Abstract
Class C G-protein-coupled receptors (GPCRs) are activated through binding of agonists to the large extracellular domain (ECD) followed by rearrangement of the transmembrane domains (TMDs). GPR156, a class C orphan GPCR, is unique because it lacks an ECD and exhibits constitutive activity. Impaired GPR156–Gi signaling contributes to loss of hearing. Here we present the cryo-electron microscopy structures of human GPR156 in the Go-free and Go-coupled states. We found that an endogenous phospholipid molecule is located within each TMD of the GPR156 dimer. Asymmetric binding of Gα to the phospholipid-bound GPR156 dimer restructures the first and second intracellular loops and the carboxy-terminal part of the elongated transmembrane 7 (TM7) without altering dimer conformation. Our findings reveal that GPR156 is a transducer for phospholipid signaling. Constant binding of abundant phospholipid molecules and the G-protein-induced reshaping of the cytoplasmic face provide a basis for the constitutive activation of GPR156.
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Data availability
Atomic coordinates and the cryo-EM map have been deposited in the the EM Data Bank (EMD) and Protein Data Bank (PDB), respectively, under the following accession numbers: EMD-35380 and PDB 8IED (GPR156-Go–scFv16), EMD-35377 and PDB 8IEB (GPR156 dimer of GPR156-Go–scFv16), EMD-35378 and PDB 8IEC (Go–scFv16 of GPR156-Go–scFv16), EMD-35390 and PDB 8IEQ (GPR156A/B/C/D), EMD-35382 and PDB 8IEI (GPR156A/B of GPR156A/B/C/D) and EMD-35389 and PDB 8IEP (GPR156C/D of GPR156A/B/C/D). Mass spectroscopy data are deposited on Figshare (https://doi.org/10.6084/m9.figshare.24212226 and https://doi.org/10.6084/m9.figshare.24715704.v1). The trajectories for GPR156-PC and GPR156-PG from the molecular dynamics simulations data are deposited on Zenodo (https://doi.org/10.5281/zenodo.8418994 and https://doi.org/10.5281/zenodo.8419006, respectively). Previously published PDBs used in this study are available under PDB accession codes 7UM5, 7MTR, 7MTS, 6UO8, 7EB2, 7C7S, 7E9H, 7EPA, 7EWL, 7M3J, 7M3F, 6WIV and 7E9G. The AlphaFold2 model is available in ModelArchive (https://www.modelarchive.org) with accession code ma-1015e. Sequence data used in the alignment for Extended Data Fig. 7 are H. sapiens GPR156, GABR1, GABR2, CaSR, mGlu1, mGlu2, mGlu3, mGlu4, mGlu5 and mGlu7 (Uniprot accession codes Q8NFN8, Q9UBS5, O75899, P41180, Q13255, Q14416, Q14832, Q14833, P41594 and Q14831, respectively). Sequence data used in the alignment for Extended Data Fig. 10d are H. sapiens Gi1, Gi2, Gi3, Gs, Gq, G12 and G13 (Uniprot accession codes P63096, P04899, P08754, P63092, P50148, Q03113 and Q14344, respectively). Source data are provided with this paper.
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Acknowledgements
We thank J. Koh for technical help and Y. Kim, C. Lee, J. Lee, K. Kim, S. H. Ryu (POSTECH), Y. Yu (Kookmin U.), M. Jin (GIST) and J. Kim (SNU) for helpful comments. This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST, No. 2021R1A2C301335711 and 2019M3E5D6066058 to Y.C.), the Bio & Medical Technology Development Program (NRF-2019M3E5D3073567 to K.P.K.), the Ministry of Science and ICT (grant number 2022R1A2C1005885 to J.H.), the BK21 program (Ministry of Education to Y.C.), Wellcome Trust Investigator Award (221795/Z/20/Z to X.Q., D.W., C.V.R.) and internal funding from the University of Southern California Dornsife College (to V.K). Cryo-EM data were acquired at the Core Research Facility, Pusan National University. Computing resources were provided by the Center for Advanced Research Computing (CARC) at the University of Southern California (https://carc.usc.edu).
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Contributions
J.S. carried out protein expression, purification and structure determination with the help of J.P. J.S. carried out data collection with the help of J.H. J.P. and J.S. performed biochemical experiments with the help of K.K. and J.-Y.L. K.P.K. and J.J. performed mass spectroscopy and phospholipid characterization analysis. X.Q., D.W. and C.V.R. performed comparative lipidomics analysis. J.H.L. and V.K. performed molecular dynamics simulations. J.S., J.P. and Y.C. designed the research; Y.C. wrote the manuscript with the help of J.S., J.P. and K.P.K.
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Nature Structural & Molecular Biology thanks Bryan Roth and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.
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Extended data
Extended Data Fig. 1 Purification of the GPR156-Go complex.
a–c, Size-exclusion chromatography profile (a), SDS-PAGE gel (b), and negative-staining microscopy analysis (c) of the purified GPR156-Go complex. Purification experiments of the GPR156-Go complex in (b) were performed independently at least three times. Negative-staining microscopy analysis (c) was performed once. d, Representative cryo-EM micrograph of the GPR156-Go complex. The cryo-EM data collection of GPR156-Go was performed once. e, 2D class averages of the Go-free GPR156. f, 2D class averages of the GPR156-Go complex. g, Comparison of constitutive activities of GPR156 and GABAB in G-protein activation, measured by BRET2 assay. The activity of GABAB was observed in the absence or presence of 100 µM GABA. Data were mean ± SEM from three independent experiments, performed in technical triplicate. Statistical differences in ΔBRET were analyzed by two-way ANOVA with Dunnett’s post hoc test. h, cAMP inhibition assay for GPR156 C-terminal truncation. Mock transfected with an empty vector was used as a negative control. cAMP production was normalized to a percent of WT activity. i, Surface expression levels of WT and GPR156 mutants in the cAMP assay, measured by ELISA. Surface expression levels of GPR156 mutants were normalized to a percent of WT surface expression level. Data in h and i were mean ± SEM from at least three independent experiments, performed in technical triplicate. Statistical differences were analyzed by one-way ANOVA with Dunnett’s post hoc test, compared to WT (NS, not significant; ***P < 0.001; ****P < 0.0001). j, k, A model of GPR156 predicted from AlphaFold2 was used for initial docking and model building. The N-terminal (1 to 39) and the C-terminal (336 to 814) regions are disordered and omitted in the figure. The model is colored according to the predicted Local Distance Difference Test (pLDDT) score (j). The Predicted-Alignment-Error (PAE) plot of the GPR156 model (k).
Extended Data Fig. 2 Flow chart of cryo-EM and data processing.
Cryo-EM processing chart of the G-protein coupled and G-protein free GPR156.
Extended Data Fig. 3 Analysis of the quality of the cryo-EM map.
a, b, Angular distributions, Fourier shell correlation curves, and globally refined cryo-EM density maps of GPR156-Go (a) and GPR156 alone (b). c–f, Angular distributions, Fourier shell correlation curves and locally refined cryo-EM density maps marked local resolution of GPR156 dimer (c), G-protein-scFv16 (d), GPR156A/B (e), and GPR156C/D (f). g, h, Fourier shell correlation curves of the model versus the map generated through PHENIX.Mtriage64 of globally and locally refined GPR156-Go (g) and GPR156 alone (h). i, j, Global fitting of the structures of GPR156-Go into the composite map (i) and GPR156 alone into the globally refined map (j). k, Cryo-EM densities and fitted atomic models. GF-GPR156, Go, GPR156A, GPR156B, and GC-GPR156 are shown in yellow, pink, salmon, cyan, and green, respectively.
Extended Data Fig. 4 Key features of GPR156.
a, Cryo-EM map of the GPR156 tetramer. b, Interface between the head-to-head dimer of GPR156. c, Interaction between TM7 of GPR156C and ICL2 of GPR156B. H-bonds (Q314-D155 and E321-V158) and hydrophobic interactions (F318-V158 and I325-I159) are highlighted. d, e, Aligned structures of the two GPR156 dimers in two views; front view (d), bottom view. Structures of the ICL2s are encircled (e). f–h, Structural comparison of GPR156B and GABAB-Gi (PDB:7EB2 ref. 5) with respect to ECL2 (f), TM7 (g), and ICLs (h). i, Cryo-EM map of the GPR156-Go complex. j, k, Aligned structures of an GPR156 alone dimer with the GPR156-Go complex in two views; front view (j), bottom view (k). l, A density on top of the ECL2 in two different views. m, Close-up view of the interactions between Y1463.55 (nGC) and H248ICL3 (GC).
Extended Data Fig. 5 Comparison of the dimeric arrangement of GPR156 with other class C GPCRs in inactive and active states.
a–i, The TMDs of Class C GPCRs were aligned with GC-GPR156 (black line) and shown in the extracellular (top) view. GPR156-Go (a), inactive GABAB (red; PDB: 7C7S ref. 33) (b), active GABAB (orange; 7EB2 ref. 5) (c), active mGlu4 (beige; 7E9H ref. 7) (d), inactive mGlu2 (yellow; 7EPA ref. 37) (e), active mGlu2 (green; 7MTS ref. 6) (f), apo GPR158 (pink; 7EWL ref. 21) (g), inactive CaSR (blue; 7M3J ref. 36) (h), active CaSR (purple; 7M3F ref. 36) (i). The gray filled line represents nGC-GPR156.
Extended Data Fig. 6 Characterization of phospholipid in GPR156.
a, Comparative lipidomics analysis of endogenous lipids bound to purified recombinant GPR156. PC and PE are enrichment in GPR156 fraction relative to total cellular lysate. Bars show mean ± standard deviation from three independent experiments (dots). b–c, GPR156 activation as measured by GTPase-Glo assay for GPR156-Gi peptidisc containing PE (b) or GPR156 in LMNG (c). A peptidisc containing GPRC5D-Gi and PE was incubated with PG as a control. Lower levels of residual GTP indicate higher level of G-protein activity. Data in (b) and (c) were mean ± SEM from three independent experiments, performed in technical duplicate. Statistical differences were analyzed by one-way ANOVA with Dunnett’s post hoc test. d‒f, Comparison of molecular dynamics simulation of GPR156 Go-coupled complexes with PG versus PC. d, Root-mean-square-fluctuation (r.m.s.f.) calculated of each subunit in the complexes; shading refers to 95 % confidence interval (n = 5). e–f, Distribution of the closest distances between the sidechain of R2796.57 and the phospholipids against the closest distances between the backbone of C216ECL2 and the phospholipids on the nGC protomer (e) and on the GC protomer (f). The shading refers to density estimated with a multivariate gaussian kernel; the marginal distribution (by count) is shown on the sidebar. The black points in the background is the datapoints collected every 0.5 ns. All distances were calculated using only the heavy atoms. The horizontal and vertical dotted line refers to 3.5 Å. g, Mutational effect on the F215ECL2 adjacent to the phospholipid head in G-protein activation, measured by BRET2 assay. Data were mean ± SEM from four independent experiments, performed in technical triplicate. Statistical differences were analyzed by two-way ANOVA with Dunnett’s post hoc test, compared to WT (NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). h‒i, Comparison of PC- and PG-bound GPR156 with representative snapshots from MD simulations of GPR156. Close-up views of a phospholipid-binding site in the simulations of PC-bound (h) and PG-bound GPR156 (i). nGC protomer and GC protomer are shown on top and bottom, respectively. Three representative snapshots (970, 985, and 1000 ns) of a simulation trajectory are displayed.
Extended Data Fig. 7 Sequence alignment of GPR156 with other human class C GPCRs.
The conserved residues are marked in yellow. The alignment was output from GPCRdb (gpcrdb.org) and edited by using snapgene (snapgene.com).
Extended Data Fig. 8 Phospholipids in GPR156.
a, In one protomer, the phenyl ring is flipped by 85° toward the dimer interface, creating space for the lateral movement of the phospholipid. We refer this conformer to as an open form. The W229 indole ring can be repositioned upon the conformational change of the F275 ring. b, In another protomer, F275 is packed against the fatty-acyl chain of the phospholipid to form a closed conformation. c, Density at the top half of the dimer interface, in which a CLR molecule is modelled. d, e, Densities near V223 (d) and W284 (e) in GPR156. f, Comparison of the phospholipid-binding in GPR156 with that of GABAB2. g, Comparison of the phospholipid-binding in GPR156 with the PAM-binding in mGlu2 and CaSR. h–j, cAMP inhibition assay for GPR156 mutated at the phospholipid binding site (h), dimer interface (i, j). Data were mean ± SEM from at least three independent experiments, performed in technical triplicate. Statistical differences were analyzed by one-way ANOVA with Dunnett’s post hoc test, compared to WT (NS, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001).
Extended Data Fig. 9 Structural transition of the cytoplasmic face of GPR156 in the Go protein-coupled state.
a–c, Comparison of the Go-free, nGC-, and GC-protomers in three different views; front (a), extracellular (b), and cytoplasmic view (c). d–e, Aligned ICL1s (d) and ICL2s (e) of the GPR156B with GC-protomers. The major structural differences in the C-terminal loop and ICLs are indicated by red arrows. f, cAMP inhibition assay for GPR156 mutated at ICL2. Data were mean ± SEM from three independent experiments. Statistical differences were analyzed by one-way ANOVA with Dunnett’s post hoc test, compared to WT. g–l, Comparison of the TMD and ICLs of GPR156 with those of other class C GPCRs–Gi; GABAB-Gi1 (PDB: 7EB2 ref. 5), mGlu2-Gi1 (7MTS ref. 6), mGlu4-Gi3 (7E9H ref. 7). Comparison of the TMD of GPR156 GC-protomer with those of other class C GPCRs bound to Gi in three different views; front (g), extracellular (h), and cytoplasmic view (i). Comparison of ICL1 (j), ICL2 (k), and ICL3 and the C-terminal loop (l). m, Mutational effect on CTL of GPR156 in G-protein activation, measured by BRET2 assay. Data were mean ± SEM from three independent experiments, performed in technical triplicate. Statistical differences were analyzed by two-way ANOVA with Dunnett’s post hoc test, compared to WT (NS, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001).
Extended Data Fig. 10 Go binding to GPR156.
a–c, Comparison of Go binding between GPR156 and other class C GPCRs: GABAB-Gi1 (PDB: 7EB2 ref. 5) (a), mGlu2-Gi1 (7MTS ref. 6) (b), mGlu4-Gi3 (7E9H ref. 7) (c). The red arrows indicate the structural differences in the receptors and the G proteins. d. Sequence alignment of the residues in the α5 helix in different human Gα proteins. Residues interacting with GPR156 are marked with a light green circle. Absolutely conserved and highly conserved (≥50%) residues are marked with orange and yellow colors, respectively.
Supplementary information
Supplementary Information
Supplementary Notes 1–7, Videos 1–3, and Tables 1–4.
Supplementary Video 1
Dynamics of TM4 and ICL2 of the GPR156B protomer revealed by 3D variability analysis.
Supplementary Video 2
Flexibility of CTL in the GPR156 GC protomer.
Supplementary Video 3
Rigid body movement of the GC protomer with respect to the G protein.
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Source Data Extended Data Fig./Table 1
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Shin, J., Park, J., Jeong, J. et al. Constitutive activation mechanism of a class C GPCR. Nat Struct Mol Biol 31, 678–687 (2024). https://doi.org/10.1038/s41594-024-01224-7
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DOI: https://doi.org/10.1038/s41594-024-01224-7