GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington’s disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52.
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The coordinates and structure factors for GPR52-Fla-apo, GPR52-Rub-apo, GPR52-Fla–c17 and GPR52–mini-Gs–Nb35 have been deposited in the PDB with accession codes 6LI1, 6LI2, 6LI0 and 6LI3, respectively. The cryo-EM 3D maps of the GPR52–mini-Gs–Nb35 complex have been deposited in the Electron Microscopy Data Bank (EMDB) with accession code EMD-0902. All other data relating to this study are available from the corresponding authors on reasonable request.
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This work was supported by the National Key Research and Development Program of China, grant 2018YFA0507000 (to F.X. and S.Z.), National Natural Science Foundation of China (31525007 to M.L., 81861128023 to F.X. and 31971178 to S.Z.), Shanghai Municipal Education Commission−Gaofeng Clinical Medicine Grant Support (20181711 to J.W.) and Shanghai Outstanding Academic Leader funding (19XD1422800 to F.X.). The diffraction data were collected at BL41XU at SPring-8 with JASRI proposal 2019A2704. We thank J. Liu, N. Chen and L. Xue of the BV facility at the iHuman Institute, ShanghaiTech University for support with protein expression; the staff of the Electron Microscopy System and Mass Spectrometry System at Shanghai Institute of Precision Medicine for technical support and assistance in data collection; M. Cao for help with the collection and analysis of cryo-EM data; Q. Sun and the Bio-EM facility at ShanghaiTech University for technical support; V. Katritch and P. Popov for suggestions on construct mutations; M. Hanson for help with processing of X-ray data; and R. C. Stevens for encouraging this work.
The authors declare no competing interests.
Peer review information Nature thanks Bryan Roth and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Schematic of the GPR52 constructs that were used for crystallization (residues 17—340). Thermostabilizing mutations (red) are A130W, A264L, W278Q, C314P, S318A, N321D and V323T. Cysteine residues (yellow), TEV cleavage site (pink) and disulfide bonds (orange dashed lines) are shown. b, Left, analytical size-exclusion chromatography of GPR52. Experiments were repeated three times with similar results. Right, superposition of GPR52-Fla-apo, GPR52-Rub-apo and GPR52–c17 structures shows the overall conserved helical arrangement. c, Crystal images of the GPR52–c17 complex (left), GPR52-Rub-apo (middle) and GPR52-Fla-apo (right). Experiments were repeated three times with similar results. d, ECL2 comparison of two apo structures (GPR52-Fla-apo, orange; GPR52-Rub-apo, cyan). The crystal packing of GPR52-Fla-apo (middle) and GPR52-Rub-apo (right) is also shown. Helix 8 of GPR52-Rub-apo is highlighted in red.
a, Basal activity of GPR52 mutants. Response-level values were compared with wild-type GPR52 by two-way ANOVA without repeated measures, followed by Dunnett’s post hoc test (****P < 0.0001). Data are mean ± s.e.m. (n = 3) b, Mapping of mutated residues (green) onto GPR52 crystal structures. c17 is shown in orange. c, Summary of functional potency (pEC50) values of c17 on the GPR52 mutants. Data are mean ± s.e.m. (n = 3). d, Relative surface expression levels of mutant constructs were monitored by a FACS staining assay (Methods) and normalized to the expression levels of wild-type GPR52. Data are mean ± s.e.m. (n = 3). NA, not available.
a, Comparison of the ECL2 ALM-occupied orthosteric binding pocket of GPR52 with agonist-bound pockets of A1R (PDB 6D9H), A2AR (PDB 6GDG) and 5-HT1BR (PDB 6G79). b, PIF motif comparison of mini-Gs-coupled GPR52 (green) and A2AR (yellow; PDB 6GDG). c, Side view (left), cytoplasmic view (middle) and extracellular view (right) of A2AR in the mini-Gs-coupled state (green, PDB 6GDG) compared with the adenosine analogue (NECA; PDB 2YDV)-bound state (pink). d, Side view (left), cytoplasmic view (middle) and extracellular view (right) of 5-HT1BR in the mini-Go-coupled state (in complex with donitriptan) (green; PDB 6G79) compared with the ergotamine-bound state (pink; PDB 4IAR). e, DRY motif of GPR52-apo, 5-HT1BR–ergotamine (PDB 4IAR) and A2AR–NECA (PDB 2YDV).
a, b, Size-exclusion chromatography profile (a) and corresponding SDS–PAGE gel (b) of the purified GPR52–mini-Gs–Nb35 complex. Experiments were repeated three times with similar results. c, Representative reference-free 2D cryo-EM average of the GPR52–mini-Gs–Nb35 complex. d, GPR52 with point mutations A130W3.41 and C314P7.50 maintained around 50% of the activity relative to the wild-type protein, according to the cAMP response level. Data are mean ± s.e.m. (n = 3). e, Representative cryo-EM micrograph of the GPR52–mini-Gs–Nb35 complex. f, Reference-free 2D averages of the GPR52–mini-Gs–Nb35 complex. g, Final 3D density map coloured according to the local resolution. h, Gold-standard FSC curves, showing the overall nominal resolution at 3.3 Å. i, Angular distribution of the particles used for the final reconstruction of the GPR52–mini-Gs–Nb35 complex.
Extended Data Fig. 5 Flow chart for the cryo-EM data processing and structure determination of the GPR52–mini-Gs–Nb35 complex.
See Methods for details. The final reconstruction has an average resolution of 3.3 Å. All the images in this figure were created in UCSF Chimera.
a, Atomic model of GPR52 transmembrane helices, ECL2 and ICL2 in the cryo-EM density map. The molecular model is shown in stick representation and the cryo-EM map as mesh. b, Stereo views of the electron density maps of ECL2. Left, the 2Fo − Fc map of ECL2 from the GPR52-Rub-apo crystal structure. Right, the electron density map of ECL2 from the GPR52–mini-Gs complex structure.
Extended Data Fig. 7 Comparison of the GPR52–mini-Gs interface with that of other GPCR–G-protein complexes.
a, b, Front view (a) and back view (b) of the GPR52–mini-Gs interface. GPR52 (centre) and mini-Gs (right) are in surface representation and coloured according to the electrostatic potential (blue, positive; red, negative). c, The GPR52–mini-Gs interface. GPR52 and mini-Gs are in cartoon representation and coloured in green and grey, respectively. A magnified view of the interface is shown on the right in surface representation. d–g, Magnified views of the interface between other receptors and G proteins in surface representation. h, Buried surface area of the interfaces between receptors and G proteins, calculated by PyMOL.
Extended Data Fig. 8 Docking position of GPR52 agonists, structural similarity network and sequence alignment of GPR52.
a, Docking position of GPR52 agonists with different scaffolds: WO-459 (left); 7m (centre) and FTBMT (right). The PR52 residues that are involved in ligand binding are shown as green sticks and c17 is shown as grey sticks, for reference. b, Structural similarity network of class-A GPCRs with reported inactive structures. c, Sequence alignment of GPR52 and GPR21 (yellow, less than 5.0 Å to ligand; green indicates key residues for structural features).
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Lin, X., Li, M., Wang, N. et al. Structural basis of ligand recognition and self-activation of orphan GPR52. Nature 579, 152–157 (2020). https://doi.org/10.1038/s41586-020-2019-0