Fig. 2: Proton recognition mode of GPR4.

a The top view of active GPR4 with the distribution of protons around ECD. The disulfide bonds are labeled by orange dashed circles and the water molecules are displayed as spheres. Colors are shown as indicated. b, c The surface of ECD in the GPR4–G_s complex at pH 6.5 by electrostatic (b) and hydrophobic (c) analyses. d The structural superposition of ECD in the GPR4–G_s complexes at pH 6.5 and pH 7.4. e–i Detailed interactions and comparisons of GPR4 at pH 6.5 and pH 7.4. The displacement of residues and polar interactions are marked by black arrows and orange dashed lines, respectively. j Effects of GPR4 mutations on the potency of pH-induced cAMP accumulation. The black bars represent the pEC₅₀ values of pH-induced responses in wild-type GPR4 (WT) and mutants, while the red bars indicate the maximum cAMP concentrations induced by pH in GPR4 WT and mutants, both normalized to WT. A decrease in pEC₅₀ indicates reduced sensitivity to pH. The original data are provided in Supplementary Fig. S9. Values are shown as means ± SEM from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way ANOVA followed by multiple comparison test, compared with WT. k The extracellular conformation distribution under pH 6.0 and pH 8.0. The side-chain minimal distance distribution of D161^ECL2–H165^ECL2, D75^2.62–H79^2.66, E170^ECL2–H269^7.36, and D81^ECL1–H165^ECL2.