Identification of a key region of kinin B1 receptor for high affinity binding of peptide antagonists

To investigate the molecular basis for the specificity of ligand recognition in human kinin B-1 (B1R) and B-2 (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 B-1 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 B-1 agonist [H-3]desArg(10)-kallidin (desArg(10)-KD) and the B-1 antagonist [H-3]desArg(10)-[Leu(9)]KD respectively, as compared with the wild-type B1R. Moreover, the substitution of the B-1 TM VII by its B-2 homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567, High affinity for desArg(10)-KD binding was fully regained when the B-2 residue Thr(287) was replaced in construct 6 by the corresponding B-1 Leu(294) residue. When the B-2 residue Tyr(295) was exchanged with the corresponding B-1 Phe(302), 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 B-2 receptor antagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residues, which may not be directly involved in the binding of B1R ligands and, hence, their Thr(287) and Tyr(295) B-2 counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.