Evolutionary study and structural basis of proton sensing by Mus GPR4 and Xenopus GPR4

Animals have evolved pH-sensing membrane receptors, such as G-protein-coupled receptor 4 (GPR4), to monitor pH changes related to their physiology and generate adaptive reactions. However, the evolutionary trajectory and structural mechanism of proton sensing by GPR4 remain unresolved. Here, we observed a positive correlation between the optimal pH of GPR4 activity and the blood pH range across different species. By solving 7-cryoelectron microscopy (cryo-EM) structures of Xenopus tropicalis GPR4 (xtGPR4) and Mus musculus GPR4 (mmGPR4) under varying pH conditions, we identified that protonation of HECL2-45.47 and H7.36 enabled polar network establishment and tighter association between the extracellular loop 2 (ECL2) and 7 transmembrane (7TM) domain, as well as a conserved propagating path, which are common mechanisms underlying protonation-induced GPR4 activation across different species. Moreover, protonation of distinct extracellular HECL2-45.41 contributed to the more acidic optimal pH range of xtGPR4. Overall, our study revealed common and distinct mechanisms of proton sensing by GPR4, from a structural, functional, and evolutionary perspective. [Display omitted] •7 apo or GPR4-Gs complex structures at different pHs were solved•Proton sensing by two conserved histidines underlies GPR4 activation across different species•The acid shift for xtGPR4 activation may facilitate adaptation to its aquatic life•xtGPR4 and mmGPR4 evolved unique proton-sensing mechanisms Structures of GPR4 from Mus musculus and Xenopus under various pH conditions are solved to elucidate the common and distinct activation mechanisms of protonation-induced GPR4 from different species. Evolutionary analysis and functional study identify that the GPR4 receptor from Xenopus has a distinct optimal pH activation profile associated with its adaptation to aquatic life.