Mutation of aspartate residues in the third extracellular loop of the rat B2 bradykinin receptor decreases affinity for bradykinin.

Two aspartates in the third extracellular loop of the rat B2 bradykinin (BK) receptor have been implicated as important residues for agonist binding. Asp268 and Asp286 were mutated to alanine residues and changes in agonist and antagonist binding affinity were examined. The IC50 value for BK as a competitor of [3H] NPC 17731 binding to the rat wild type receptor was 1.1 nM, while the Ala268 and Ala286 receptor mutants exhibited IC50 values of 19 nM and 28 nM, respectively. The Ala268Ala268 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 [3H] NPC 17731 binding. Electrophysiological examination of Xenopus oocytes expressing wild type or Ala268 Ala286 receptors confirmed the importance of the Asp268 and Asp286 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.