The N terminus of bradykinin when bound to the human bradykinin B2 receptor is adjacent to extracellular Cys20 and Cys277 in the receptor
(1996) In Journal of Biological Chemistry 271(47). p.29746-29751- 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... (More)
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.
(Less)
- author
- Herzig, Maryanne C.S. ; Nash, Norman R. ; Connolly, Maureen ; Kyle, Donald J. and Fredrik Leeb-Lundberg, L. M. LU
- publishing date
- 1996-11-30
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 271
- issue
- 47
- pages
- 29746 - 29751
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- scopus:0029860809
- pmid:8939910
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.271.47.29746
- language
- English
- LU publication?
- no
- id
- 0a810e68-81fe-4e97-917c-3420f61b63e6
- date added to LUP
- 2019-06-10 11:15:02
- date last changed
- 2024-01-01 09:27:19
@article{0a810e68-81fe-4e97-917c-3420f61b63e6,
abstract = {{<p>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, Cys<sup>20</sup>, Cys<sup>103</sup>, Cys<sup>184</sup>, and Cys<sup>277</sup>, in the receptor were mutated to serines, and single and double mutants were transfected into COS-7 cells. The Ser<sup>20</sup> and Ser<sup>277</sup> single mutants and the Ser<sup>20</sup>/Ser<sup>277</sup> double mutant bound [<sup>3</sup>H]BK and the antagonist [<sup>3</sup>H]NPC17731 with pharmacological profiles identical to the wild- type B2 receptor. In contrast, the Ser<sup>103</sup> and Ser<sup>184</sup> 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 Ser<sup>20</sup> and Ser<sup>277</sup> single mutant receptors, whereas these reagents were unable to cross-link BK to the Ser<sup>20</sup>/Ser<sup>277</sup> double mutant. These results show that Cys<sup>103</sup> and Cys<sup>184</sup> are both required for expression of high affinity agonist and antagonist binding sites in the human B2 receptor, while Cys<sup>20</sup> and Cys<sup>277</sup> 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 Cys<sup>277</sup> in extracellular domain 4 and Cys<sup>20</sup> in extracellular domain 1 of the receptor.</p>}},
author = {{Herzig, Maryanne C.S. and Nash, Norman R. and Connolly, Maureen and Kyle, Donald J. and Fredrik Leeb-Lundberg, L. M.}},
issn = {{0021-9258}},
language = {{eng}},
month = {{11}},
number = {{47}},
pages = {{29746--29751}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{Journal of Biological Chemistry}},
title = {{The N terminus of bradykinin when bound to the human bradykinin B2 receptor is adjacent to extracellular Cys<sup>20</sup> and Cys<sup>277</sup> in the receptor}},
url = {{http://dx.doi.org/10.1074/jbc.271.47.29746}},
doi = {{10.1074/jbc.271.47.29746}},
volume = {{271}},
year = {{1996}},
}