EXTRACELLULAR DOMAINS OF THE BRADYKININ B2 RECEPTOR INVOLVED IN LIGAND-BINDING AND AGONIST SENSING DEFINED BY ANTIPEPTIDE ANTIBODIES

Many of the physiological functions of bradykinin are mediated via the B2 receptor, Little is known about binding sites for bradykinin on th e receptor, Therefore, antisera against peptides derived from the puta tive extracellular domains of the B2 receptor were raised, The antibod ies strongly reacted with their corresponding antigens and cross-react ed both with the denatured and the native B2 receptor, Affinity-purifi ed antibodies to the various extracellular domains were used to probe the contact sites between the receptor and its agonist, bradykinin or its antagonist HOE140, Antibodies to extracellular domain 3 (second lo op) efficiently interfered, in a concentration-dependent manner, with agonist and antagonist binding and vice versa, Antibodies to extracell ular domain 4 (third loop) blocked binding of the agonist but not of t he antagonist, whereas antibodies to extracellular domains 1 and 2 or to intracellular domains failed to block ligand binding, Antibodies to ectodomain 3 competed with agonistic anti-idiotypic antibodies for B2 receptor binding, Further, affinity-purified antibodies to the amino- terminal portion of extracellular domain 3 transiently increased intra cellular free Ca2+ concentration and thus are agonists, The Ca2+ signa l was specifically blocked by the B2 antagonist HOE140, By contrast, a ntibodies to the carboxyl-terminal segment of extracellular domain 4 f ailed to trigger Ca2+ release, The specific effects of antibodies to t he amino-terminal portion of extracellular domain 3 suggest that this portion of the B2 receptor may be involved in ligand binding and in ag onist function.