Identification of a Key Region of Kinin B1 Receptor for High Affinity Binding of Peptide Antagonists

Abstract To investigate the molecular basis for the specificity of ligand recognition in human kinin B1(B1R) and B2 (B2R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B2R domains by their B1 counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B2R (construct 6) displayed 7- and 20- fold decreased affinities for the B1 agonist [3H]desArg10-kallidin (desArg10-KD) and the B1 antagonist [3H]desArg10-[Leu9]-KD respectively, as compared with the wild-type B1R. Moreover, the substitution of the B1 TM VII by its B2homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg10-KD binding was fully regained when the B2 residue Thr287was replaced in construct 6 by the corresponding B1Leu294 residue. When the B2 residue Tyr295 was exchanged with the corresponding B1Phe302, high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B1R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B2 receptor antagonist, Hoe140. Therefore we proposed that Leu294 and Phe302residues, which may not be directly involved in the binding of B1R ligands and, hence, their Thr287 and Tyr295 B2 counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.