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Free to read. Author contributions: S. Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction.
In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap VFT domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane 7TM domains responsible for G protein activation.
These two domains are interconnected by a cysteine-rich domain CRD. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators.
These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation. Most cell surface receptors are multimeric complexes of which each subunit is produced through the association of different domains throughout evolution 1 — 5.
The activation of such receptor complexes is a result of coordinated conformational changes or movement of these different domains.
Although an increasing amount of 3D crystal structures are becoming available, there is still limited information available on the structural basis of interdomain communication. These receptors are obligatory dimers, either homo- or heterodimers, made by the association of two domains over evolutionary time; an extracellular bilobate venus flytrap VFT domain associated with a G protein activating 7 transmembrane 7TM domain Fig.
The VFTs are evolved from certain types of bacterial periplasmic binding proteins, especially those of the leucine-isoleucine-valine binding protein family involved in the transport of amino acids, sugars, or ions. Accordingly, class C GPCRs represent exciting new targets for drug development for both the pharmaceutical and food industries, as illustrated by the number of drugs targeting these receptors already on the market the GABA B receptor agonist baclofen, umami compounds, and various sweeteners such as aspartame, and cinacalcet, a positive allosteric modulator of the CaSR , and those in clinical trials 9 — 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.
The precise mechanisms of how agonist binding in the VFTs leads to the 7TM conformational changes that are required for G protein activation remain unknown. Structural and mutagenesis studies indicated that the closure of the VFT resulting from agonist binding in the cleft represents a key step in receptor activation 12 — It is assumed that this VFT conformational change is associated with a reorientation of the two VFTs in these dimeric receptors 15 , 16 , leading to a relative movement of the two 7TMs and the activation of one of them Fourth, sweet proteins such as brazzein have been proposed to activate the sweet taste receptor by interacting with the CRD In the present study, we further examined the role of the CRD in mGlu receptor activation.
We first confirm its critical role in the allosteric coupling between the VFT and the 7TM domains, but most importantly we show that a precise association of the two CRDs within mGlu dimers leads to full receptor activation.
Our study thus provides a clear demonstration that dimerization and structural rearrangement at the CRD dimer interface is responsible for mGlu receptors activation. The crystal structure of the mGlu3 extracellular domain revealed that the CRD is stabilized by four intradomain disulfide bridges Fig.
In the present study, we have analyzed the importance of each of these disulfide bridges by changing the conserved cysteines to alanines in the rat mGlu2 CRD. We found that all these mutations abolished agonist-induced responses Fig. S1 A , bound agonist with a wild-type affinity Fig. S1 B , and are still able to form dimers as revealed by cell surface time-resolved fluorescence resonance energy transfer FRET measurements Fig. In addition, the mutations did not prevent the 7TM domain from activating G proteins.
Taken together, our results suggest that the structure of the CRD in its entirety, stabilized by the four disulfide bridges, is required for the transduction from agonist binding to G protein activation in the mGlu2 receptor. 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.
However, whereas the mutation of Cys produced a mutant that can be activated by LY, this PAM was incapable of activating the receptor in which the partner Cys Cys was mutated. Additional experiments revealed that the CA 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.
This explains why no ligand-induced calcium signals could be measured with this receptor Fig. The full constitutive activity of the mutant CA is further supported by the absence of potentiation of inositol accumulation by the agonist and the PAM Fig. A Inositol-phosphate IP accumulation in cells that express the wild-type mGlu2 and the indicated mutants without basal or after stimulation with glutamate or LY, in conditions where the chimeric Gqi9 protein is coexpressed. However, it was not inhibited by the competitive antagonist, LY, 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.
Altogether, these data revealed that the CA mutation stabilizes the 7TM in an active state, even though the VFT can remain open, suggesting that the mutation likely affects a key element involved in the signal transfer from the VFT to the 7TM. We investigated whether the constitutive activity could result from an aberrant intrasubunit disulfide bridge between the VFT and the CRD. By inserting a thrombin cleavage site between the VFT and the CRD to examine whether a DTT sensitive link exists between these two domains, as this can be detected after thrombin cleavage Fig.
S3 B 24 , we showed that a disulfide bridge can indeed form between Cys and Cys, when the two other Cys are mutated Cys and Cys Fig.
However, this mutant receptor did not display constitutive activity Fig. S3 D , suggesting that such a disulfide bridge is unlikely responsible for the constitutive activity of the CA mutant. Given that a Cys appears necessary at position in the CA mutant for constitutive activity, and because mGlu receptors are well-established dimers, we speculated that the Cys may form an intersubunit disulfide bridge, then stabilizing an active form of the mGlu dimer.
We verified that only the mGlu2-C1—mGlu2-C2 heterodimer could indeed reach the cell surface, whereas the two homodimers are retained in the endoplasmic reticulum Fig. This system allowed us to show that only a receptor dimer carrying the CA mutation in both subunits displays a high constitutive activity, whereas any dimer carrying the mutation in a single subunit did not Fig.
This is consistent with the Cys being involved in an intersubunit disulfide bridge. The constitutive activity of mGlu2 CA mutant results from an intersubunit disulfide bridge. B Western blot in nonreducing or reducing conditions for wild-type and indicated mutants of mGlu2.
The mutation CA results in a receptor deleted of the intersubunit disulfide bridge that could be resolved as monomer see arrows both in the absence and presence of DTT. Addition of the mutation CA in the CA mutant produces a receptor with a new intersubunit disulfide bridge and in this double mutant the monomer could be resolved only in the presence of DTT. To firmly demonstrate this possibility, we examined whether a DTT sensitive covalent link can be observed between both subunits of the CA mutant in which the natural intersubunit disulfide bridge has been removed by mutating Cys into Ala.
As expected, monomers of the CA mutant can be detected on a Western blot under nonreducing condition, whereas only the dimer can be detected in the wild-type and the double mutant CA—CA Fig.
These data demonstrate that the mutation CA leads to an intersubunit disulfide bridge likely involving Cys and this is likely responsible for the constitutive activity of this mutant. To firmly demonstrate that a covalent linkage between the CRDs within an mGlu dimer could be sufficient to stabilize an active state of the receptor, we examined whether introducing additional Cys residues within the CRD could generate constitutively active receptors.
A series of 13 Cys mutants were produced on the basis of the known 3D structure of the mGlu3 CRD, at positions possibly corresponding to the dimer interface 15 , 16 , 18 Fig.
Western blot analysis of the CA—LC double mutant confirmed that the additional Cys could form an intersubunit disulfide bridge Fig. Cysteine scanning in the putative CRD's interface produces mGlu2 with constitutive activity. A Molecular model of mGlu2 where the residues mutated into Cys are in yellow or orange. The positions where the presence of a Cys leads to constitutive activity are highlighted in orange. B IP accumulation in cells that express the wild-type mGlu2 and the indicated mutants without basal or after stimulation with glutamate, in the presence of the chimeric Gqi9 protein.
No monomers in denaturating conditions in the absence of DTT were observed for the fully constitutively active mutant LC 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. However, all of the mutations at the interface do not result in a cross-linking between the subunits, for example the EC mutant Fig.
Interestingly, the appearance of a disulfide bridge between the two subunits does not necessarily stabilize the active state of the receptor; indeed, cross-linking between the subunits can be observed with most mutants such as QC, PC, EC, and GC, even though none of them display constitutive activity.
We investigated whether similar mutations in the CRD of other mGlu receptors could also generate constitutively active mutants. Similar constitutive mutants in mGlu4 and mGlu5 receptors. A IP accumulation in cells that express the wild-type mGlu4 or mGlu5 and the indicated mutants without basal or after stimulation with the indicated agonists, in the presence of the chimeric Gqi9 protein.
As observed with mGlu2, the mutation of the first cysteine in the CRD of mGlu4 and -5, C and C, respectively, also result in constitutively active receptors that could not be further stimulated by the full agonists, l -AP4 and quisqualate, respectively Fig. The weaker constitutive activity of mGlu4 CA may be explained by its lower expression level at the cell surface Fig. Also as observed with mGlu2, introduction of a Cys at the equivalent position of L H and T in mGlu4 and -5, respectively also generated constitutively active mutants, whereas no such activity was measured when these residues were mutated into Ala Fig.
In the present study we provide important information on the role of the CRD in the activation process of the mGlu receptors. We further illustrate its important contribution for the allosteric coupling between the ligand binding VFT and the G protein coupling 7TM domains, and most importantly, we show that a precise orientation of the two CRDs within an mGlu dimer is associated with a full activity of the receptor. Because these key data were reproduced with three distinct mGlu receptors, the proposed mechanism is likely general to all class C GPCRs containing a CRD, then including the sweet and umami taste receptors 31 , the CaSR 32 , and the GPRC6a recently shown to be involved in the effect of osteocalcin Many things were expected from the resolution of the crystal structure of the extracellular domain of mGlu receptors for the understanding of the activation mechanism of these complex dimeric receptors 15 , 16 , Although these studies revealed that the VFT closure represents a first step in the activation process, how this conformational change is transmitted to the 7TM to activate the G protein remains to be elucidated.
However, recent structures are not consistent with this proposal. Indeed active and resting orientations were observed with antagonist- and agonist-bound VFTs, respectively 7 , Indeed, these structures were all solved with five different bound agonists 18 and correspond to what was previously proposed to be the resting orientation with the two CRDs far apart, whereas both VFTs are closed.
Our data show that a precise association of the two CRDs is sufficient for full mGluR activation, indicating that the two CRDs are likely contacting each other in the active state. Such a proposal is consistent with a 3D model of the dimeric extracellular domain in which either one or both VFTs are in the closed state and their association in the active orientation Acc or Aco; Fig. In such models, all residues that, when mutated into Cys, lead to an intersubunit disulfide bridge and a constitutively active receptor are facing each other at a distance consistent with a disulfide bond formation.
It is interesting to note that even though intersubunit cross-linking was obtained with other residues such as Glu , such mutant receptors did not display constitutive activity, indicating that only a specific and precise association of the CRDs leads to G protein activation. 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 , 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 The C termini of the CRD arrowheads , the positions and red that cross-link between the two subunits then providing a constitutively active receptor, and the positions orange that cross-link without producing constitutive activity are highlighted.
The effects of competitive and noncompetitive antagonists reveal that some flexibility exists in the mGlu receptor for the interconnection between the VFT and 7TM domains. The constitutive activity of the cross-linked receptor was inhibited by negative allosteric modulators acting in the 7TM domains, thereby indicating that the constitutive activity involves conformational changes in the 7TM domain.
This action of NAMs also indicates that the 7TM domains can be maintained in an inactive conformation even when the CRDs are linked together in their active association. In contrast, the constitutive activity of the cross-linked mutant is not affected by the orthosteric antagonists known to prevent VFT closure.
Such absence of inhibition is observed even though the antagonist binds to the receptor, then suggesting that even when both VFTs are in the open state, the CRDs can still be in their active orientation the Aoo conformation , as observed with the mGlu1 structure bound to a LY 7.
This is well illustrated by the requirement of the natural disulfide bridge that links these two domains for the functional coupling between the VFT and the 7TM Such a constraint is likely important for the VFTs to control the relative position of the CRDs in the dimeric mGlu, thereby controlling receptor activity.
Accordingly, it is likely that a change in the relative orientation of the VFTs resulting from agonist binding and VFT closure can bring the CRDs in close contact, leading to the activation of one 7TM. However, the real amplitude of movement between the VFTs remains to be identified.
Indeed these data revealed a close proximity between the 7TM domains, closer that what is expected on the basis of the resting orientation of the VFTs. Accordingly, either the CRD move relative to the VFT or most likely the changes of the VFT relative position occurring during activation are not as important as those observed in the crystal structures.
Consistent with this proposal, a precise association mode of the CRDs is required for receptor activation. In conclusion, due to their key role in the mechanism of activation of the receptors, the CRDs may constitute the site of action of molecules that modulate the activity of the class C GPCRs.
For instance, it was suggested that the mode of action of the sweet proteins such as brazzein was through the binding to CRD of the T1R3 subunit of the heteromeric sweet taste receptor As such, this study brings unique views on how the genetic association during evolution of proteins able to bind amino acids, sugars, or ions, was successful in generating GPCRs activated by such ligands, through the use of an intermediate CRD.
LY 2S amino[ 1S,2S carboxy-cyclopropyl] xanthyl propanoic acid and LY 2,2,2-trifluoro- N -[4- 2-methoxyphenoxy phenyl]- N - 3-pyridinylmethyl -ethanesulfonamide were purchased from Tocris Cookson. Human thrombin CalBiochem was obtained from Merck. Detection of the HA-tagged constructs at the cell surface by Western blotting or ELISA with or without thrombin treatment were performed as previously described 24 , Final models were built using Modeler 7.
We thank Dr. This article contains supporting information online at www. Read article at publisher's site DOI : Nature , , 16 Jun Cited by: 4 articles PMID: Front Cell Dev Biol , , 10 May RNA , 27 10 , 08 Jul Cited by: 0 articles PMID: Nature , , 30 Jun Cited by: 1 article PMID: Cited by: 2 articles PMID: This data has been text mined from the article, or deposited into data resources.
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By using the site you are agreeing to this as outlined in our privacy notice and cookie policy. Search articles by 'Siluo Huang'. Huang S 1 ,. Jianhua Cao Search articles by 'Jianhua Cao'. This conformation of the bulky indole group is accommodated by G 5. However, in other mGlus except mGlu 5 , residues at position 5. Critical FITM-receptor interactions are revealed by mutations and structure activity relationships.
Previous mutagenesis studies have proposed at least one common allosteric site for the mGlu family within the 7TM domain. S2 and selectivity for mGlu 1 over mGlu 5 Fig. S12 9. Examination of the contact residues in the binding pocket reveals only four residues of mGlu 1 that differ from mGlu 5 : V 3.
Therefore, we mutated these four residues to their corresponding amino acid in mGlu 5 Fig. Methionine substitution of T 7. Thus, T 7. In addition, we assessed mutations known to influence the allosteric modulation of other mGlu subtypes that had not previously been explored in mGlu 1.
T 6. Location of P 5. Interestingly, multiple mGlu 5 modulator scaffolds are known to be sensitive to mutations of two non-conserved residues, S 6. However, both of these residues contribute to a small pocket separated from the FITM pocket by the Y 3.
Given that S 3. S13 9 into the crystal structure. S13, A-C and analyzing the binding energy contribution per residue Fig. S13D revealed that T 7. S13, C and D. Compound 17 lacks not only the hydrogen bond with T 7. S11A and Table S2 , may compensate for this loss and account for the retained activity at 10 nM. The 3-pyridyl analog compound 14, Fig.
Compound 28 exhibits approximately 10 fold lower potency and differs from FITM by the introduction of a methyl group to the amine on the pyrimidine ring. Docking compound 28 reveals a major energy penalty that arises from the loss of a polar interaction and the introduction of steric clash with T 7.
Compound 28 also lacks a polar interaction with Q 3. Compound 22 Fig. Collectively, by comparing the binding of FITM with those of other less active or inactive compounds, we attribute the superior potency observed for FITM to the polar interaction between T 7.
One of the interactions apparently stabilizing this conformation is a salt bridge between the K 3. In addition to the salt bridge between K 3. Thus, polar interactions within the intracellular crevice may be involved in the regulation of G protein binding and receptor activation in both class A and C GPCRs, though through distinct residue positions. The intracellular crevice in NAM-bound mGlu 1 adopts a closed conformation. A A cartoon demonstrating agonist triggered opening of the intracellular cavity for G protein binding.
D Side and E intracellular views of the mGlu 1 receptor, the side chains of residues involved in a hydrogen bond network that stabilize the receptor in an inactive conformation are shown as white carbons. This observation raises a possibility that this interaction network might contribute to the communication between the ECD and the 7TM domain during receptor activation.
In addition, part of the linker residues e. W, a residue conserved in all mGlus insert into the lipid bilayer, where they form extensive contacts with cholesterol molecules that mediate the observed dimerization of the 7TM domain Fig. A Shown in cyan is the extracellular part of the mGlu 1 7TM. ECL2 residues MI are shown as white carbons, while the linker region residues IE are shown as yellow carbons. Hydrogen bond interactions between ECL2 and the linker region are shown as dashed lines.
The current model probably does not capture the specific conformation and interaction between CRD and 7TM domain, and a more tightly packed domain interaction is very likely. Several dimeric structures of mGlu receptor VFDs have been solved in different conformations: putative active A or resting R state defined by the relative orientation between the VFD protomers as well as closed c or open o states defined by the conformation of each VFD Comparing different conformations, the distance between the C-terminal ends of the ECDs within a dimer changes dramatically 3.
In our crystal structure of the 7TM domain, we observed a parallel dimer mediated by interactions of helix I and cholesterols. If this is a conformation that can be adopted by the full-length receptor dimer, the CRDs of each protomer should also be in close proximity. Disulfide bond crosslinking experiments suggested that the CRDs of each protomer may form close contact in an activated receptor dimer Although our structure is solved in complex with a NAM and the 7TM domain appears to be in an inactive state, there is evidence supporting the existence of a glutamate-bound, but signaling incapable state, in the full-length mGlu dimer Moreover, there is evidence that cholesterol can positively modulate glutamate responses by recruiting mGlus to lipid rafts 31 , 36 , consistent with the observation that the close proximity of the N-terminus of the 7TM domain results from a dimer conformation mediated by multiple cholesterol molecules.
This model might represent a glutamate bound, but signaling incapable, conformation of mGlu 1. While consistent with the currently available experimental data, we acknowledge that this model is only one of the several possible explanations for the biological role of the 7TM domain dimer we observed, and needs to be tested in future studies.
We further acknowledge that the 7TM domain dimer conformation might vary in different states of the receptor and may be modulated by several factors in biological systems, such as membrane lipid content or other protein-protein interactions. As noted for the recently solved class B and F GPCR structures, and now for class C, despite a lack of sequence and motif conservation, the architecture of the 7TM bundle is generally preserved.
Furthermore, while class C GPCRs are known to form obligate dimers via the ECDs, the observed 7TM dimer suggests additional points of communication between protomers, mediated by multiple cholesterol molecules and direct protein-protein interactions. Moreover, as a robust structural template, the mGlu 1 7TM domain structure will likely provide insights into pharmacology of small molecule allosteric modulators for class C GPCRs.
We would like to thank Dr. Emmitte and P. Velasquez for help on molecular biology; T. Trinh and M. Chu for help on baculovirus expression; K. Kadyshevskaya for assistance with figure preparation; A. Walker for assistance with manuscript preparation; J. Smith, R. Fischetti, and N. This version has not undergone final editing. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS.
Supporting Online Material. National Center for Biotechnology Information , U. Author manuscript; available in PMC Apr 4. Cho , 2 Yan Xia , 4 Colleen M. Jeffrey Conn , 2 and Raymond C. Karen J. Hyekyung P. Colleen M. Jeffrey Conn. Raymond C. Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Science. See other articles in PMC that cite the published article.
Abstract The excitatory neurotransmitter glutamate induces modulatory actions via the metabotropic glutamate receptors mGlus , which are class C G protein-coupled receptors GPCRs.
Open in a separate window. Determinants of subtype selectivity within the common allosteric site Previous mutagenesis studies have proposed at least one common allosteric site for the mGlu family within the 7TM domain. Supplementary Material Supplementary Materials Click here to view. S1 to S13 References 38— References and Notes 1. Structural diversity of G protein-coupled receptors and significance for drug discovery.
Nat Rev Drug Discov. Pharmacol Ther.
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