Study of GPR81, the Lactate Receptor, from Distant Species Identifies Residues and Motifs Critical for GPR81 Functions

Receptors from distant species may have conserved functions despite significant differences in protein sequences. Whereas the noncritical residues are often changed in distant species, the amino acids critical in receptor functions are often conserved. Studying the conserved residues between receptors from distant species offers valuable information to probe the roles of residues in receptor function. We identified two zebrafish receptors (zGPR81-1 and zGPR81-2) that show approximately 60% identity to human GPR81, GPR109a, and GPR109b but respond only to L-lactate and not to the GPR109a ligands. Protein sequence comparison among zebrafish GPR81s, mammalian GPR81s, GPR109a, and GPR109b identified a common structure (six Cys residues at the extracellular domains that potentially form three disulfide bonds) in this subfamily of receptors. In addition, a number of residues conserved in all GPR81s but not in GPR109s have been identified. Furthermore, we identified a conserved motif, C165-E166-S167-F168, at the second extracellular loop of GPR81. Using site-directed mutagenesis, we showed that Arg71 at the transmembrane domain 2 is very critical for GPR81 function. In addition, we demonstrated that the C165-E166-S167-F168 motif at the second extracellular loop is critical for GPR81 function, and the conserved six Cys residues at the extracellular regions are necessary for GPR81 function. It is important to mention that for those residues important for GPR81 function, the corresponding residues or motifs in GPR109a are also critical for GPR109a function. These findings help us better understand the interaction between lactate and GPR81 and provide useful information for GPR81 ligand design.