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The sixth transmembrane domains of the human B1 and B2 bradykinin receptors are structurally compatible and involved in discriminating between subtype-selective agonists

Leeb, Tosso ; Mathis, Sandra A. and Leeb-Lundberg, L. M.Fredrik LU (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
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
272
issue
1
pages
311 - 317
publisher
ASBMB
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
2020-01-13 01:58:50
@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    = {ASBMB},
  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},
}