist effect on the mGlu5C1: mGlu1XC2 combination (Fig. 4). These results are not because of a particularity of Ro01-6128 itself, because similar results were obtained using XITA, another mGlu1-selective PAM (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To generate mGlu dimers possessing a single site for a PAM, we first mutated the mGlu5 receptor to make it sensitive to the mGlu1 PAM Ro01-6128. In agreement with published data the three mutations introduced in TM3 and TM5 were sufficient to allow Ro01-6128 to enhance agonist potency on the mGlu5 receptor. However, these three mutations did not prevent the effect of the mGlu5-selective PAM DFB, indicating that the residues responsible for the selectivity of Ro01-6128 and DFB are not at the same position in the mGlu1 and mGlu5 HD. Accordingly, either Ro01-6128 and DFB bind at different sites, or they bind differently in a similar binding pocket. By examining in more details the divergence between mGlu1 and mGlu5 receptors at the level of the proposed Ro01-6128 binding pocket, additional positions can be identified that may possibly be involved in the selective action of DFB.
As previously reported, the exchange of the C-terminal tail of mGlu receptors by that of either GABAB1 (C1) or GABAB2 (C2), allows to largely increase the proportion of receptor dimers composed of two defined subunits at the cell surface. Indeed, the receptors possessing the C1 tail reach the cell surface only when associated with the receptor chimera containing the C2 tail. Such heterodimers can be detected at the cell surface with the HTRF approach, and because a disulfide bridge linked the subunits of a mGlu dimer, such heterodimers are not expected to dissociate at the cell surface. Indeed, no homodimers of subunits bearing the C1 tail can be detected with the HTRF technology. Of interest, this approach also allows the formation of mGlu1:mGlu5 heterodimers. This is in contrast with what has been reported with the full-length wild-type subunits (4). This suggests that the C-terminal tails of these receptors play a major role in their selective interaction. In agreement with this proposal, mGlu1 and mGlu5 receptors mostly differ at the level of their C-terminal tails. Whereas the sequence identity between the ECDs and HDs of these two receptors is 75%, it is less than 30% for the C-terminal tails. Further supporting the involvement of the C-terminal tail in the control of mGlu receptor dimer formation, the mGlu1a and mGlu1b receptor splice variants that differ in their C-terminal tail only have been shown not to form heterodimers (38). However, we cannot exclude the possibility that the formation of the mGlu1:mGlu5 dimer is the consequence of a strong coiled-coil interaction between the GABAB1 and GABAB2 C-tails.
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Because PAMs have been shown to stabilize the active conformation of the HD of either mGlu5 (6) or GABAB2 (26) receptors, and because a single PAM per dimer appears sufficient for the full activity, one may propose that a single HD per dimer is turned into an active conformation during activation of these dimeric receptors. This is consistent with our previous study showing that two antagonists interacting in the HD are required to inhibit activity of mGlu dimers (29). This plus other data described below nicely fit with the proposal that the dimer of HD functions in an asymmetrical way, a single HD being activated at a time. As illustrated in Fig. 5 using the mGlu1: mGlu5 heterodimer as an example, activation of this dimeric receptor with agonists leads to only one of the HD, either that of mGlu1 or that of mGlu5, in an active state. Accordingly, the binding of Ro01-6128 should favor the activation of the mGlu1 HD, whereas DFB should favor the activation of the mGlu5 HD. This is consistent with the enhancing effect of a PAM acting in mGlu5 when the mGlu1 HD is made unable to activate G-proteins by a point mutation in the third intracellular loop. Conversely, this model nicely explains the non-competitive action of PAMs acting in the subunit bearing the third intracellular loop mutation. Indeed, by binding in this subunit, the PAM is expected to favor the formation of the active conformation of this subunit and so to prevent activation of the other HD, leading to a smaller response. Further supporting this conclusion, an inverse agonist binding in the HD mutated in the third intracellular loop favors the activation of the associated subunit and as such acts as a enhancer, as described previously (29).
Taken together, our data strongly support the asymmetric functioning of the dimer of HDs in mGlu receptors that we previously proposed (29). Such an observation is surprising considering 1) that the mGlu receptors are homodimers, and as such, symmetrical complexes and 2) that the dimer of ECDs functions in a symmetrical way, the activation of both ECDs being required for full activation of the receptor (28). Accordingly, one has to imagine some hindrance at the level of the dimer of HDs that prevents both HDs to reach an active state at a time. This may result from some specificity of the dimer interface at that level of the receptor dimer. However, one attractive possibility is that the heterotrimeric G-protein is responsible for this effect, because this protein is not symmetric. Although highly speculative, this idea is supported by recent data on the class A GPCRs that show that a single heterotrimeric G-protein interacts with a dimer of HDs, the 
-subunit contacting a first HD via its C-terminal end, whereas the second HD contacts both the N-terminal -helix of the -subunit as well as the 
-
dimer (3, 39–41).
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FOOTNOTES |
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Supported by a fellowship from the Fondation pour la Recherche Médicale.
¶ Supported by a bourse de Docteur Ingénieur from the CNRS.
Supported by both CisBio International and the French government (CIFRE fellowship).
¶¶ To whom correspondence should be addressed: Institut de Génomique Fonctionnelle; Département de Pharmacologie Moléculaire; 141 Rue de la Cardonille, Montpellier F-34094 Cedex 5, France. Tel.: 33-467-14-2988; Fax: 33-467-54-2432; E-mail: jppin{at}igf.cnrs.fr.
1 The abbreviations used are: GPCR, G-protein-coupled receptor; HD, heptahelical domain; ECD, extracellular domain; PAM, positive allosteric modulator; mGlu, metabotropic glutamate receptor; GABAB, -aminobutyric acid receptor, type B; Ro01-6128, diphenylacetylcarbamic acid ethyl ester; XITA, 9H-xanthene-9-carboxylic acid (2-isopropyl-2H-tetrazol-5-yl)-amide; DFB, 3,3'-difluorobenzaldazine; ELISA, enzyme-linked immunosorbent assay; FRET, fluorescence resonance energy transfer; TR-FRET, time-resolved FRET; HA, hemagglutinin A, HEK293, human embryonic kidney 293; HTRF, homogeneous time-resolved fluorescence.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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