MUTATIONS IN THE B-2 BRADYKININ RECEPTOR REVEAL A DIFFERENT PATTERN OF CONTACTS FOR PEPTIDIC AGONISTS AND PEPTIDIC ANTAGONISTS

The B-2 bradykinin receptor, a seven-helix transmembrane receptor, bin ds the inflammatory mediator bradykinin (BK) and the structurally rela ted peptide antagonist HOE-140, The binding of HOE-140 and the binding of bradykinin are mutually exclusive and competitive. Fifty-four site -specific receptor mutations were made, BK's affinity is reduced 2200- fold by F261A, 490-fold by T265A, 60-fold by D286A, and 3-10-fold by N 200A, D268A, and Q290A, In contrast, HOE-140 affinity is reduced less than 7-fold by F254A, F261A, Y297A, and Q262A. The almost complete dis cordance of mutations that affect BK binding versus HOE-140 binding is surprising, but it was paralleled by the effect of single changes in BK and HOE-140. [Ala(9)]BK and [Ala(6)]BK are reduced in receptor bind ing affinity 27,000- and 150-fold, respectively, while [Ala(9)]HOE-140 affinity is reduced 7-fold and [Ala(6)]HOE-140 affinity is unchanged. NMR spectroscopy of all of the peptidic analogs of BK or HOE-140 reve aled a beta-turn at the C terminus. Models of the receptor-ligand comp lex suggested that bradykinin is bound partially inside the helical bu ndle of the receptor with the amino terminus emerging from the extrace llular side of helical bundle, In these models a salt bridge occurs be tween Arg(9) and Asp(286); the models also place Phe(8) in a hydrophob ic pocket midway through the transmembrane region. Models of HOE-140 b inding to the receptor place its beta-turn one alpha-helical turn deep er and closer to helix 7 and helix 1 as compared with bradykinin-recep tor complex models.