THE 6TH TRANSMEMBRANE DOMAINS OF THE HUMAN B1 AND B2 BRADYKININ RECEPTORS ARE STRUCTURALLY COMPATIBLE AND INVOLVED IN DISCRIMINATING BETWEEN SUBTYPE-SELECTIVE AGONISTS

In order to investigate the molecular basis for the ability of the hum an B1 and B2 bradykinin (BK) receptor subtypes to discriminate between subtype-selective ligands, we constructed chimeric proteins in which the sixth transmembrane domains (TM-VI) of these receptors were exchan ged, The pharmacological profiles of the constructs were analyzed by r adioligand binding in particulate preparations of transiently transfec ted HEK293 cells using the agonist [H-3]des-Arg(10)-kallidin and the a ntagonist [H-3]NPC17731. The ability of these constructs to transmit a n intracellular signal was measured in transiently transfected A10 cel ls, a vascular smooth muscle cell line, by single cell Ca2+ imaging. S ubstitution of B1 TM-VI into the B2 receptor (B2(B1VI)) dramatically r educed the affinity of the B2-selective agonist BK, whereas the affini ty of the B2-selective antagonist NPC17731 was unaltered. High affinit y BK binding was fully regained when two residues, Tyr(259) and Ala(26 3), near the extracellular surface of TM-VI in B2(B1VI), were replaced with the corresponding residues in the wild-type B2 receptor, which a re Phe(259) and Thr(263). The construct B1(B2VI), produced by substitu tion of B2 TM-VI into the B1 receptor, did not support high affinity b inding of the B1-selective agonist des-Arg(10)-kallidin. In contrast t o BK and des-Arg(10)-kallidin, the binding of the less subtype-selecti ve agonist kallidin showed little sensitivity to TM-VI exchange. These results show that TM-VI in the human B1 and B2 BK receptor subtypes, although only 36% identical, are structurally compatible. Furthermore, this domain contributes significantly to the ability of these recepto rs to discriminate between the subtype-selective agonists BK and des-A rg(10)-kallidin.