Comparative analysis reveals molecular adaptation of mammalian HCA2 to microbial metabolites

Metabolite-sensing G protein-coupled receptors (GPCRs), such as hydroxycarboxylic acid receptor 2 (HCA₂), translate endogenous and microbial signals into physiological responses, regulating metabolism and immunity, yet the extent of HCA₂ functional diversification across mammals remain unclear. Here, comparative pharmacology, metabolomics, evolutionary analysis, and structural mapping of mammalian HCA₂ orthologs reveal extensive functional diversification across mammals, especially in odd-toed ungulates. Notably, African rhinoceros HCA₂ exhibits multiple HCA₃-like substitutions, resulting in loss of responsiveness to HCA₂ agonists and increased sensitivity to HCA₃-specific ligands. Positive selection analyses and metabolomic profiling of fecal extracts implicate microbiome-derived metabolites, particularly phenylpropionic and trans-cinnamic acid, as potential drivers of this adaptive shift. Mutagenesis experiments identified key amino acid substitutions in extracellular and transmembrane regions that modulate ligand potency and efficacy. These findings demonstrate that mammalian HCA₂ receptors have undergone lineage-specific molecular evolution shaped by host-microbe metabolic interactions, highlighting how ecological contexts drive receptor adaptation and functional diversification.