The sixth transmembrane domains of the human B1 and B2 bradykinin receptors are structurally compatible and involved in discriminating between subtype-selective agonists
(1997) In Journal of Biological Chemistry 272(1). p.311-317- Abstract
In order to investigate the molecular basis for the ability of the human B1 and B2 bradykinin (BK) receptor subtypes to discriminate between subtype- selective ligands, we constructed chimeric proteins in which the sixth transmembrane domains (TM-VI) of these receptors were exchanged. The pharmacological profiles of the constructs were analyzed by radioligand binding in particulate preparations of transiently transfected HEK293 cells using the agonist [3H]des-Arg10-kallidin and the antagonist (3H]NPC17731. The ability of these constructs to transmit an intracellular signal was measured in transiently transfected A10 cells, a vascular smooth muscle cell line, by single cell Ca2+ imaging.... (More)
In order to investigate the molecular basis for the ability of the human B1 and B2 bradykinin (BK) receptor subtypes to discriminate between subtype- selective ligands, we constructed chimeric proteins in which the sixth transmembrane domains (TM-VI) of these receptors were exchanged. The pharmacological profiles of the constructs were analyzed by radioligand binding in particulate preparations of transiently transfected HEK293 cells using the agonist [3H]des-Arg10-kallidin and the antagonist (3H]NPC17731. The ability of these constructs to transmit an intracellular signal was measured in transiently transfected A10 cells, a vascular smooth muscle cell line, by single cell Ca2+ imaging. Substitution of B1 TM-VI into the B2 receptor (B2(B1VI)) dramatically reduced the affinity of the B2-selective agonist BK, whereas the affinity of the B2-selective antagonist NPC17731 was unaltered. High affinity BK binding was fully regained when two residues, Tyr259 and Ala263, near the extracellular surface of TM-VI in B2(B1VI), were replaced with the corresponding residues in the wild-type B2 receptor, which are Phe259 and Thr263. The construct B1(B2VI), produced by substitution of B2 TM-VI into the B1 receptor, did not support high affinity binding of the B1-selective agonist des-Arg10-kallidin. In contrast to BK and des-Arg10-kallidin, the binding of the less subtype-selective agonist kallidin showed little sensitivity to TM-VI exchange. These results show that TM-VI in the human B1 and B2 BK receptor subtypes, although only 36% identical, are structurally compatible. Furthermore, this domain contributes significantly to the ability of these receptors to discriminate between the subtype-selective agonists BK and des-Arg10-kallidin.
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- author
- Leeb, Tosso ; Mathis, Sandra A. and Leeb-Lundberg, L. M.Fredrik LU
- publishing date
- 1997-01-18
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 272
- issue
- 1
- pages
- 311 - 317
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- pmid:8995263
- scopus:0031027029
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.272.1.311
- language
- English
- LU publication?
- no
- id
- a7701921-8a11-44ef-8322-eccacb1bf1c5
- date added to LUP
- 2019-06-10 11:11:10
- date last changed
- 2024-01-01 09:38:14
@article{a7701921-8a11-44ef-8322-eccacb1bf1c5,
abstract = {{<p>In order to investigate the molecular basis for the ability of the human B1 and B2 bradykinin (BK) receptor subtypes to discriminate between subtype- selective ligands, we constructed chimeric proteins in which the sixth transmembrane domains (TM-VI) of these receptors were exchanged. The pharmacological profiles of the constructs were analyzed by radioligand binding in particulate preparations of transiently transfected HEK293 cells using the agonist [<sup>3</sup>H]des-Arg<sup>10</sup>-kallidin and the antagonist (<sup>3</sup>H]NPC17731. The ability of these constructs to transmit an intracellular signal was measured in transiently transfected A10 cells, a vascular smooth muscle cell line, by single cell Ca<sup>2+</sup> imaging. Substitution of B1 TM-VI into the B2 receptor (B2(B1VI)) dramatically reduced the affinity of the B2-selective agonist BK, whereas the affinity of the B2-selective antagonist NPC17731 was unaltered. High affinity BK binding was fully regained when two residues, Tyr<sup>259</sup> and Ala<sup>263</sup>, near the extracellular surface of TM-VI in B2(B1VI), were replaced with the corresponding residues in the wild-type B2 receptor, which are Phe<sup>259</sup> and Thr<sup>263</sup>. The construct B1(B2VI), produced by substitution of B2 TM-VI into the B1 receptor, did not support high affinity binding of the B1-selective agonist des-Arg<sup>10</sup>-kallidin. In contrast to BK and des-Arg<sup>10</sup>-kallidin, the binding of the less subtype-selective agonist kallidin showed little sensitivity to TM-VI exchange. These results show that TM-VI in the human B1 and B2 BK receptor subtypes, although only 36% identical, are structurally compatible. Furthermore, this domain contributes significantly to the ability of these receptors to discriminate between the subtype-selective agonists BK and des-Arg<sup>10</sup>-kallidin.</p>}},
author = {{Leeb, Tosso and Mathis, Sandra A. and Leeb-Lundberg, L. M.Fredrik}},
issn = {{0021-9258}},
language = {{eng}},
month = {{01}},
number = {{1}},
pages = {{311--317}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{Journal of Biological Chemistry}},
title = {{The sixth transmembrane domains of the human B1 and B2 bradykinin receptors are structurally compatible and involved in discriminating between subtype-selective agonists}},
url = {{http://dx.doi.org/10.1074/jbc.272.1.311}},
doi = {{10.1074/jbc.272.1.311}},
volume = {{272}},
year = {{1997}},
}