Advanced

Agonist-promoted trafficking of human bradykinin receptors : Arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells

Lamb, Maria E; De Weerd, Willem F C and Leeb-Lundberg, Fredrik LU (2001) In Biochemical Journal 355(3). p.741-750
Abstract

In this study, we analysed the agonist-promoted trafficking of human B2 (B2R) and B1(B1R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B2R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B2R using radioligand binding and by imaging of B2R-GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B1R was sequestered to a considerably lesser extent upon binding of des-Arg10-kallidin.... (More)

In this study, we analysed the agonist-promoted trafficking of human B2 (B2R) and B1(B1R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B2R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B2R using radioligand binding and by imaging of B2R-GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B1R was sequestered to a considerably lesser extent upon binding of des-Arg10-kallidin. B2R sequestration was rapid (half-life ∼ 5 min) and reached a steady-state level that was significantly lower than that of BK sequestration. B2R sequestration was minimally inhibited by K44A dynamin (22.4±3.7%), and was insensitive to arrestin-(319-418), which are dominant-negative mutants of dynamin I and β-arrestin respectively. Furthermore, the B2R-mediated sequestration of BK was completely insensitive to both mutants, as was the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the β2-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2±16.3%) and by arrestin-(319-418) (36.9±4.4%). Our results show that B2R is sequestered to a significantly greater extent than is B1R upon agonist treatment in HEK293 cells. Furthermore, B2R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and β-arrestin-independent manner and, at least in part, involves caveolae.

(Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
keywords
Caveolae, Clathrin-coated pit, G-protein-coupled receptor, Internalization, Peptide
in
Biochemical Journal
volume
355
issue
3
pages
741 - 750
publisher
Portland Press Limited
external identifiers
  • scopus:0035339549
ISSN
0264-6021
DOI
10.1042/bj3550741
language
English
LU publication?
no
id
cb231a6d-a605-42ce-9902-11142b31b97c
date added to LUP
2017-04-07 09:22:06
date last changed
2018-01-07 11:58:22
@article{cb231a6d-a605-42ce-9902-11142b31b97c,
  abstract     = {<p>In this study, we analysed the agonist-promoted trafficking of human B<sub>2</sub> (B<sub>2</sub>R) and B<sub>1</sub>(B<sub>1</sub>R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B<sub>2</sub>R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B<sub>2</sub>R using radioligand binding and by imaging of B<sub>2</sub>R-GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B<sub>1</sub>R was sequestered to a considerably lesser extent upon binding of des-Arg<sup>10</sup>-kallidin. B<sub>2</sub>R sequestration was rapid (half-life ∼ 5 min) and reached a steady-state level that was significantly lower than that of BK sequestration. B<sub>2</sub>R sequestration was minimally inhibited by K44A dynamin (22.4±3.7%), and was insensitive to arrestin-(319-418), which are dominant-negative mutants of dynamin I and β-arrestin respectively. Furthermore, the B<sub>2</sub>R-mediated sequestration of BK was completely insensitive to both mutants, as was the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the β<sub>2</sub>-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2±16.3%) and by arrestin-(319-418) (36.9±4.4%). Our results show that B<sub>2</sub>R is sequestered to a significantly greater extent than is B<sub>1</sub>R upon agonist treatment in HEK293 cells. Furthermore, B<sub>2</sub>R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and β-arrestin-independent manner and, at least in part, involves caveolae.</p>},
  author       = {Lamb, Maria E and De Weerd, Willem F C and Leeb-Lundberg, Fredrik},
  issn         = {0264-6021},
  keyword      = {Caveolae,Clathrin-coated pit,G-protein-coupled receptor,Internalization,Peptide},
  language     = {eng},
  month        = {05},
  number       = {3},
  pages        = {741--750},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Agonist-promoted trafficking of human bradykinin receptors : Arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells},
  url          = {http://dx.doi.org/10.1042/bj3550741},
  volume       = {355},
  year         = {2001},
}