MUTATION OF ASPARTATE RESIDUES IN THE 3RD EXTRACELLULAR LOOP OF THE RAT B-2 BRADYKININ RECEPTOR DECREASES AFFINITY FOR BRADYKININ

Two aspartates in the third extracellular loop of the rat B-2 bradykinin (BK) receptor have been implicated as important residues for agonist binding. Asp(268) and Asp(286) were mutated to alanine residues and changes in agonist and antagonist binding affinity were examined. The IC50 value for BK as a competitor of [H-3] NPC 17731 binding to the rat wild type receptor was 1.1 nM, while the Ala(268) and Ala(286) receptor mutants exhibited IC50 values of 19 nM and 28 nM, respectively. The Ala(268)Ala(286) receptor mutant exhibited an IC50 for BK of 500 nM. These mutations had little effect on binding affinity when NPC 17761, a BK antagonist, was used to compete [H-3] NPC 17731 binding. Electrophysiological examinationPof Xenopus oocytes expressing wild type or Ala(268)Ala(286) receptors confirmed the importance of the Asp(268) and Asp(286) residues for BK recognition. BK activated the mutant receptor with comparable efficacy relative to the wild type receptor, but a 1750-fold reduction in potency was observed. (C) 1994 Academic Press, Inc.