Abstract
Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6–TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6–TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
All data generated or analysed in this study are included in this article and the Supplementary Information. The cryo-EM density maps and corresponding coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB), respectively, under the following accession codes: EMD-23994 and 7MTQ (inactive-state mGlu2), EMD-23995 and 7MTR (Glu/ago-PAM-bound mGlu2), and EMD-23996 and 7MTS (mGlu2–Gi complex).
References
Koehl, A. et al. Structural insights into the activation of metabotropic glutamate receptors. Nature 566, 79–84 (2019).
Attwood, T. K. & Findlay, J. B. Fingerprinting G-protein-coupled receptors. Protein Eng. 7, 195–203 (1994).
Weis, W. I. & Kobilka, B. K. The molecular basis of G protein-coupled receptor activation. Annu. Rev. Biochem. 87, 897–919 (2018).
Niswender, C. M. & Conn, P. J. Metabotropic glutamate receptors: physiology, pharmacology, and disease. Annu. Rev. Pharmacol. Toxicol. 50, 295–322 (2010).
Conn, P. J., Lindsley, C. W. & Jones, C. K. Activation of metabotropic glutamate receptors as a novel approach for the treatment of schizophrenia. Trends Pharmacol. Sci. 30, 25–31 (2009).
Chappell, M. D. et al. Discovery of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride (LY3020371·HCl): a potent, metabotropic glutamate 2/3 receptor antagonist with antidepressant-like activity. J. Med. Chem. 59, 10974–10993 (2016).
Monn, J. A. et al. Synthesis and pharmacological characterization of C4-(thiotriazolyl)-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY2812223), a highly potent, functionally selective mGlu2 receptor agonist. J. Med. Chem. 58, 7526–7548 (2015).
Galici, R., Echemendia, N. G., Rodriguez, A. L. & Conn, P. J. A selective allosteric potentiator of metabotropic glutamate (mGlu) 2 receptors has effects similar to an orthosteric mGlu2/3 receptor agonist in mouse models predictive of antipsychotic activity. J. Pharmacol. Exp. Ther. 315, 1181–1187 (2005).
Kingston, A. E. et al. LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors. Neuropharmacology 37, 1–12 (1998).
Bollinger, K. A. et al. Design and synthesis of mGlu2 NAMs with improved potency and CNS penetration based on a truncated picolinamide core. ACS Med. Chem. Lett. 8, 919–924 (2017).
Papasergi-Scott, M. M. et al. Structures of metabotropic GABAB receptor. Nature 584, 310–314 (2020).
Farinha, A. et al. Molecular determinants of positive allosteric modulation of the human metabotropic glutamate receptor 2. Br. J. Pharmacol. 172, 2383–2396 (2015).
Lundström, L. et al. Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mechanistic studies with new potent negative allosteric modulators. Br. J. Pharmacol. 164 (2b), 521–537 (2011).
Christopher, J. A. et al. Fragment and structure-based drug discovery for a class C GPCR: discovery of the mGlu5 negative allosteric modulator HTL14242 (3-chloro-5-[6-(5-fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile). J. Med. Chem. 58, 6653–6664 (2015).
Gao, Y. et al. Asymmetric activation of the calcium sensing receptor homodimer. Nature https://doi.org/10.1038/s41586-021-03691-0 (2021).
Xue, L. et al. Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer. Nat. Chem. Biol. 11, 134–140 (2015).
Thibado, J. K. et al. Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors. eLife 10, e67027 (2021).
Goudet, C. et al. Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like receptors. Proc. Natl Acad. Sci. USA 101, 378–383 (2004).
Trzaskowski, B. et al. Action of molecular switches in GPCRs - theoretical and experimental studies. Curr. Med. Chem. 19, 1090–1109 (2012).
Maeda, S., Qu, Q., Robertson, M. J., Skiniotis, G. & Kobilka, B. K. Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes. Science 364, 552–557 (2019).
Thal, D. M. et al. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 531, 335–340 (2016).
Pérez-Benito, L. et al. Molecular switches of allosteric modulation of the metabotropic glutamate 2 receptor. Structure 25, 1153–1162.e4 (2017).
Fukuda, J. et al. Identification of a novel transmembrane domain involved in the negative modulation of mGluR1 using a newly discovered allosteric mGluR1 antagonist, 3-cyclohexyl-5-fluoro-6-methyl-7-(2-morpholin-4-ylethoxy)-4H-chromen-4-one. Neuropharmacology 57, 438–445 (2009).
Mühlemann, A. et al. Determination of key amino acids implicated in the actions of allosteric modulation by 3,3′-difluorobenzaldazine on rat mGlu5 receptors. Eur. J. Pharmacol. 529, 95–104 (2006).
Liu, J. et al. Allosteric control of an asymmetric transduction in a G-protein-coupled receptor heterodimer. eLife 6, e26985 (2017).
Goudet, C. et al. Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators. J. Biol. Chem. 280, 24380–24385 (2005).
Hlavackova, V. et al. Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR. EMBO J. 24, 499–509 (2005).
Kniazeff, J. et al. Closed state of both binding domains of homodimeric mGlu receptors is required for full activity. Nat. Struct. Mol. Biol. 11, 706–713 (2004).
Ellaithy, A., Gonzalez-Maeso, J., Logothetis, D. A. & Levitz, J. Structural and biophysical mechanisms of class C G-protein-coupled receptor function. Trends Biochem. Sci. 45, 1049–1064 (2020).
Dror, R. O. et al. Structural basis for nucleotide exchange in heterotrimeric G proteins. Science 348, 1361–1365 (2015).
Maeda, S. et al. Development of an antibody fragment that stabilizes GPCR/G-protein complexes. Nat. Commun. 9, 3712 (2018).
Koehl, A. et al. Structure of the µ-opioid receptor–Gi protein complex. Nature 558, 547–552 (2018).
Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011).
Petersen, T. N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786 (2011).
Stoveken, H. M., Hajduczok, A. G., Xu, L. & Tall, G. G. Adhesion G-protein-coupled receptors are activated by exposure of a cryptic tethered agonist. Proc. Natl Acad. Sci. USA 112, 6194–6199 (2015).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Ilca, S. L. et al. Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus. Nature 570, 252–256 (2019).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Preprint at https://doi.org/10.1101/2021.01.12.426430 (2021).
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Daniel, A., Eugene, P. & Cheng, Y. asarnow/pyem: UCSF pyem v0.5. https://doi.org/10.5281/zenodo.3576630 (2019).
Krishna Kumar, K. et al. Structure of a signaling cannabinoid receptor 1–G protein complex. Cell 176, 448–458.e12 (2019).
Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46 (W1), W296–W303 (2018).
Robertson, M. J., van Zundert, G. C. P., Borrelli, K. & Skiniotis, G. GemSpot: a pipeline for robust modeling of ligands into cryo-EM maps. Structure 28, 707–716.e3 (2020).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Laurent, B. et al. Epock: rapid analysis of protein pocket dynamics. Bioinformatics 31, 1478–1480 (2015).
Friesner, R. A. et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 49, 6177–6196 (2006).
Vedel, L., Bräuner-Osborne, H. & Mathiesen, J. M. A cAMP biosensor-based high-throughput screening assay for identification of Gs-coupled GPCR ligands and phosphodiesterase inhibitors. J. Biomol. Screen. 20, 849–857 (2015).
Christopher, J. A. et al. Structure-based optimization strategies for G protein-coupled receptor (GPCR) allosteric modulators: a case study from analyses of new metabotropic glutamate receptor 5 (mGlu5) X-ray structures. J. Med. Chem. 62, 207–222 (2019).
Doré, A. S. et al. Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain. Nature 511, 557–562 (2014).
Michel, J., Tirado-Rives, J. & Jorgensen, W. L. Prediction of the water content in protein binding sites. J. Phys. Chem. B 113, 13337–13346 (2009).
Acknowledgements
We thank E. Montabana at the Stanford-SLAC cryo-EM facility for support with data collection and J.-P. Aubry of the FACS facility (University of Geneva) for assistance with the FACS experiments. We also thank G. Eskici for discussions and support with coding. This work was supported, in part, by T32GM089626 (J.G.M.), R01 NS092695 (G.S., B.K.K. and J.M.M.) and R01 NS028471 (B.K.K.). B.K.K. is a Chan Zuckerberg Biohub Investigator.
Author information
Authors and Affiliations
Contributions
A.B.S. expressed and purified receptors, prepared cryo-EM samples, collected cryo-EM datasets, processed cryo-EM data and performed modelling. X.B.-A. purified G-protein complexes and assisted with modelling. M.d.L. performed and analysed ago-PAM binding-pose validation experiments. J.G.M. purified scFv16. M.J.R. assisted in modelling calculations of the ligand pose. R.M.N. assisted in mutagenesis experiments. M.M.P.-S. performed the GTPγS activity assay and assisted with manuscript and figure preparation. J.-P.R. developed the chemical series leading to the identification of the ago-PAM. D.S. designed and analysed ago-PAM binding-pose validation experiments. J.M.M. performed and analysed cell-based in vitro functional G-protein-coupling assays. Y.G. assisted with data analysis. C.Z. assisted with the initial screening of cryo-EM samples. B.K.K. provided advice for G-protein complex formation and analysed results. A.B.S. and G.S. wrote the manuscript with input from B.K.K., J.M.M., M.M.P.-S. and Y.G. G.S. supervised the project.
Corresponding authors
Ethics declarations
Competing interests
B.K.K. is a co-founder of and consultant for ConfometRx, Inc.
Additional information
Peer review information Nature thanks Karen Gregory, Martin Lohse and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Preparation of cryo-EM samples.
a–c, Size-exclusion chromatography profiles of purified inactive-state mGlu2 (a), Glu/ago-PAM-bound state mGlu2 (b) and the mGlu2–Gi complex (c), repeated three times with similar results. Inset in c shows the size-exclusion profile of purified Gi heterotrimer. d, Gi protein nucleotide exchange stimulated by purified mGlu2 preparations in (1) inactive state, (2) Glu/ago-PAM-bound state and (3) active state preparation used for cryo-EM studies of the mGlu2–Gi complex, as determined in a GTPγS binding assay. mGlu2 purified in the presence of antagonist LY341495 and NAM VU6001966 did not produce a substantial increase in Gαi GTPγS binding above the intrinsic binding of Gi alone. By contrast, mGlu2 purified in the presence of the agonist glutamate and ago-PAM ADX55164 produced a roughly 3.5-fold increase in Gi GTPγS binding. Data represent mean ± s.e.m. of reactions performed in triplicate. e, Representative cryo-EM micrograph of mGlu2–Gi–scFv complex from a single dataset with 45,371 micrographs.
Extended Data Fig. 2 Cryo-EM processing summary of mGlu2 in its inactive and Glu/ago-PAM-bound states.
a, Cryo-EM data processing workflow for the mGlu2 inactive state. b, Fourier shell correlation (FSC) curves for the mGlu2 inactive state cryo-EM maps of the ECD focused refinement and the global refinement. c, Angular distribution heat map of particles for reconstruction of the mGlu2 inactive state. d, Cryo-EM data processing workflow for the Glu/ago-PAM-bound state of mGlu2. e, FSC curves of the Glu/ago-PAM bound state of mGlu2 for the VFT focused refinement and the global refinement. f, Angular distribution heat map of particle projections in reconstruction of the Glu/ago-PAM-bound state of mGlu2. FSC curves and local refinement spectra were determined using CryoSPARC. Dashed lines represent the resolution at 0.143 FSC. All the processing steps were performed with Relion 3.1 (red) or CryoSPARC 3.1 (blue).
Extended Data Fig. 3 Cryo-EM processing summary for the mGlu2–Gi complex.
a, Cryo-EM data processing workflow for the mGlu2–Gi complex. b, FSC curves for the locally refined maps of the Gβγ, 7TM–Gαiβγ, CRD–7TM and VFT–CRD. Dashed lines represent the resolution at 0.143 FSC. c, Angular distribution heat map of particles for the mGlu2–Gi global reconstruction. FSC curves and local refinement spectra were calculated using CryoSPARC. All the processing steps were performed with Relion 3.1 (red) or CryoSPARC 3.1 (blue).
Extended Data Fig. 4 Agreement between cryo-EM map and model.
a, EM density and model for the 7TM of the mGlu2 inactive state. The 7TM model of mGlu2 from the mGlu2–Gi complex is rigid-body-docked to the mGlu2 inactive-state map (7TMA; green, 7TMB; coral, additional density inside the allosteric pocket of the 7TM; purple). b, Magnified view of a density inside the allosteric pocket of 7TMB that may correspond to negative allosteric modulator VU6001966. c, EM density, and model for the mGlu2–Gi complex; transmembrane helices of mGlu2 G-protein-coupled protomer (GC), transmembrane helices of non-G-protein-coupled protomer (NGC), ECL2, glutamate, ago-PAM ADX55164, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine as a representative phospholipid and α5 helix of Gαi. Densities were visualized with UCSF ChimeraX and zoned at 4 Å with a uniform threshold.
Extended Data Fig. 5 Comparison of structures across family C GCPRs.
The overall architecture of mGlu2 is similar to that of other family C GCPRs. The differences in the angle between VFT and CRD, and the angle between CRD and 7TMs leads to variations in the 7TM configuration. a–d, Models of single protomers of family C GPCRs are overlaid based on VFT superposition. Comparison of the inactive-state model of mGlu2 from this study with the mGlu5 apo state model (PDB: 6N52) (a), the mGlu5 active state model (PDB: 6N51) (b), the CaSR inactive state model (c) and the CaSR active state model (d, see ref. 15). e, Top-down view of mGlu2 7TM bundles shows that TM6 helices are distal to the 7TM interface in the inactive state (left) but form the active-state interface (right). f−h, The inactive state configuration of 7TMs of family C GPCRs are variable. Top-down view of the 7TMs of inactive-state (left) and active-state (right) family C GPCRs: mGlu5 (apo PDB: 6N52, active PDB: 6N51) (f), GABAB (inactive PDB: 6W2X, active PDB: 7C7Q) (g) and CaSR (h, ref. 15). The receptors display variable 7TM configuration.
Extended Data Fig. 6 Ago-PAM modulation of mGlu2.
a, The mGlu2 ago-PAM ADX55164, used to stabilize the active receptor conformation, potentiates the functional response of mGlu2 to l-glutamate. Receptor activation is measured by co-transfection of the mGlu2 with the neuronal glutamate transporter EAAT3 to remove extracellular glutamate and a chimeric Gq/o5 to enable intracellular calcium release as a readout. b, In a similar assay, mutation of the ECL2 tip (residues 712–714; ERR-AAA) and the Y767A mutation on the TM6–TM6 interface blunt glutamate-induced signalling (left) and compared to wild-type mGlu2, the ago-PAM ADX55164 has higher potency and higher Emax for the ERR-AAA mutant, consistent with a partial uncoupling of the ECD from the 7TM (right). c, Schematic of interactions between mGlu2 residues and ago-PAM ADX55164 bound within the 7TM core. Green, hydrophobic; blue, polar; purple, positively charged; magenta arrow, hydrogen bond; green line, π–π stacking; grey, glycine. d, e, Magnified views of the cryo-EM map of G-protein-coupled active-state mGlu2 GC (coral) (d) and NGC (green) (e) protomers show that ago-PAM (blue) binds only to the GC protomer, whereas the analogous pocket is not accessible by the membrane (pocket opening highlighted with orange box) on the NGC protomer. A phospholipid density between two 7TMs is shown in grey. The head group of the lipid molecule does not seem to interact with mGlu2 or Gi and the density might represent a lipid molecule with different head groups. All family C GPCR dimeric structures display elongated densities between two protomers, most likely corresponding to either cholesterol or phospholipids. Additionally, GABAB structures displayed a phospholipid molecule inside the 7TM, indicating the importance of lipid molecules in family C GPCR dimerization and activity. Data in a and b represent mean ± s.e.m. from four and five independent experiments measured in duplicate, respectively.
Extended Data Fig. 7 Ago-PAM binding-pose validation for mGlu2.
Residues within the binding pocket of ago-PAM ADX55164 were mutated to study their role in PAM activity. a, Mutant and wild-type receptor responses to increasing concentrations of glutamate in the absence (brown) or presence (green) of 200 nM ADX55164 were tested in an intracellular calcium release assay following co-transfection with the EAAT3 and Gq/o5. Responses were normalized to the maximum response of the wild-type receptor. The concentrations of glutamate are plotted on the x axis [log (M)]. The amount of receptor DNA transfected was increased up to tenfold to obtain mutant expression levels similar to that of the wild-type; however, for some mutants, such expression levels could not be reached. Data shown represent the mean ± s.e.m. from five independent experiments. b, Mutant and wild-type receptor surface expression levels were monitored by fluorescence microscopy using an N-terminal Flag-tag present on all constructs and an anti-Flag Cy3 antibody. Data in b represent images from three independent experiments. R720A, S731A and L732A mutants did not produce glutamate or ADX55164 responses and did not show surface expression in immunofluorescence studies (not shown). c, The change in pEC50 of mutant and wild-type receptors upon addition of 200 nM ADX55164 plotted from individual experiments along with individual data points for estimation of surface expression of mutants compared to the wild-type (100%) by flow cytometry. Statistics were derived from at least 4 independent experiments by one-way ANOVA and comparison of each mutant to the wild-type. A statistical difference from the wild-type is indicated by an asterisk (*). P values were corrected for multiple comparisons using Dunnett’s test and are provided in Supplementary Table 1.
Extended Data Fig. 8 Comparison of water coordination, 7TM activation and the G-protein interface across GPCRs.
a, EM density for the region of the observed water molecule coordinated inside the allosteric pocket of PAM-less 7TM of mGlu2 (labelled residues are conserved in mGlu5, and blue dashed lines represent hydrogen bonds between the water molecule and mGlu2). b, Comparison of the water molecule coordinated inside the allosteric pocket of mGlu5 (PDB: 4OO9, yellow; water, brown) and the PAM-less 7TM of mGlu2 (mGlu2–Gi structure, green; water, red). The homologous Tyr647 residue in mGlu5 (Tyr659) coordinates a water molecule in the NAM-stabilized mGlu5 allosteric pocket14,55,56 and has a role in ligand pharmacology by affecting allosteric modulator cooperativity23. The mGlu2–Gi map also reveals a density that corresponds to a water molecule within the PAM-less 7TM bundle, coordinated between residues Tyr647, Thr769, Ser801 and Gly802, similar to the water-molecule coordination in the allosteric pocket of NAM-stabilized mGlu5 structures14,55,56. The PAM-bound 7TM bundle shows a weaker density for this water molecule. Using JAWS simulations57, a statistical thermodynamics-based approach to determine water-molecule positioning, a water molecule was observed to be bound in the pocket containing the putative water site in both protomers. The conservation of these four residues and resolution of water molecules in mGlu2 and mGlu5 indicates the importance of water for mGlu ligand pharmacology. c, Superposition of M1 muscarinic receptor (M1R) inactive (PDB: 5CXV) and active (PDB: 6OIJ) states reveals TM6 movement (agonist: iperoxo, orange). d, Tryptophan toggle switch in M1R. e, f, Comparison of the overall Gi protein coupling arrangement on the cannabinoid receptor 1 (CB1), a representative family A GPCR (e) and mGlu2 (f). The non-G-protein-coupled protomer model is not shown for clarity. g, Magnified view of cryo-EM map of G-protein-coupled active-state mGlu2.
Extended Data Fig. 9 Functional analysis of mGlu2 mutants for Gi activation.
a, Truncation of the mGlu2 C terminus or mutation of critical residues of mGlu2 involved in the formation of the observed G-protein interface substantially decreases receptor activation by l-Glu as tested in an intracellular calcium release assay following co-transfection with the EAAT3 and Gq/o5. Data in a represent mean ± s.e.m. from five independent experiments. b, An Epac1-based FRET cAMP biosensor capable of reporting intracellular cAMP levels was used to investigate the effect of mutating critical residues of mGlu2 in the Gi-interacting interface. FRET between the fluorescent proteins decreases after cAMP binding to a fusion protein consisting of an Epac1-domain (residues 14–881) flanked by fluorescent proteins mCerulean and mCitrine. c, Representative kinetic traces of LY354740 (mGlu2 agonist)-induced inhibition of forskolin-stimulated cAMP formation through Gi activation by wild-type mGlu2 and mutants. The effect of the NAM MNI-137 and non-transfected cell traces are shown for comparison. Indicated are time points for mGlu2 ligand addition and for taking cAMP values for generation of the concentration-response curves (CRC) shown in Fig. 3b. Data in c represent mean ± s.d. from one representative experiment performed in triplicate.
Extended Data Fig. 10 Intermediate states of mGlu2–Gi activation.
a, Cryo-EM maps of mGlu2 in two distinct intermediate states with open-closed VFTs and inactive 7TM conformation. The protomer contributing TM3 to the interface (7TMA) displays an open VFT and the protomer contributing TM4 to the interface (7TMB) displays a closed VFT in one intermediate state (left), and the opposite in the other intermediate state (right). b, Reference-free cryo-EM 2D class average of the mGlu2–Gi protein complex shows heterotrimeric G-protein pre-coupling to mGlu2 in the inactive state (top), compared to an average of the nucleotide-free Gi coupled to mGlu2 (bottom). The α-helical domain of Gα is ordered in the pre-coupled state, indicating a GDP-bound G protein. This observation suggests that VFT activation and the approximately 180° rearrangement of the 7TMs to establish the TM6–TM6 interface would be necessary for G-protein activation. c, Cryo-EM model of G-protein-coupled active state model of mGlu2 and G protein overlaid to either of the 7TMs of mGlu2 in the inactive-state model. The αN helix of the Gα protein in both conformations would clash with the membrane, while Gβ would also clash with the adjacent protomer if the Gi is bound to TMB of the inactive state (clash represented by red stars).
Supplementary information
Rights and permissions
About this article
Cite this article
Seven, A.B., Barros-Álvarez, X., de Lapeyrière, M. et al. G-protein activation by a metabotropic glutamate receptor. Nature 595, 450–454 (2021). https://doi.org/10.1038/s41586-021-03680-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-021-03680-3
This article is cited by
-
Constitutive activation mechanism of a class C GPCR
Nature Structural & Molecular Biology (2024)
-
Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor
Nature (2024)
-
Structure, function and drug discovery of GPCR signaling
Molecular Biomedicine (2023)
-
G protein-coupled receptors in neurodegenerative diseases and psychiatric disorders
Signal Transduction and Targeted Therapy (2023)
-
Steering G protein activation by mGlu heterodimer
Cell Research (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
