The N terminus of bradykinin when bound to the human bradykinin B2 receptor is adjacent to extracellular Cys²⁰ and Cys²⁷⁷ in the receptor

Chemical cross-linking combined with site-directed mutagenesis was used to evaluate the role of extracellular cysteines and their positions relative to the binding site for the agonist bradykinin (BK) in the human BK B2 receptor. All extracellular cysteines, Cys²⁰, Cys¹⁰³, Cys¹⁸⁴, and Cys²⁷⁷, in the receptor were mutated to serines, and single and double mutants were transfected into COS-7 cells. The Ser²⁰ and Ser²⁷⁷ single mutants and the Ser²⁰/Ser²⁷⁷ double mutant bound [³H]BK and the antagonist [³H]NPC17731 with pharmacological profiles identical to the wild- type B2 receptor. In contrast, the Ser¹⁰³ and Ser¹⁸⁴ single mutants were unable to bind either of the two radioligands. However, these mutants were still expressed as determined by immunoblotting with anti-B2 receptor antibodies. Previous studies on the bovine B2 receptor showed that bifunctional reagents, which are reactive to amines at one end and to free sulfhydryls in the opposite end, cross-link the N terminus of receptor-bound BK to a sulfhydryl in the receptor (Herzig, M. C. S., and Leeb-Lundberg, L. M. F. (1995) J. Biol. Chem. 270, 20591-20598). Here, we show that m- maleimidobenzoyl-N-hydroxysuccinimide ester and 1,5-difluoro-2,4- dinitrobenzene cross-linked BK to the wild-type human B2 receptor and the Ser²⁰ and Ser²⁷⁷ single mutant receptors, whereas these reagents were unable to cross-link BK to the Ser²⁰/Ser²⁷⁷ double mutant. These results show that Cys¹⁰³ and Cys¹⁸⁴ are both required for expression of high affinity agonist and antagonist binding sites in the human B2 receptor, while Cys²⁰ and Cys²⁷⁷ are not required. Furthermore, the results provide direct biochemical evidence that the N terminus of BK, when bound to the B2 receptor, is adjacent to Cys²⁷⁷ in extracellular domain 4 and Cys²⁰ in extracellular domain 1 of the receptor.