Abstract
The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.
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Data availability
Coordinates for the GPR161–miniGs–Gβγ–Nb35 protein complex have been deposited in the RCSB PDB under accession code 8SMV. EM density maps for GPR161–miniGs–Gβγ–Nb35 complex have been deposited in the Electron Microscopy Data Bank under accession code EMD-40603. The molecular dynamics simulation trajectories have been deposited in the Zenodo database under https://doi.org/10.5281/zenodo.7887650. Publicly available PDB entries used in this study are 6LI3, 4LDO, 3SN6, 7Y89 and 8HMV. Protein sequence data for sequence alignments are available from the National Center for Biotechnology Information’s RefSeq. Sequences used in the alignment in Extended Data Fig. 7 are NP_001254539, NP_001074595, XP_004938289, XP_041427552, NP_001007200, XP_019638841, XM_002731669 and XP_782439. Mass spectrometry data and are available online. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Institutes of Health (NIH) grant nos. R01GM108799 (A.S.E.), 1R35GM149287 (A.S.E.), Grant 1 P50 MH122379 (D.F.C.), R01AR054396 and R01HD089918 (J.F.R.), R35GM144136 (S.M.) and R01GM138992 (R.O.D. and A.M.). Additional support came from a National Science Foundation Graduate Research Fellowship (M.K.) and Human Frontier Science Program Long-Term Fellowship grant no. LT000916/2018-L (C.-M.S.). Cryo-EM equipment at UCSF is partially supported by NIH grant nos. S10OD020054 and S10OD021741. Some of this work was performed at the Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the NIH Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (grant no. U24 GM129541). We thank C. Hecksel at S2C2 personnel for invaluable support and assistance. We are indebted to J. Eggenschwiler for his generous gift of anti-Gli2 antibody. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. A.M. acknowledges support from the Edward Mallinckrodt, Jr. Foundation and the Vallee Foundation. A.M. is a Chan Zuckerberg Biohub Investigator.
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Contributions
N.H., S.H. and I.D. cloned, expressed, and biochemically optimized the purification of GPR161 complex constructs for structural studies. N.H., S.H. and I.D. performed cryo-EM data collection, with help from the SLAC Cryo-EM Center and data processing. N.H., S.H., I.D. and A.M. built and refined models of GPR161. N.H. and S.H. generated receptor constructs and determined expression levels by flow cytometry and performed signaling studies, complementation assays and analyzed the data. M.K. and C.-M.S. performed and analyzed molecular dynamics simulations under the supervision of R.O.D. N.H. prepared samples for, performed and analyzed SPA data with A.M. Z.C. performed and analyzed mass spectrometry experiments using reagents provided by D.F.C. under the supervision of A.S.E. S.-H.H., V.R.P. and S.M. prepared constructs, performed and analyzed GPR161 localization and Hedgehog pathway repression experiments. S.P.B. performed phylogenetic analysis under the supervision of D.S.M. and additional phylogenetic analysis was provided by J.F.R. P.T., D.R. and E.S. analyzed GPR161 variants. All authors contributed to figures. N.H., S.H., A.M. and S.M. wrote the paper, with edits and approval from all authors. A.M. supervised the overall project.
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A.M. is a founder of Epiodyne and Stipple Bio, consults for Abalone and serves on the scientific advisory board of Septerna. R.O.D. serves on the scientific advisory board of Septerna. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Biochemical preparation of GPR161-miniGs complex.
a) Cartoon depiction of GPR161 stabilization, solubilization, and purification. b) Size-exclusion chromatogram (left) and SDS-PAGE gel (right) of purified GPR161-Gs complex with Nb35. Purification and SDS-PAGE gel were done once and not repeated. Chromatogram and SDS-PAGE gel are shown for preparation used for cryo-EM analysis.
Extended Data Fig. 2 Cryogenic electron microscopy processing of GPR161.
a) A representative motion-corrected cryogenic electron microscopy (cryo-EM) micrograph obtained from a Titan Krios microscope (n = 8,294). b) A subset of highly populated, reference-free 2D-class averages. c) Schematic showing the cryo-EM data processing workflow. Initial processing was performed using UCSF MotionCor2 and cryoSPARC. Particles were transferred using the pyem script package to RELION for alignment-free 3D classification. Finally, particles were processed in cisTEM using the manual refinement job type with a 7TM mask followed by a full particle mask. Dashed boxes indicated selected classes. d) Gold-standard Fourier Shell Correlation (GSFSC) curve for final refined and sharpened map computed in cryoSPARC. e) Euler angle distribution of final refined map computed in cryoSPARC.
Extended Data Fig. 3 Cryo-EM local density.
a) Orthogonal views of local resolution for the sharpened, final map of GPR161-Gs complex computed with local resolution in cryoSPARC. b) Close up of local resolution for sterol density. c) Isolated cryo-EM densities from the unsharpened, final map of GPR161 complex. Shown are the transmembrane (TM) helices, extracellular loops, and cholesterol-like density.
Extended Data Fig. 4 Comparison to additional GPCR structures.
a) Structural comparison of GPR161 heterotrimer complex and β2AR heterotrimer complex (PDB ID: 3SN6) (ref. 28). GPR161 has the same hallmarks of GPCR activation as the prototypical receptor, β2AR. b) View of the GPR161 ECL2 inside the canonical Class A GPCR binding site. ECL2 makes multiple hydrophobic interactions deep within the pocket. The superficial part of the pocket harbors ionic interactions between ECL2 and the binding pocket. c) Structural comparison of GPR161 to other orphan GPCRs with self-activating ECL2, including GPR17 (PDB ID: 7Y89) and GPR21 (PDB ID: 8HMV)31,32. The cis-interaction of ECL2 with the canonical ligand-binding site is seen across self-activating orphan GPCRs but the precise loop conformation changes between receptors. d) Luminescence for β-arrestin recruitment in the PRESTO-Tango assay when compared across 314 GPCRs (data replotted from Kroeze WM et al.22, n = 4 for each target, shown as mean ± s.e.m. of technical replicates). GPR21 yields a signal slightly above the median. GPR52 yields a signal one order of magnitude above the median. GPR161 and GPR17 yield a signal about two orders of magnitude above the median.
Extended Data Fig. 5 GPR161 molecular dynamics simulation trajectories.
a) Time traces of ECL2 position in all six unrestrained simulations of GPR161 with miniGs removed. ECL2 position is represented by distance between W182 and T189. b) Time traces of distance between cholesterol and GPR161 residue W3277.56 in all six unrestrained simulations of GPR161 with miniGs removed. c) Time traces of distance between cholesterol and GPR161 residue W3277.56 in all six simulations where GPR161 residues that contact miniGs are restrained to their miniGs bound conformation. d) A comparison of the cryo-EM structure (green and magenta) to a representative snapshot from an unrestrained simulation of GPR161 with miniGs removed shows that, in the absence of miniGs, the intracellular ends of TM6 and TM7 move inwards, obstructing the Gs binding site.
Extended Data Fig. 6 Surface expression of GPR161 mutants.
a) Representative flow cytometry surface expression histograms for receptors and mutants used in cell-based assays. b) Surface expression of receptors and mutants quantified by anti-FLAG-A647 median fluorescence intensity ± sd from n = 3 (for L465PC-term) or n = 4 (for rest) biologically independent samples.
Extended Data Fig. 7 Phylogenetic analysis of GPR161.
a) BLAST search results for Human GPR161 (Uniprot: Q8N6U8). Sequences are plotted from highest confidence (E-Value) and highest sequence identity (% identity) to lowest. Representative organisms spanning the full range of homologous GPR161 sequences are listed. b) Full sequence alignment of eight GPR161 model organism sequences identified in BLAST search.
Extended Data Fig. 8 Photolabeling with LKM238 and mass spectrometry sequence coverage.
a) Product ion spectrum of LKM238-labeled GPR161-miniGs with peptides mapped to TM6. This peptide is modified with a mass consistent with LKM238 at position K2676.32. Red brackets and peaks indicate product ions that contain the LKM238 adduct. b) Mass spectrometric sequence coverage of GPR161-miniGs. Underlined segments indicate transmembrane spanning helices, red font indicates peptides identified by tandem MS analysis and gray font indicates glycosylation sites.
Extended Data Fig. 9 GPR161 localization and repression of GLI2 ciliary trafficking.
a) Representative images of GPR161 mutants on ciliary localization and GLI2 repression in ciliary tips in NIH 3T3 cells. NIH 3T3 Flp-In CRISPR based Gpr161−/− cells stably expressing untagged mouse wild-type or Gpr161 mutants were starved for 24 h upon confluence and were treated for further 24 h ± SAG (500 nM). After fixation, cells were immunostained with anti-GLI2 (red), anti-GPR161 (green), anti-acetylated, and centrosome (AcTub; PCNT purple) antibodies. Whole cell images with an arrow indicating imaged cilia. Scale bar, 5 µm. b) Quantification of GPR161 positive cilia indicating trafficking and egress of GPR161 from cilia in the pathway off and on state, respectively. ECL2 mutants do not traffic to cilia suggesting impaired biogenesis. GPR161-V129E3.54 does not egress from cilia following pathway activation and GPR161-L465PC-term has reduced egress compared to GPR161. (*P < 0.05; ns, not significant; two-way ANOVA followed by Šidák’s multiple comparison tests; Adjusted P values for DMSO vs. SAG: NIH3T3, Gpr161−/− + WT, +AAA7.52, 7.56, 8.51, +L465PC-term, + AAA7.52, 7.56, 8.51 L465PC-term = <0.0001, Gpr161−/−, Gpr161−/− + W182RECL2, +W182GECL2 = > 0.9999, Gpr161−/− + V129E3.54 = 0.997) c) Quantification of GLI2 positive cilia indicating Hedgehog pathway activation. ECL2 mutants and GPR161-V129E3.54 do not rescue, similar to Gpr161−/−. For b,c, data are shown from n = 3 independent experiments from images taken from 2-3 different regions/experiment and counting 15-30 cells/region. Data are mean ± s.d. (*P < 0.05; ns, not significant; two-way ANOVA followed by Šidák’s multiple comparison tests; Adjusted P values for DMSO vs. SAG: NIH3T3, Gpr161−/− + WT = < 0.0001, Gpr161−/− = >0.9999, Gpr161−/− + W182RECL2 = 0.9705, Gpr161−/− + W182GECL2 = 0.9724, Gpr161−/− + V129E3.54 = 0.9882, Gpr161−/− + AAA7.52, 7.56, 8.51 + L465PC-term = 0.9917). d) Transcript abundance of wild-type and mutant Gpr161 constructs stably expressed in Gpr161−/− NIH3T3 cells quantified by quantitative RT-PCR. e) GPR161-V129E3.54 has reduced recruitment of miniGs compared to WT. Nanoluc complementation assay for receptor recruitment of miniGs. Data are mean ± s.d., n = 2 (for V129E3.54) or n = 4 (for GPR161) biologically independent samples (*P < 0.05; ns, not significant; Unpaired two-tailed t test with Welch’s correction; Adjusted P value: GPR161 vs. V129E3.54 = 0.0008). f) GPR161-V129E3.54 has similar recruitment of PKA-RI compared to GPR161. Nanoluc complementation assay for receptor recruitment of PKA-RI. Data are mean ± s.d., n = 2 (for V129E3.54) or n = 5 (for GPR161) biologically independent samples (*P < 0.05; ns, not significant; Unpaired two-tailed t test with Welch’s correction; Adjusted P value: GPR161 vs. V129E3.54 = 0.9406).
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Source Data Figs. 2–5 and Extended Data Figs. 1, 6, 8 and 9.
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Source Data Extended Data Fig. 1
Unprocessed gel from Extended Data Fig.1.
Source Data Extended Data Fig. 6
Flow cytometry expression data and unprocessed gel from Extended Data Fig. 6.
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Hoppe, N., Harrison, S., Hwang, SH. et al. GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway. Nat Struct Mol Biol 31, 667–677 (2024). https://doi.org/10.1038/s41594-024-01223-8
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DOI: https://doi.org/10.1038/s41594-024-01223-8
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