A SINGLE POSITION IN THE 3RD TRANSMEMBRANE DOMAINS OF THE HUMAN B1 AND B2 BRADYKININ RECEPTORS IS ADJACENT TO AND DISCRIMINATES BETWEEN THEC-TERMINAL RESIDUES OF SUBTYPE-SELECTIVE LIGANDS

In order to identify agonist-and antagonist-binding epitopes in the hu man B1 and B2 bradyknin (BK) receptors, we exploited the ability of th ese receptors to discriminate between peptide Ligands that differ only by the absence (B1) and presence (B2) of a C-terminal Arg, This was d one by constructing chimeric proteins in which specific domains were e xchanged between these receptors as recently described by us (Leeb, T. , Mathis, S. A. and Leeb-Lundberg, L. M. F. (1997) J. Biol. Chem. 272, 311-317). The constructs were then expressed in HEK293 and A10 cells and assayed by radioligand binding and by agonist-stimulated inositol phospholipid hydrolysis and intracellular Ca2+ mobilization. Substitut ion of the third transmembrane domain (TM-III) of the B1 receptor in t he B2 receptor (B2(B1III)) dramatically reduced the affinities of Ba-s elective peptide ligands including both the agonist BK and the antagon ist NPC17731. High affinity binding of both ligands to B2(E1III) was f ully regained when one residue, Lys(111), in TM-III of this chimera wa s replaced with the corresponding wild-type (WT) B2 receptor residue, Ser (B2(B1IIIS(111))). Replacement of Ser(111) with Lys in the WT B2 r eceptor decreased the affinities of BK and NPC17731 and increased the affinity of the B1-selective des-Arg(10) analog of NPC17731, NPC18565. The results show that the C-terminal residue of peptide agonists and antagonists when bound to the B2 receptor is adjacent to Ser(111) in t he receptor. A Lys at this position, as is the case in the WT B1 recep tor, provides a positive charge that repels the C-terminal Arg in Ba-s elective peptides and attracts the negative charge of the C terminus o f B1-selective peptides, which lack the C-terminal Arg. Therefore, the residues at this one single position are crucial in determining the p eptide selectivity of B1 and B2 BK receptors.