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 bradykin in (BK) receptor have been implicated as important residues for agonis t 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 ra t wild type receptor was 1.1 nM, while the Ala(268) and Ala(286) recep tor mutants exhibited IC50 values of 19 nM and 28 nM, respectively. Th e 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. Electro physiological examinationPof Xenopus oocytes expressing wild type or A la(268)Ala(286) receptors confirmed the importance of the Asp(268) and Asp(286) residues for BK recognition. BK activated the mutant recepto r with comparable efficacy relative to the wild type receptor, but a 1 750-fold reduction in potency was observed. (C) 1994 Academic Press, I nc.