Cryo-EM structure of the bicarbonate receptor GPR30
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan
- Department of Ion Signaling and Response, The Sakaguchi Laboratory, Keio University School of Medicine, Japan
- Department of Biochemistry, Juntendo University Graduate School of Medicine, Japan
- Department of Signal Exploration, The Sakaguchi Laboratory, Keio University School of Medicine, Japan
Figures
Figure 1 with 2 supplements
Overall structure of the G-protein-coupled receptor 30 (GPR30)-miniGsqiβ1γ2-scFv16 complex.
(A) Unsharpened cryo-electron microscopy (cryo-EM) density map of the GPR30-miniGsqiβ1γ2-scFv16-Nb35 complex, with the components individually colored. (B) The refined structure of the complex is shown as a ribbon representation.
Figure 1—figure supplement 1
Sample purification.
(A) Schematic representation of the fusion-G system. (B) Fluorescence detection size exclusion chromatography (FSEC) analysis of complex formation by G-protein-coupled receptor 30 (GPR30). The trace from solubilized cells expressing only GPR30 is orange, and that from cells co-expressing GPR30 and G-protein is blue. (C) Size exclusion chromatography of the GPR30-G-protein complex on a Superose 6 Increase column. The fraction shaded in blue was collected. (D) SDS-PAGE gel of samples after size exclusion chromatography, stained with Coomassie Brilliant Blue.
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Figure 1—figure supplement 1—source data 1
Original SDS-PAGE data corresponding to Figure 1—figure supplement 1, panel D.
- https://cdn.elifesciences.org/articles/99874/elife-99874-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
SDS-PAGE data corresponding to Figure 1—figure supplement 1, panel D.
- https://cdn.elifesciences.org/articles/99874/elife-99874-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Cryo-electron microscopy (cryo-EM) analysis.
Flowchart of the cryo-EM data processing for the G-protein-coupled receptor 30 (GPR30)-Gq complex, including particle projection selection, classification, and 3D density map reconstruction. The 3D density map was refined with a mask on the receptor. Details are provided in the Methods section.
Figure 2 with 2 supplements
Receptor structure.
(A–C) Overall structure of the receptor, viewed from the membrane plane (A), intracellular side (B), and extracellular side (C).
Figure 2—figure supplement 1
ECL2 density.
(A–C) Top view of density map around disulfide bond between ECL2 and TM3 at counter levels 8.5 (A), 7.25 (B), and 6 (C). (D–F) Side view of density map around disulfide bond between ECL2 and TM3 at counter levels 8.5 (D), 7.25 (E), and 6 (F).
Figure 2—figure supplement 2
Structural comparison with AlphaFold-predicted structure.
(A–C) Superimposition of the cryo-electron microscopy (cryo-EM) (orange) and AF3 (khaki) structures. (A) Overall view of the receptor, (B) focused on the intracellular side, and (C) focused on the extracellular side.
Figure 3 with 5 supplements
Architecture of the extracellular pocket.
(A) Molecular surface of the extracellular side. (B) Cross section of the pocket. (C–E) Residues facing pocket B (C), pocket C (D), and pocket A (E). In panel (E), only residues with reduced bicarbonate responses are highlighted. (F) Calcium assay using stable HEK293 cell lines expressing the N-terminal HA-tagged wild-type (WT) and mutant GPR30. The mutants D1112.56A, N2766.52A, Q296ECL3A; E121ECL1A, R122ECL1A, S1343.29A, Q2155.39A, E2185.42A; D210ECL2A, Q1383.33A; and D125ECL1A, C207ECL2A, P711.44A are highlighted in red, blue, green, and purple, respectively. The cells were stimulated by the indicated concentrations of NaHCO3 and 50 µM ATP at the timepoint of t=20 s. The Y-axis indicates the difference between the maximum and minimum fluorescent values during 15–60 s, normalized by those with ATP stimulation. Nonlinear regression (four-parameter) was used for curve fitting. (G–H) X-Y plot of parameters calculated by fluorescence-activated cell sorting (FACS) analysis and Ca assay. D1112.56A; D210ECL2A, Q1383.33A; D125ECL1A, C207ECL2A, P711.44A; and mock are colored in red, green, purple, and gray, respectively (G). Cell surface (x-axis) and total (y-axis) expression of G-protein-coupled receptor 30 (GPR30) (H). X-Y plot of cell surface (x-axis) and normalized maximum responses (y-axis) (I). X-Y plot of cell surface (x-axis) and EC50 (y-axis). EC50 was calculated using the data shown in panel (F).
Figure 3—figure supplement 1
Cell surface expression of stable wild-type (WT) and G-protein-coupled receptor 30 (GPR30) mutants.
Flow cytometric-based assay to analyze WT and mutant GPR30 cell surface expression. Cell surface expression, defined as the HA-positive subset (red) compared with isotype control staining (blue), is indicated on each histogram.
Figure 3—figure supplement 2
Flow cytometric gating criteria to analyze G-protein-coupled receptor 30 (GPR30) cell surface expression.
(A, B) Gating used in the analysis. (C, D) Cell surface expression is defined as the HA-positive subset (red) compared with isotype control staining (blue). (C) Mock cells, (D) wild-type (WT) GPR30-expressing cells.
Figure 3—figure supplement 3
Flow cytometric-based assay to analyze the whole-cell expression of wild-type (WT) G-protein-coupled receptor 30 (GPR30) and its mutants.
(A) Gating used in the analysis. (B) Whole-cell expression is defined as the HA-positive subset (red) compared with isotype control staining (blue).
Figure 3—figure supplement 4
Cell surface expression and bicarbonate-induced activation of HEK293 cells transiently expressing G-protein-coupled receptor 30 (GPR30) mutants.
(A, B) TGFα shedding assay using HEK293 cells transfected with HA-tagged hGPR30. The mutants D1112.56A; D125ECL1A, C207ECL2A, P711.44A, and H3077.36A; and S1343.29A, D210ECL2A, and Q2155.39A are highlighted in red, blue, and purple, respectively. (C) Whole-cell expression of HA-tagged mutants, analyzed by western blotting with 20 μg of whole-cell lysate per lane. (D) Cell surface expression of HA-tagged mutants, using cell surface biotinylation and avidin immunoprecipitation, analyzed by western blotting with 1.5 μg of cell surface protein per lane. (E, F) Quantitative analysis of HA expression in (C and D). Statistical analysis: $p=0.01, §p<0.001 compared to HA-hGPR30 cells stimulated with vehicle, #p<0.0001 compared to HA-hGPR30 cells stimulated with 11 mM NaHCO3, using two-tailed unpaired t-test with Bonferroni’s correction after two-way ANOVA. Data are presented as mean values (A) and mean values ± SEM (B).
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Figure 3—figure supplement 4—source data 1
Uncropped western blotting membrane photos for (C) Whole cell and (D) Cell surface expression of HA-tagged mutants.
- https://cdn.elifesciences.org/articles/99874/elife-99874-fig3-figsupp4-data1-v1.zip
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Figure 3—figure supplement 4—source data 2
Uncropped western blotting membrane photos for (C) Whole cell and (D) Cell surface expression of HA-tagged mutants.
- https://cdn.elifesciences.org/articles/99874/elife-99874-fig3-figsupp4-data2-v1.zip
Figure 3—figure supplement 5
Conservation of the G-protein-coupled receptor 30 (GPR30) homologs.
Amino acid alignment of representative GPR30 homologs.
Figure 4
G-protein coupling.
(A) Hydrogen-bonding interactions between the C-terminal α5-helix residues and the receptor. (B) Electrostatic and hydrogen-binding interactions between ICL3 and the α5-helix. (C–H) Interface between ICL2 and Gq, with residues involved in hydrophobic interactions represented by CPK models. G-protein-bound G-protein-coupled receptors (GPCRs) used in the comparison are as follows: H1R-Gq (PDB 7DFL, gray), B2R-Gq (PDB 7F6I, yellow-green), MRGPRX2-Gq (PDB 7S8L, green), 5-HT2A-Gq (PDB 6WHA, gray), and GPR103-Gq (PDB 8ZH8, red). (I) Comparison of the angles and positions of α5h and αN relative to the receptor. (J) Superimposition of the Gα subunits.
Figure 5 with 1 supplement
Structural comparison with related G-protein-coupled receptors (GPCRs).
(A) Structural comparison of G-protein-coupled receptor 30 (GPR30) and type 2 angiotensin II receptor (AT2R) (PDB 5UNF). (B) Interactions around P711.44 in TM1 of GPR30. (C–F) Conformational changes of TM1 upon agonist binding in CB1 (C), A2AR (D), and ETB (E). The agonist-bound states are colored with the respective colors, while the inactive states are colored gray. The PDB codes used in this figure are CB1-active (PDB 5XRA), CB1-inactive (PDB 5TGZ), A2AR-active (PDB 6GDG), A2AR-inactive (PDB 3EML), ETB-active (PDB 8IY5), and ETB-inactive (PDB 5X93).
Figure 5—figure supplement 1
Sequence alignment of G-protein-coupled receptor 30 (GPR30) and type 2 angiotensin II receptor (AT2R).
Figure 6
Structural comparison with G-protein-coupled receptor 30 (GPR30) bound to Lys05.
(A, B) Superimposition of bicarbonate-GPR30, Lys05-binding GPR30 (PDB 8XOF), and apo-GPR30 (PDB 8XOG) structures, viewed from the extracellular side. (C) Comparison of the angles and positions of α5h and αN relative to the receptor. (D) Superimposition of the Gα subunits.
Tables
Table 1
Cryo-electron microscopy (cryo-EM) data collection, refinement, and validation statistics.
| Data collection | GPR30-Gq complex | ||
|---|---|---|---|
| Microscope | Titan Krios (Thermo Fisher Scientific) | ||
| Voltage (keV) | 300 | ||
| Electron exposure (e-/Å2) | 50.660 | ||
| Detector | Gatan K3 Summit camera (Gatan) | ||
| Magnification | ×105,000 | ||
| Defocus range (μm) | –0.8 to 1.6 | ||
| Pixel size (Å/pix) | 1.16 | ||
| Number of movies | 9824 | ||
| Symmetry | C1 | ||
| Picked particles | 10,148,422 | ||
| Final particles | 522,404 | ||
| Map resolution (Å) | 3.15 | ||
| FSC threshold | 0.143 | ||
| Model refinement | |||
| Atoms | |||
| R.m.s. deviations from ideal | |||
| Bond lengths (Å) | 0.003 | ||
| Bond angles (°) | 0.528 | ||
| Validation | |||
| Clashscore | 9.56 | ||
| Rotamers Outliers(%) | 0.23 | ||
| Ramachandran plot | |||
| Favored (%) | 96.32 | ||
| Allowed (%) | 3.68 | ||
| Outlier (%) | 0 | ||
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/99874/elife-99874-mdarchecklist1-v1.docx
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Supplementary file 1
EC50 and maximum Ca response to bicarbonate by the mutant and Wild-type GPR30.
- https://cdn.elifesciences.org/articles/99874/elife-99874-supp1-v1.docx
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Cryo-EM structure of the bicarbonate receptor GPR30
eLife 13:RP99874.
https://doi.org/10.7554/eLife.99874.4
