Abstract
β-Arrestins (βarrs) mediate the desensitization and internalization of activated G-protein-coupled receptors (GPCRs). The molecular mechanism by which dimeric family C GPCR members recruit arrestins remains elusive. Here we report two structures of metabotropic glutamate receptor subtype 3 (mGlu3) coupled to βarr1, with stoichiometries of 2:1 and 2:2. The l-glutamate-bound mGlu3 dimer adopts an inactive state, with both Venus flytrap domains closed, engaging βarr1 either asymmetrically or symmetrically. The transmembrane domain of the mGlu3 protomer interacts with βarr1 through a binding pocket formed by three intracellular loops and an ordered C-terminal region. Three phosphorylation sites (pS857, pS859 and pT860) on the C-terminal tail of mGlu3 engage the N domain of βarr1. βarr1 stabilizes mGlu3 in an inactive conformation, characterized by a TM3/TM4–TM3/TM4 dimeric interface, previously observed in the negative allosteric modulator-bound structure of mGlu3. Our findings provide important insights into βarr-mediated inactivation of family C GPCRs.
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Data availability
The data that support this study are available in a publicly accessible repository. Atomic coordinates have been deposited in the PDB under accession numbers 9II3 and 9II2 for mGlu3–βarr1-one and mGlu3–βarr1-two, respectively. Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (mGlu3–βarr1-one: EMD-60938 for the global complex, EMD-60940 for 2TMD–βarr1–scFv30, EMD-60939 for ECD and EMD-62655 for 1TMD–βarr1–scFv30; mGlu3–βarr1-two: EMD-60941 for the global complex, EMD-60942 for ECD and EMD-60943 for TMD–βarr1–scFv30). The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD059170. Source data are provided with this paper.
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Acknowledgements
We are grateful for technical assistance from the Center of Cryo-Electron Microscopy, Zhejiang University on Cryo-EM, for data acquisition, J. Lu for electron microscopy support at Nankai University and S. Chen and L. Li from the Proteomics Center at the National Institute of Biological Sciences. This work was supported by National Natural Science Foundation of China (grant 32271288 to X.Y. and grants 32471269 and 32071231 to Y.S.), the China Postdoctoral Science Foundation (grant 2024M761516 to T.W.), Innovation Talent Program by Haihe Laboratory of Synthetic Biology (grant 22HHSWSS00009 to X.Y.), Fundamental Research Funds for the Central Universities, Nankai University (grant 63241207 to X.Y.) and Tianjin Science and Technology Major Project (grant 24ZXZSSS00170 to X.Y.).
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X.Y. and Y.S. conceived the project. T.W. and M.D. performed protein expression and purification, sample preparation and cryo-EM data collection. T.W. performed all BRET experiments and cell surface expression and western blot experiments. T.W. and N.L. performed the mass spectrometry experiments. Y.L., N.J., X.L., S.C., X.Z. and X.Y. collected cryo-EM data. X.Y. conducted the cryo-EM reconstruction. T.W., X.Y. and Y.S. analyzed the data, designed the study and wrote the paper. All authors discussed the results and contributed to paper preparation.
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Nature Chemical Biology thanks Kaavya Krishna Kumar, Arun Shukla and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Structural comparison of mGlu3 protomers and dimers in mGlu3-βarr1 complexes.
(a) Structural alignment of βarr1-coupled mGlu3 protomers from the mGlu3-βarr1-one (blue) and mGlu3-βarr1-two (green) complexes. Red arrows indicate notable differences between two protomers. (b) Structural comparison of mGlu3 dimers from the mGlu3-βarr1-one (blue) and mGlu3-βarr1-two (green) complexes, based on the alignment of βarr1-coupled mGlu3 protomers. The mGlu3 dimer from the mGlu3-βarr1-two complex is depicted as a surface representation. (c-e) Structural comparison between the βarr1-coupled mGlu3 protomer (blue) and the non-βarr1-coupled mGlu3 protomer (pink). Zoomed-in views of the CRD (d) and TMD (e) regions are displayed. Red arrows highlight notable differences in the comparison.
Extended Data Fig. 2 Structural comparison of different receptor-arrestin interfaces.
(a) Superimposition of βarr1 (blue) from the mGlu3-βarr1 complex with arrestins (yellow) from various receptor-arrestin complexes. The TMD of mGlu3, serving as a guide for membrane positioning, is colored grey. Arrows indicate the position of the FLR of arrestins. (b) Superimposition of the TMD from the mGlu3-βarr1 complex with TMDs from other receptor-arrestin complexes. βarr1 from the mGlu3-βarr1 complex and arrestins from other receptor-arrestin complexes are colored blue and yellow, respectively. Dash lines (cyan and orange) represent the approximate long-axis positions of the arrestin molecules. The two arrestin molecules are not positioned on the same two-dimensional planes, so the angles between two dash lines on the paper plane are estimated and labeled. The following PDB codes were used: V2R-βarr1 (7R0C); Rhodopsin-Varr (5W0P); 5-HT2BR-βarr1 (7SRS); GCGR-βarr1 (8JRV); β1AR-βarr1 (6TKO); M2R-βarr1 (6U1N); CB1-βarr1 (8WU1); NTSR1-βarr1-1 (6UP7); NTSR1-βarr1-2 (6PWC).
Extended Data Fig. 3 BRET assays of Gi dissociation.
(a,c,e) Dose-dependent L-glutamate stimulated mGlu3 receptor response, measured using BRET-based Gi activation assay. The mGlu3 wild type or mutations were used. Data points are presented as mean ± s.e.m. (n = 3 biological replicates). (b,d,f) Cell surface expression levels of mGlu3 wild type or mutants, measured by flow cytometry. A one-way ANOVA with 5% confidence intervals was used for data analysis. ns: no significance. *: P < 0.05, **: P < 0.01. P values are shown on the plot. (g) FACS sequential gating and sorting strategy. (Left panel) Gating strategy for live cells (P1), based on forward scatter (FSC-A) and side scatter (SSC-A) parameters. (Right panel) Cells with FITC fluorescence (P2) are selected, based on fluorescence intensity. The equation for cell surface expression (% of wild type) is shown, where Mut represents mGlu3 mutants, and WT represents mGlu3 wild type.
Extended Data Fig. 4 BRET assays of βarr1 recruitment by mGlu3.
(a-d) Interactions of βarr1 with wild type mGlu3 and its mutants in the ICL1 (a), ICL2 (b), ICL3 (c) and CTR (d) regions in response to L-glutamate. (e-h) Interactions of mGlu3 with wild type βarr1 and its mutants in response to L-glutamate. The βarr1 mutants involved in engagement with ICL1 (e), ICL2 (f), ICL3 (g) and CTR (h) were assayed. Data points are presented as mean ± s.e.m. (n = 3 biological replicates). Statistical analysis and P values are provided in Supplementary Table 1.
Extended Data Fig. 5 Mass spectra for detecting the mGlu3-βarr1-scFv30 protein complex mediated by GRK2.
Representative spectra of peptides with phosphorylated residues are shown. Phosphorylation was observed at residues S857 (a), S859 (b), and T860 (c).
Extended Data Fig. 6 Interaction of arrestins with the phosphorylation pattern of the C-tail of different GPCRs.
Cartoon representation of the N-domain of arrestins is shown in blue. The first β-strand of arrestins is labeled S1. The C-tails or ICL3 of various GPCRs are depicted in a green stick model. Oxygen, nitrogen, and phosphorus atoms are colored red, blue, and orange, respectively. The phosphorylated sites are drawn and labeled according to the coordinate files deposited in the PDB database. The following PDB codes were used: V2R-βarr1 (7R0C); Rhodopsin-Varr (5W0P); 5-HT2BR-βarr1 (7SRS); M2R-βarr1 (8JAF); M2R-βarr1 (6U1N); β1AR-βarr1 (6TKO); CB1-βarr1 (8WU1); GCGR-βarr1 (8JRV); NTSR1-βarr1-1 (6UP7); NTSR1-βarr1-2 (6PWC).
Extended Data Fig. 7 Structural comparison of mGlus dimers in the inactive state.
The superimposition involves one protomer from the mGlu3-βarr1-two complex (green) with one protomer from various mGlu dimer in the inactive state: the mGlu2-mGlu3 hetero-dimer colored cyan (a, PDB code 8JD1) in the Rco state; the mGlu2 homo-dimer colored red (b, PDB code 7EPA) in the Roo state; the mGlu7 homo-dimer colored yellow (c, PDB code 7EPC) in the Roo state; the mGlu1 homo-dimer colored pink (d, PDB code 7DGD) in the Roo state; the mGlu5 homo-dimer colored purple (e, PDB code 6N52) in the Roo state and the mGlu5 homo-dimer colored grey (f, PDB code 8T7H) in the Rcc state. These two superimposed protomers are displayed in surface representation, while the other two protomers are shown in cartoon form.
Extended Data Fig. 8 Putative model of signaling supercomplex of mGlus.
(a-c) Construction of Gi- and βarr1-coupled supercomplexes of the mGlu2-mGlu3 hetero-dimer, mGlu2 homo-dimer and mGlu4 homo-dimer. The TMD of the βarr1-coupled protomer from the mGlu3-βarr1-one complex is superimposed onto the TMD of the non-G protein-coupled protomer from the Gi-coupled mGlu2-mGlu3 hetero-dimer (a, PDB code 8JD3), from the Gi-coupled mGlu2 homo-dimer (b, PDB code 7MTS), or from the Gi-coupled mGlu4 homo-dimer (c, PDB code 7E9H). Gi and βarr1 are depicted as blue and cyan surfaces, respectively. mGlu2, mGlu3 and mGlu4 are shown in yellow, pink and green cartoons, respectively. Both side view (top) and bottom view (bottom) of each putative supercomplex are displayed. (d) Conformational conflict of the mGlu3 homo-dimer in the Rcc state when engaging with βarr1 and Gi. The TMD of Gi-coupled mGlu2 protomer in the mGlu2-Gi complex (PDB code 7MTS) is superimposed onto the TMD of the non-βarr1-coupled mGlu3 protomer in the mGlu3-βarr1-one complex. The mGlu3 homo-dimer, βarr1 and Gi are depicted in blue cartoon, red cartoon with a transparent surface and cyan surface, respectively. βarr1 overlaps with the beta subunit of Gi.
Supplementary information
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Source Data Extended Data Fig. 3 (download PDF )
Source data for flow cytometry.
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Statistical source data.
Source Data Extended Data Fig. 4 (download PDF )
Source data for flow cytometry.
Source Data Extended Data Fig. 5 (download XLSX )
Mass spectrometry analysis of GRK2-phosphorylated mGlu3–βarr1–scFv30 samples expressed in Sf9 cells.
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Wen, T., Du, M., Lu, Y. et al. Molecular basis of β-arrestin coupling to the metabotropic glutamate receptor mGlu3. Nat Chem Biol 21, 1262–1269 (2025). https://doi.org/10.1038/s41589-025-01858-8
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DOI: https://doi.org/10.1038/s41589-025-01858-8
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