The B 2 bradykinin receptor, a seven-helix transmembrane receptor, binds the inflammatory mediator bradykinin (BK) and the structurally related 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 N200A, D268A, and Q290A. In contrast, HOE-140 affinity is reduced less than 7-fold by F254A, F261A, Y297A, and Q262A. Bradykinin is a central initiator of acute and chronic inflammation and the associated pain and edema. Most of the acute and many of the chronic responses to bradykinin are mediated by B 2 bradykinin receptors (1, 2), while some of the chronic responses to bradykinin are mediated by B 1 bradykinin receptors (3). B 1 and B 2 bradykinin receptors are members of the G-protein-coupled seven-transmembrane (GPC-7TM) 1 receptor superfamily (4 -7). For B 1 receptors, [des-Arg 9 ]bradykinin is a more potent agonist than bradykinin, whereas B 2 receptors bind and respond to bradykinin about 10,000-fold more effectively than [des-Arg 9 ]bradykinin. Several peptidic B 2 bradykinin antagonists have been identified; these compounds reduce pain and inflammation (8 -12). Peptidic antagonists also reduce death from experimental shock (13-15). Bradykinin receptor antagonists are potentially useful in the treatment of pain, acute and chronic inflammation, shock, allergic or infectious rhinitis, and asthma. The peptidic antagonists are useful tools; but, to date, these compounds have made poor human therapeutic agents because of their poor bioavailability and formulation difficulties (16,17). The discovery of a nonpeptide antagonist of bradykinin would improve the prospects of treating bradykinin-instigated inflammation, pain, or edema. Thus, we have focused on a molecular understanding of the bradykinin receptor ligand binding site, believing that this information may help in the discovery and design of nonpeptidic antagonists.We used the results from molecular modeling studies of B 2 bradykinin receptors and NMR studies of peptidic agonists and antagonists to generate a number of models for agonist binding to the B 2 BKR. These models were tested by site-directed mutagenesis of the receptor and by making single amino acid changes in agonist and antagonist peptides. The data reveal a disparity between the way peptidic agonists and antagonists bind to the BKR. We attempt to reconcile the disparities by proposing new models of the BKR-ligand complex. -140 (18, 19). The product was HPLC-purified and then characterized by HPLC and mass spectrometry as to its chemical purity (Ն96%), identity, and specific activity (56.5 Ci/mmol). Media and other cell culture additives were from Life Technologies, Inc. Biochemicals and enzymes were from Boehringer Mannheim. Common reagents were from Sigma. EXPERIMENTAL PROCEDURES Materials MethodsStandard molecular biological and cell culture methods were used except as speci...