Overall structure of the GPR30-miniGsqiβ1γ2-scFv16 complex.

A Unsharpened 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.

Cryo-EM data collection, refinement and validation statistics

Receptor structure.

A–C Overall structure of the receptor, viewed from the membrane plane (A), intracellular side (B), and extracellular side (C). D Extracellular view of the ribbon model overlaid the cryo-EM map.

Architecture of the extracellular pocket.

A Interactions between the ECLs. Black dashed lines indicate hydrogen bonds. B Molecular surface of the extracellular side. C–F Residues facing pocket A (C), pocket B (D), pocket C (E), and pocket D (F).

Mutational analysis.

A, B TGFα shedding assay using HEK293 cells transfected with HA-tagged hGPR30. The mutants D111A; C207A, P71A, H307A; and D125A, S134A, D210A, Q215A are highlighted in red, blue, and purple, respectively. Statistical analysis: $ p = 0.0001, # p < 0.0001 compared to mock cells using two-tailed unpaired t-test with Bonferroni’s correction after two-way ANOVA. ns indicates no significant difference. Data are presented as mean values (A) and mean values ± SEM (B). C Residues that were subjected to mutant analysis are mapped to the structure. Only residues with reduced bicarbonate responses are colored.in magenta. D Density corresponding to bicarbonate. E Putative binding mode of bicarbonate. Black dashed lines indicate hydrogen bonds.

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 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.

Structural comparison with related GPCRS.

A Structural comparison of GPR30 and AT2R (PDB 5UNF). B Interactions around P70 in TM1 of GPR30. C–F Conformational changes of TM1 upon agonist binding in CB1 (C), β2AR (D), A2AR (E), and ETB (F). 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), β2AR-active (PDB 3SN6), β2AR-inactive (PDB 2RH1), A2AR-active(PDB 6GDG), A2AR-inactive (PDB 3EML), ETB-active (PDB 8IY5), and ETB-inactive (PDB 5X93).