A different molecular interaction of bradykinin and the synthetic agonist FR190997 with the human B2 receptor: evidence from mutational analysis

摘要

class="enumerated" style="list-style-type:decimal">Binding affinity at the [³H]-BK binding site and activity as inositol phosphate (IP) production by the peptide bradykinin (BK) and the nonpeptide were studied at wild-type or point-mutated human B2 receptors (hB2R) expressed in CHO cells.The effect of the following mutations were analyzed: E47A (TM1), W86A and T89A (TM2), I110A, L114A and S117A (TM3), T158A, M165T and L166F (TM4), T197A and S211A (TM5), F252A, W256A and F259A (TM6), S291A, F292A, Y295A and Y295F (TM7), and the double mutation W256A/Y295F.As the wild-type receptor-binding affinity of was 40-fold lower than BK, whereas their agonist potency was comparable, both agonists produced similar maximal effects (Emax). Mutations were evaluated as affecting the affinity and/or efficacy of compared with BK.Two mutations were found to impair the agonist affinity of both agonists drastically: W86A and F259A. BK agonist affinity (pEC50) was reduced by 1400- and 150-fold, and that of was reduced by 400- and 25-fold, at the W86A and F259A mutant B2 receptors, respectively.Contrary to BK, the affinity of was selectively decreased at I110A, Y295A, and Y295F mutants (>10³-fold), and a different efficacy was measured at the Y295 mutants, being devoid of the capability to trigger IP production at Y295A mutant.L114A, F252A, and W256A selectively impaired the efficacy of , whereas its binding affinity was not affected. As a consequence, behaved as a high-affinity antagonist in blocking the IP production induced by BK.The lack of capability of to activate or to bind the double mutant W256A/Y295F suggests that these residues are part of the same binding site, which is also important for receptor activation by the nonpeptide ligand.Overall, by means of mutational analysis, we indicate an hB2R recognition site for the nonpeptide agonist (between TM3, 6, and 7), different from that of BK, and show that in the same binding crevice some mutations (L114, W256, and F252) are selectively responsible for the agonist properties of only .