Alterations of further conserved and nonconserved residues also affect ligand potency at GPR35. Residue 4.60 is an arginine in both human and rat orthologs of GPR35 and was converted to methionine, whereas residue 4.62, leucine in human but arginine in rat was swapped between species. Arginine 164 in ECL2 of human GPR35a was converted to serine, whereas the equivalent residue, 161 in rat GPR35, was altered to arginine. These mutants were then compared with the wild-type sequences in BRET-based GPR35 β-arrestin-2 interaction studies to explore effects on the potency of lodoxamide (A), bufrolin (B), zaprinast (C), and cromolyn disodium (D). Human GPR35a is shown on the left side of the figure, whereas rat GPR35 is shown on the right.