Identification of a key region of kinin B-1 receptor for high affinity binding of peptide antagonists

To investigate the molecular basis for the specificity of ligand recognitio n 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 term inus, the human B2R domains by their B-1 counterparts. The chimeric constru ct 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 antag onist [H-3]desArg(10)-[Leu(9)]KD respectively, as compared with the wild-ty pe B1R. Moreover, the substitution of the B-1 TM VII by its B-2 homologue T M 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 corresp onding 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 a ntagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residue s, which may not be directly involved in the binding of B1R ligands and, he nce, their Thr(287) and Tyr(295) B-2 counterparts, are localized in a recep tor region, which plays a pivotal role in the binding selectivity of the pe ptide or pseudopeptide kinin ligands.