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Structures of the ADGRG2–Gs complex in apo and ligand-bound forms

Nature Chemical Biology volume 18, pages 1196–1203 (2022)Cite this article

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Abstract

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA–ADGRG2–Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

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Fig. 1: Cryo-EM structure of apo-ADGRG2 in complex with the DNGs.
Fig. 2: Identification of DHEA as agonist and DOC as antagonist of ADGRG2.
Fig. 3: Cryo-EM structure of DHEA-bound ADGRG2.
Fig. 4: Interaction modes of DHEA or DOC with ADGRG2.

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Data availability

All data generated in this study are included in the main text or supplementary materials. Source data are provided with this paper. The atomic coordinates and the cryo-EM density maps have been deposited in PDB and Electron Microscopy Data Bank (EMDB) databases under accession codes 7XM6 and EMD-30658 for apo-ADGRG2–Gs complex; 7XKD and EMDB-33248 for DHEA–ADGRG2–βT-Gs complex (DHEA upper state), 7XKF and EMDB-33250 for DHEA–ADGRG2–βT-Gs complex (DHEA lower state); 7XKE and EMDB-33249 for DHEA–ADGRG2-FL-Gs complex, respectively. All other data are available upon request to the corresponding authors. Publicly available datasets used in this study are PDB IDs 3SN6 and 7D77.

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Acknowledgements

This work was supported by the National Key R&D Program of China (grant nos. 2018YFC1003600 to X.Y., 2019YFA0904200 to J.-P.S. and P.X.), the National Natural Science Foundation of China (grant nos. 92057121 to X.Y., 31971195 to P.X., 82090024 and 91949202 to F.Y. and 32000850 to Z.-M.L), the National Science Fund for Distinguished Young Scholars (grant no. 81825022 to J.-P.S.), the Major Fundamental Research Program of Natural Science Foundation of Shandong Province, China (grant nos. ZR2021ZD18 to X.Y. and ZR2020ZD39 to J.-P.S.), Shandong Provincial Natural Science Fund for Excellent Young Scholars (grant no. ZR2021YQ18 to P.X.), the Key Research and Development Program of Shandong Province (grant nos. 2021ZLGX02 to J.-P.S. and GG201709260059 to P.X.), the Key Research Project of the Natural Science Foundation of Beijing, China (Z20J00129 to J.-P.S.), the Rolling program of ChangJiang Scholars and Innovative Research Team in University (grant no. IRT_17R68 to X.Y.), the Fundamental Research Funds for the Central Universities (grant no. 2021JCG020 to J.-P.S. and P.X.). We thank the Cryo-Electron Microscopy Center of Southern University of Science and Technology for supporting our project and we are grateful to X. Ma, Y. Gao and L. Fu for their technical help during cryo-EM data collection. We thank the HPC Cloud Platform of Shandong University and HPC-Service Station in the Cryo-EM center at the Southern University of Science and Technology for data processing support. We also thank Y. Yu at Translational Medicine Core Facility of Advanced Medical Research Institute, Shandong University for her assistance with EnVision multimode plate reader (PerkinElmer).

Author information

Author notes

  1. These authors contributed equally: Hui Lin, Peng Xiao, Rui-Qian Bu, Shengchao Guo, Zhao Yang, Daopeng Yuan.

Authors and Affiliations

  1. Key Laboratory of Experimental Teratology of the Ministry of Education, Department of Physiology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China

    Hui Lin, Zhao Yang, Chang-Hao Liu, Shaohui Huang & Xiao Yu

  2. Department of Clinical Laboratory, The Second Hospital, and Advanced Medical Research Institute, Cheeloo College of Medicine, Shandong University, Jinan, China

    Hui Lin, Peng Xiao & Jin-Peng Sun

  3. Key Laboratory of Experimental Teratology of the Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China

    Hui Lin, Peng Xiao, Shengchao Guo, Zhao Yang, Qing-Tao He, Chao Zhang, Yu-Qi Ping, Ru-Jia Zhao, Dan Jiang & Yue-Tong Xi

  4. Key Laboratory of Molecular Cardiovascular Science of the Ministry of Education, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Beijing, China

    Hui Lin & Jin-Peng Sun

  5. Department of Biology, Southern University of Science and Technology, Shenzhen, China

    Rui-Qian Bu, Chen-Yang Xue, Bai-Sheng Yang & Zhongmin Liu

  6. School of Medicine, Tsinghua University, Beijing, China

    Daopeng Yuan

  7. School of Life Sciences, University of Science and Technology of China, Hefei, China

    Zhong-Liang Zhu

  8. Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, China

    Chuan-Xin Zhang, Han Zhao & Jin-Peng Sun

  9. School of Pharmacy, Binzhou Medical University, Yantai, China

    Chuan-Shun Ma & Dao-Lai Zhang

  10. Institute of Reproductive Medicine, School of Medicine, Nantong University, Nantong, China

    Xiao-Ning Zhang & Xu-Hui Zeng

  11. Key Laboratory of Male Reproductive Health, National Research Institute for Family Planning, National Health and Family Planning Commission, Beijing, China

    Jian-Yuan Li

  12. Department of Urology, Peking University Third Hospital, Beijing, China

    Hao-Cheng Lin

  13. Department of Obstetrics/Gynecology, Joint Laboratory of Reproductive Medicine (SCU-CUHK), Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, China

    Wen-Ming Xu

  14. Key Laboratory of Infection and Immunity of Shandong Province, Department of Pharmacology, School of Basic Medical Sciences, Shandong University, Jinan, China

    Fan Yi

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  1. Hui Lin
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  2. Peng Xiao
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Contributions

J.-P.S. and X.Y. conceived, designed and supervised the overall project. Z.L. and J.-P.S. designed and supervised the cryo-EM sample preparation, data collection and structural refinement. J.-P.S., X.Y., F.Y., Z.L., P.X., Z.Y., S.G. and H.L. participated in data analysis and interpretation. S.G., P.X. and D.Y. generated the ADGRG2 insect cell expression construct, established the ADGRG2–Gs complex formation strategy and prepared protein samples for cryo-EM. S.G. and P.X. generated viruses and infected Sf9 cells for large quantitative protein production. H.L. performed steroid ligands screening assay for ADGRG2. S.G. and Y.-Q.P. prepared negative staining. R.-Q.B. evaluated the sample by negative-stain EM. R.-Q.B., C.-Y.X. and B.-S.Y. prepared the cryo-EM grids, collected the cryo-EM data and performed cryo-EM map calculations under the supervision of Z.L. R.-Q.B. and Z.L. performed the model building and refinement with assistance from Z.-L.Z., D.Y. and Q.-T.H. C.Z. performed molecular dynamics stimulations. J.-P.S., X.Y., P.X., S.-C.G., Z.Y. and H.L. designed all the mutations for determination of ligand-binding sites. H.L., S.-C.G., C.-H.L., D.J. and D.-L.Z. generated all constructs and mutants for the cell-based assays. H.L., S.-C.G., R.-J.Z., S.H., C.-S.M. and Y.-T.X. performed ELISAs and cAMP accumulation assays. Z.Y. performed FlAsH–BRET assays. C.-X.Z., X.-N.Z., H.-C.L., X.-H.Z. and H.Z. performed the western blot assay. S.G., H.L., P.X., Z.Y., J.-Y.L. and W.-M.X. prepared the figures. J.-P.S., X.Y. and Z.L. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Fan Yi, Zhongmin Liu, Jin-Peng Sun or Xiao Yu.

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Nature Chemical Biology thanks Bryan Roth, Jean-Ju Chung 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 Reconstitution of ADGRG2-Gs complex.

a, SDS-PAGE of samples of in vitro reconstituted apo-ADGRG2-AA-120-CT in complex with wild-type Gs heterotrimer, dominant negative Gs (DNGs) heterotrimer, or DNGs heterotrimer stabilized with Nb35 after Flag-tagged antibody pulling down. Full SDS-PAGE scans were shown. Experiments were repeated three times with similar results. b, ADGRG2-120-AA-CT (120-CT) showed higher expression yield in Sf9-insect cell expression system compared with wild-type ADGRG2. Full SDS-PAGE scans were shown. Experiments were repeated three times with similar results. c, Representative concentration dependent curve of the VPM-p15-induced cAMP accumulation in HEK293 cells overexpressing similar levels (as shown in Extended Data Fig. 1d) of wild-type ADGRG2 or ADGRG2-120-AA-CT using Glosensor assay. Values are the mean ± SEM of three independent experiments performed in triplicate (n = 3). d, ELISA experiments showing similar expression levels of wild-type ADGRG2 and ADGRG2-120-AA-CT on HEK293 cell surface when indicated amounts of plasmids were transiently transfected. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). Comparison between ADGRG2-WT and ADGRG2-AA-CT was determined by two-sided one-way ANOVA with Tukey’s test. ns, no statistical significance (P = 0.7149). e-f, Representative elution profile and SDS-PAGE of the size-exclusion chromatography peak of apo-ADGRG2-AA-120-CT-DNGs (apo-ADGRG2-FL-DNGs) (e) or DHEA-ADGRG2-βT-120-CT-DNGs (DHEA-ADGRG2-βT-DNGs) (f) complex. Full SDS-PAGE scans were shown. Experiments were repeated three times with similar results. g, Two-dimensional class averages of negative staining sample showing distinct N-terminal structural features of the apo-ADGRG2-FL-DNGs complex. Scale bar: 10 nm. h-j, Representative cryo-EM micrograph (scale bar: 20 nm) and 2D class averages (scale bar: 50 nm) of the apo-ADGRG2-FL-DNGs (h), DHEA-ADGRG2-βT-DNGs (i), and DHEA-ADGRG2-AA-120-CT-mini-Gs (DHEA-ADGRG2-FL-mini-Gs) (j) complexes. The 2D class average for apo-ADGRG2-FL-DNGs complex was shown in Fig. 1b. Representative Cryo-EM micrographs from 9,997 movies (h) or 5,222 movies (i) or 4,780 movies (j) and representative two-dimensional class averages determined using approximately 3.46 million (i) or 0.66 million (j) particles from 2D classification were shown. k-m, Schematic flow chart of the image processing steps for the apo-ADGRG2-FL-DNGs (k), DHEA-ADGRG2-βT-DNGs (l), and DHEA-ADGRG2-FL-mini-Gs (m) complexes.

Source data

Extended Data Fig. 2 Local resolution of cryo-EM map and biochemical validation of the apo-ADGRG2-FL-DNGs, DHEA-ADGRG2-βT-DNGs, and DHEA-ADGRG2-FL-mini-Gs complex structures.

a-c, Local resolution map of the final 3D reconstruction of the apo-ADGRG2-FL-DNGs (a), DHEA-ADGRG2-βT-DNGs (b), and DHEA-ADGRG2-FL-mini-Gs (c) complexes estimated using RESMAP. d-f, Gold-standard Fourier shell correlation (FSC) and cross-validation of model to cryo-EM density maps corresponding to apo-ADGRG2- DNGs (d), DHEA-ADGRG2-βT-DNGs (e), or DHEA-ADGRG2- FL-mini-Gs (f) complexes, respectively. g-h, Cryo-EM density maps and the models of apo-ADGRG2-FL -DNGs (g) and DHEA-ADGRG2-FL- mini-Gs (h) complexes are shown for all transmembrane helices, helix 8, DHEA (h), and α-helix 5 of Gαs. Notably, the EM density between the I755 and P762 could not be defined. The density map of DHEA-ADGRG2-βT-DNGs complexes is shown in Supplementary Fig. 2.

Extended Data Fig. 3 Detailed descriptions of ECL2 region of ADGRG2.

a, Cryo-EM density maps of ECL2 region in apo-ADGRG2-FL DNGs, DHEA-ADGRG2-FL- mini-Gs and DHEA-ADGRG2-βT-DNGs complex structures, respectively. b, Structural presentation of the ECL2 of ADGRG2 in apo-ADGRG2- FLDNGs, DHEA-ADGRG2-FL- mini-Gs and DHEA-ADGRG2-βT-DNGs complex structures, respectively.

Extended Data Fig. 4 Key structural elements in TM6 and TM7 of ADGRG2.

a-b, Structural comparison of DNGs-coupled apo-ADGRG2 (slate) with Gs-coupled β2AR (PDB: 3SN6, gray) at the “PIF motif” region (a). Note that ADGRG2 does not contain similar residues of the triad I3.40aP5.50aF6.44a motif in Class A GPCR subfamily, and the equivalent residues at the I3.40aP5.50aF6.44a motif of ADGRG2 were separated from each other without tight packing (b). c, Cryo-EM densities of the conserved “upper quaternary core (UQC)” of ADGRG2 consisting of F7013.44, F7865.43, M7043.47 and W8386.53. d, Cryo-EM densities of the key residues stabilizing the TM6-TM7 kinks in apo-ADGRG2-DNGs complex. e, Sequence alignment of residues forming the TM6-TM7 kinks in ADGRG subfamily members and selected receptors from other adhesion GPCR subfamilies. The conserved residues are highlighted with colors. f, ELISA experiments showing similar expression levels of wild-type (WT) ADGRG2 and its mutants on HEK293 cell surface when indicated amounts of plasmids were transiently transfected. Related to Extended Data Fig. 4g. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). Comparison between wild-type ADGRG2 and its mutants was determined by two-sided one-way ANOVA with Tukey’s test. n.s., no statistical significance. (P = 0.5819, 0.5777, 0.4031 from left to right) g, Representative dose response curves of VPM-p15 induced cAMP accumulation in HEK293 cells over-expressing wild-type (WT) ADGRG2 or its mutants. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). h, Structural comparison of the TM6 (upper) and TM7 (lower) helices of Apo-ADGRG2-DNGs (slate) with those of ADGRF1 (PDB: 7WU3, pink), ADGRD1 (PDB: 7EPT, yellow-orange), ADGRG5 (PDB: 7EQ1, sky-blue), ADGRG1 (PDB: 7SF8, pale-cyan), ADGRL3 (PDB: 7SF7, gray), ADGRG4 (PDB: 7WUJ, pale-green) and ADGRG2-β (PDB: 7WUQ, yellow) (from left to right).

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Extended Data Fig. 5 Identification of DHEA as the agonist of ADGRG2.

a, Vertical cross section of the putative ligand binding pocket in apo-ADGRG2 (left panel). The net-shaped deep layer of the pocket is outlined, and the surrounding residues are indicated (right panel). b, EM densities of the putative ligand binding pocket residues of DNGs-coupled apo ADGRG2. Data are correlated to a. c, Comparison between pocket residues in structures of DNGs-coupled apo ADGRG2 and Go-coupled ADGRG3-Cortisol (PDB: 7D77). Apo ADGRG2 was depicted in light blue and ADGRG3 was depicted in yellow. d-l, Representative dose response curves of the steroid ligands induced cAMP accumulation in HEK293 cells over-expressing wild-type mADGRG2 or hADGRG2 using Glosensor assay. Note that the 0.1% DMSO was used as the control. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). m, ELISA experiments showing similar expression levels of mADGRG2 and human GPR126 on HEK293 cell surface when indicated amounts of plasmids were transiently transfected. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). ns, no statistical significance. Comparison between ADGRG2 and GPR126 was determined by two-sided one-way ANOVA with Tukey’s test. (P = 0.6177) n-o, Representative dose response curves of VPM-p15, DHEA or DHEAS induced cAMP accumulation (n) or PrPc induced cAMP accumulation (o) in HEK293 cells over-expressing mADGRG2 or GPR126. The transiently transfected with indicated amount of mADGRG2 and GPR126 plasmid were shown in m. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). p, Bar graph presentation of the efficacies of VPM-p15, DHEA or DHEAS toward mADGRG2 and PrPc toward GPR126. The efficacies generated from data shown in n and o are normalized and presented as the response percentage for VPM-p15. The similar expression levels of mADGRG2 and GPR126 (ADGRG6) were validated by ELISA (as shown in m). Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). ***P < 0.001; **P < 0.01, DHEA or DHEAS-induced ADGRG2 activation or PrPc-induced GPR126 activation compared with VPM-p15-inducedADGRG2 activation using two-sided one-way ANOVA with Tukey’s test (P = 0.0004, 0.001, 0.0003 from left to right).

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Extended Data Fig. 6 Screening of steroid hormone for ADGRG2-Gq coupling and the ligand induced-conformational changes within ADGRG2 extracellular domains.

a, Heatmap represents the Gq signaling activation induced by steroid ligands (100 μM) or VPM-p15 (100 μM) in mADGRG2- (left panel) or hADGRG2- (right panel) -overexpressing HEK293 cells. The 0.1% DMSO was used as the control, VPM-p15 was applied as a positive control. b, Concentration dependent Gq activation measured by G trimer dissociation BRET assay in HEK293 cells overexpressing ATIR in response to AII stimulation. Representative curves from three independent experiments were shown (n = 3). c, Concentration dependent Gq activation measured by G trimer dissociation BRET assay in HEK293 cells overexpressing mADGRG2-FL in response to DHEA or VPM-p15 stimulation. Representative curves from three independent experiments were shown (n = 3). d, Schematic representation of the FlAsH-BRET assay design. NanoLuc (Nluc) was inserted at the N-terminus of ADGRG2-β, and the FlAsH motif (CCPGCC) was incorporated in the designated positions at the extracellular loops (ECLs) of the receptor. e, Detailed description of the FlAsH motif incorporation sites at the ECLs of ADGRG2 according to 3-dimensional structure shown in b. f, ELISA experiments showing similar expression levels of the wild-type ADGRG2 and six FlAsH-BRET sensors. Data are normalized to the expression level of the wild-type ADGRG2. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). n.s., no statistical significance; Comparison between wild-type ADGRG2 and six FlAsH-BRET sensors was determined by two-sided one-way ANOVA with Tukey’s test (P = 0.9776, 0.821, 0.5814, 0.3245, 0.2459, 0.9261 from left to right). g-h, Representative dose response curve of six ADGRG2 FlAsH-BRET sensors in response to DHEA (e) or DOC (f) stimulation. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). i-j, The maximal response of ADGRG2 FlAsH-BRET sensors upon DHEA (g) or DOC (h) stimulation. Data are derived from the dose-response curves in e and f. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). **P < 0.01; ***P < 0.001; ND, not detected; Comparison between FlAsH-BRET sensors stimulated with DHEA or DOC and those stimulated with control vehicle. were determined by two-sided one-way ANOVA with Tukey’s test (g: P = 0.0004, <0.0001; h: P = 0.0002, 0.0015, 0.0013, 0.0006 from left to right).

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Extended Data Fig. 7 Interaction modes of DHEA with ADGRG2.

a, Schematic diagrams showing the sequence features of the ADGRG2 constructs used for the reconstitution of DHEA-ADGRG2-DNGs complex. The N-terminal Stachel sequence truncated ADGRG2-β subunit construct (ADGRG2-βT) was used. The C-tail was replaced with that of GPR120. b, Representative concentration dependent curve of the DHEA-induced cAMP accumulation in HEK293 cells overexpressing ADGRG2-βT or control (pcDNA3.1) using Glosensor assay. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). c-d, Orthogonal views of and structural models for the DHEA-ADGRG2-βT-120CT-DNGs-Nb35 (c) and DHEA-ADGRG2-AA-120CT-miniGs-Nb35 (d) complexes. The DHEA densities were highlighted with red dashed squares. e, Fitting of the DHEA (Right panel) or CHS (Left panel) into the EM density of the ligand binding pocket in the DHEA-ADGRG2-βT-DNGs complex structure. The DHEA can be fit into the EM density while CHS cannot. f, Representative concentration dependent curve of the DHEA-induced cAMP accumulation in ADGRG2-βT-overexpressed HEK293 cells in the absence or presence of 100 μM CHS. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). g, Diagram of DHEA interactions in the ligand binding pocket of ADGRG2. Hydrogen bonds are depicted as red dashed lines. h, Assignment of DHEA (yellow) in the ligand binding pocket of ADGRG2 (green). DHEA could be fit into the density in two conformational states. In the ‘upper’ state, DHEA forms a hydrogen bond (red dashed line) with a water molecule via its 3-hydroxyl group. i, Superposition of the structures of DHEA-ADGRG2-FL and DHEA-ADGRG2-βT complexes indicate that DHEA adopt a similar pose in the orthosteric ligand pocket of ADGRG2. For clarity, only the vertical cross section of ADGRG2-FL was shown. DHEA in DHEA-ADGRG2-FL is pink while in DHEA-ADGRG2-βT is yellow. j, Clear EM densities enabled unambiguous assignment of DHEA, a water molecule, and surrounding residues in the ligand binding pocket of ADGRG2-FL. The hydrogen bond formed between DHEA and water molecule is shown in red dashed line.

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Extended Data Fig. 8 Effects of mutations of putative binding pocket residues of ADGRG2 on DHEA binding.

a-c, Representative dose response curves of DHEA induced cAMP accumulation in HEK293 cells over-expressing wild-type ADGRG2 or its mutants using Glosensor assay. The dashed lines depicting data of wild-type ADGRG2 in (a) are represented again in (b) and (c) for comparison. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). The similar expression levels of wild-type receptors and mutants were validated by ELISA (as shown in Supplementary Fig. 5a-b). d and h, The efficacy changes of DHEA-induced cAMP accumulation forwild-type and mutant ADGRG2 (d) and the maximal response of ADGRG2 FlAsH-BRET sensor S3 (annotated as WT) and the sensor-based alanine mutants to DHEA stimulation (h). Data are correlated to a-c and e-g. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). **P < 0.01; *P < 0.05; n.s., no statistical significance; ND, not detected; Comparison between wild-type ADGRG2 (d) or FlAsH-BRET sensor S3 (h) and their mutants were determined by two-sided one-way ANOVA with Tukey’s test (d: P = 0.0139, 0.0101, 0.0088, 0.0416, 0.4114, 0.8433, 0.2144, 0.0061; h: P = 0.0195, 0.1243, 0.0073, 0.0846, 0.0327 from left to right). e-g, Representative concentration dependent curve of ADGRG2 FlAsH-BRET sensor S3 (annotated as WT) and the sensor-based alanine mutants in response to DHEA stimulation. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). The similar expression levels of wild-type receptors and mutants were validated by ELISA (as shown in Supplementary Fig. 5c). i, Representative dose response curves of VPM-p15 induced cAMP accumulation in HEK293 cells over-expressing wild-type ADGRG2 or its mutants using Glosensor assay. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). j, The effect of mutations on the efficacy of VPM-p15-induced cAMP accumulation. The bar graph was generated from data shown in i. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). **P < 0.01; ***P < 0.01; ND, not detected; Comparison between wild-type ADGRG2 and its mutants was determined by two-sided one-way ANOVA with Tukey’s test (P = 0.0009, 0.0012 from top to bottom).

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Extended Data Fig. 9 Comparison of ligand binding pocket between DHEA-ADGRG2-FL and Cortisol-ADGRG3.

a, Comparison of ligand binding pocket between DHEA-ADGRG2-FL and Cortisol-ADGRG3 (PDB: 7D77). Compared with Cortisol, the DHEA has a 30˚ tilting toward TM5-TM6. The angles for DHEA-ADGRG2-FL TM6-TM7 kinks were labeled by two-way arrows in red. b, Structural representation of three conserved large hydrophobic residues forming direct contacts with ligands shared by both DHEA-ADGRG2-FL and Cortisol-ADGRG3. c, Sequence alignment of conserved pocket residues involved in ligand recognition in DHEA-coupled ADGRG2-FL and Cortisol-bound ADGRG3.

Extended Data Fig. 10 Effects of mutations of putative binding pocket residues of ADGRG2 on DOC binding.

a, RMSD analysis of DOC-ADGRG2 during 200-ns molecular dynamics simulation trajectories by GROMACS. RMSDs of DOC (lower panel) or DOC binding pocket residues (upper panel) are shown. The initial modeled complex state after equilibration (0 ns) was used for calculation. b, Binding mode of DOC in ADGRG2 according to computational simulation. c, Diagram of DOC interactions in the ligand binding pocket of ADGRG2. Hydrogen bonds are depicted as red dashed lines. d-f, Representative dose response curve of ADGRG2 FlAsH-BRET sensor S3 (annotated as WT) and the sensor-based alanine mutants in response to DOC stimulation. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). The expression levels of ADGRG2 mutants were normalized to equivalent levels of their wild-type (WT) receptors by adjusting the amount of plasmid transfected according to ELISA assay data shown in Supplementary Fig. 5c. g, The maximal response of ADGRG2 FlAsH-BRET sensor S3 (annotated as WT) and the sensor-based alanine mutants to DOC stimulation. Data are derived from the dose-response curves in Extended Data Fig. 10d-f and are normalized to the maximal response of the sensor S3. Values are mean ± SEM from three independent experiments performed in triplicates (n = 3). *P < 0.05; n.s., no significant difference; ND, not detected; FlAsH-BRET sensor S3 based alanine mutants were compared to the sensor S3. All data were analyzed by two-sided one-way ANOVA with Tukey’s test (P = 0.0673, 0.0220, 0.2403, 0.3698, 0.1591, 0.2406, 0.7043, 0.1264, 0.2, 0.8616 from left to right).

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Lin, H., Xiao, P., Bu, RQ. et al. Structures of the ADGRG2–Gs complex in apo and ligand-bound forms. Nat Chem Biol 18, 1196–1203 (2022). https://doi.org/10.1038/s41589-022-01084-6

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