Molecular mechanism of pH sensing and activation in GPR4 reveals proton-mediated GPCR signaling

Abstract

Maintaining pH homeostasis is critical for cellular function across all living organisms. Proton-sensing G protein-coupled receptors (GPCRs), particularly GPR4, play a pivotal role in cellular responses to pH changes. Yet, the molecular mechanisms underlying their proton sensing and activation remain incompletely understood. Here we present high-resolution cryo-electron microscopy structures of GPR4 in complex with G proteins under physiological and acidic pH conditions. Our structures reveal an intricate proton-sensing mechanism driven by a sophisticated histidine network in the receptor's extracellular domain. Upon protonation of key histidines under acidic conditions, a remarkable conformational cascade is initiated, propagating from the extracellular region to the intracellular G protein-coupling interface. This dynamic process involves precise transmembrane helix rearrangements and conformational shifts of conserved motifs, mediated by strategically positioned water molecules. Notably, we discovered a bound bioactive lipid, lysophosphatidylcholine, which has positive allosteric effects on GPR4 activation. These findings provide a comprehensive framework for understanding proton sensing in GPCRs and the interplay between pH sensing and lipid regulation, offering insights into cellular pH homeostasis and potential therapies for pH-related disorders.

Fig. 1. Overall structures of GPR4–G_s/G_q complexes at acidic and physiological pH conditions.

a Schema diagram of GPR4 activation of G_s/G_q pathways at various pH conditions. b Curves showing pH-dependent cAMP accumulation in cells overexpressing GPR4 or pcDNA3.1. c LPC-induced activation of GPR4 at pH 7.4 measured in a dose-dependent manner using a cAMP accumulation assay. Values are represented as means ± SEM of three independent experiments (n = 3). d–f Overall structures and EM-density maps for GPR4 complexes. The receptors are colored blue (GPR4–G_s at pH 6.5), green (GPR4–G_s at pH 7.4), and salmon (GPR4–G_q at pH 7.4), respectively. Gα_s subunit is colored wheat and Gα_q subunit is colored purple. Colors of the Gβγ subunits are shown as indicated. Red balls refer to water molecules in our structures.

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.

Fig. 3. Proton recognition mode of GPR4–G_q complex at physiological pH.

a The preserved interaction between H17^1.32 and H80^2.67. b The interaction between H79^2.66 and the common water molecule. c The extended polar network of H165^ECL2 and H269^7.36. The rotational direction of residues is labeled by a black arrow indicating the rotation of H165^ECL2, compared with H165^ECL2 in the GPR4–G_s complexes, and polar interactions are shown as orange dashed lines. d, e The special networks involving the water molecules in G_q-coupled GPR4. The water molecules are displayed as green spheres. The polar interactions are shown as orange dashed lines. f, g Effects of mutations on cAMP and IP1 accumulation responses. ΔpEC₅₀ represents the difference between pEC₅₀ values of GPR4 WT and mutants. U.D. means undetectable because the maximum activation level is below 50% to determine pEC₅₀ values (f). E_max values represent the maximum cAMP or IP1 accumulation induced by various pH conditions in GPR4 WT and mutants, which are normalized to WT (g). Heat map is generated on the basis of the pEC₅₀ or E_max. Values are shown as means of three independent experiments. The original data are provided in Supplementary Fig. S11.

Fig. 4. Involvement of neutral phenylalanine and tyrosine residues in proton sensing.

a–c Detailed interactions of phenylalanine residues in GPR4 complexes. The GPR4–G_s complexes are colored blue (pH 6.5) and green (pH 7.4), respectively. The GPR4–G_q complex at pH 7.4 is colored salmon. d–f Detailed interactions of tyrosine residues in GPR4 complexes. Polar interactions are marked by orange dashed lines. g Effects of mutations on GPR4-induced cAMP accumulation. Colors are shown as indicated. The original data are provided in Supplementary Fig. S9. Values are shown as means ± SEM from three independent experiments. h Conformation distribution under pH 6.0 and pH 8.0. The side-chain minimal distance distributions of E170^ECL2–Y76^2.63 (upper) and E170^ECL2–Y98^3.33 (lower) are shown.

Fig. 5. Proton-induced activation state of GPR4.

a Overall structural comparison of the experimentally obtained GPR4–G_s complex at pH 6.5, the AlphaFold2 (AF2)-predicted GPR4 structure, the inactive structure of β₂AR (PDB: 2RH1), and the simulated GPR4 at pH 8.0. b–d The conformational changes of classical motifs in GPR4, including DRY (b), PIV (c), and DPxxY (d) motifs, compared with the inactive β₂AR. e Conformational changes of ECD upon activation. N-terminus and ECLs assemble to transduce activation signals. The transparent ones refer to their original positions and the normal ones refer to their positions after activation. The black dashed arrows indicate displacement directions. f, g Detailed conformational changes of residues related to activation of GPR4–G_s at pH 6.5 compared to the simulated GPR4 at pH 8.0. The former is colored in blue, and the latter is colored in gray. h, i Structural comparisons between the active and the inactive (simulated) GPR4. j The potential propagation path for signal transduction. Related residues are highlighted. Black arrows represent the movements of the receptor and specific residues. k Schematic diagram showing the proton-induced activation mechanism of GPR4. Direct interactions induced by protons are displayed by black arrows, and black dashed arrows indicate the connection of residues and conformational rearrangements of residues.

Fig. 6. Novel lipid regulation of GPR4.

a–c The overall view of LPC-binding sites around GPR4–G_s at pH 6.5 (a) and pH 7.4 (b), and those around GPR4–G_q at pH 7.4 (c), respectively. LPCs and water molecules are shown as sticks and red balls, respectively. d The engaged residues between LPCs and TM3/5 of GPR4. The polar interactions are displayed by orange dashed lines. e The effect of S200A mutation on LPC positive allosteric activity measured by pH-induced cAMP accumulation in GPR4 WT or mutants with or without 100 μM LPC. Values are represented as means ± SEM of three independent experiments (n = 3). f, g Structural superposition of GPR4 and ADHRF1 (PDB: 7WU3).