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

The Journal of Biological Chemistry
M C HerzigL M Leeb-Lundberg

Abstract

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 t...Continue Reading

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May 14, 2004·European Journal of Pharmacology·Francesca BellucciCarlo Alberto Maggi
Sep 23, 2008·Chembiochem : a European Journal of Chemical Biology·Artur GieldonHarald Schwalbe
Feb 29, 2000·The Journal of Biological Chemistry·S BastianJ L Paquet
Nov 20, 2015·Biological Chemistry·Rafael Filippelli-SilvaSuma I Shimuta

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