Agonist-promoted trafficking of human bradykinin receptors : Arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells
(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.
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- author
- Lamb, Maria E ; De Weerd, Willem F C and Leeb-Lundberg, Fredrik LU
- publishing date
- 2001-05-01
- 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
- external identifiers
-
- pmid:11311137
- 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
- 2024-01-13 18:13:39
@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}},
keywords = {{Caveolae; Clathrin-coated pit; G-protein-coupled receptor; Internalization; Peptide}},
language = {{eng}},
month = {{05}},
number = {{3}},
pages = {{741--750}},
publisher = {{Portland Press}},
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}},
doi = {{10.1042/bj3550741}},
volume = {{355}},
year = {{2001}},
}