THE AGONIST BINDING-SITE ON THE BOVINE BRADYKININ B2 RECEPTOR IS ADJACENT TO A SULFHYDRYL AND IS DIFFERENTIATED FROM THE ANTAGONIST BINDING-SITE BY CHEMICAL CROSS-LINKING

Chemical cross-linking was used to analyze the binding sites for the a gonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on the bovine B2 bradykinin receptor. [H-3]BK and [H-3]NPC17731 bound with hi gh affinity to the same B2 receptor in bovine myometrial membranes as determined by the total number of specific binding sites and pharmacol ogical specificity of the binding of these two radioligands. Cross-lin king experiments were done using a series of bifunctional reagents rea ctive either primarily to amines (homobifunctional) or reactive to ami nes in one end and to sulfhydryls in the opposite end (heterobifunctio nal). All the heterobifunctional reagents plus the homobifunctional ar ylhalide 1,5-difluoro-2,4-dinitrobenzene were effective in cross-linki ng the [H-3]BK N terminus specifically to a sulfhydryl in the receptor , and this crosslinking occurred at 5-100 mu M reagent, In contrast, t he homobifunctional N-hydroxysuccinimide ester reagents, at less than or equal to 1 mM, were only able to cross-link [H-3]BK to membrane pro teins nonspecifically. The sulfhydryl reagents N-ethylnaleimide, iodoa cetamide, and phenylarsine oxide blocked cross linking, whereas these re agents did not inhibit reversible specific [H-3]BK binding. Immunob lotting with anti-BK antiserum revealed that low concentrations of BK (5-50 mu M) were cross-linked to a receptor-specific species of 65 kDa . All cross-linking of [H-3]NPC17731 was nonspecific with both homobif unctional and heterobifunctional reagents. The 65 kDa receptor specifi c species was observed on anti-HOE140 immunoblots, but only when prote ins were cross linked with very high concentrations of HOE140 (greater than or equal to 500 nM). Our results provide direct biochemical evid ence that the binding site for the agonist BK in the bovine B2 recepto r is adjacent to a cysteine and is differentiated from the binding sit e(s) for the antagonists NPC17731 and HOE140.