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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

12 Issues per year


IMPACT FACTOR 2016: 3.273

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1437-4315
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Volume 385, Issue 9 (Sep 2004)

Issues

Inhibition of sequestration of human B2 bradykinin receptor by phenylarsine oxide or sucrose allows determination of a receptor affinity shift and ligand dissociation in intact cells

Alexander Faussner
  • Ludwig-Maximilians-Universität München, Abteilung für Klinische Chemie und Klinische Biochemie, Nussbaumstrasse 20, D-80336 München, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Steffen Schuessler
  • Ludwig-Maximilians-Universität München, Abteilung für Klinische Chemie und Klinische Biochemie, Nussbaumstrasse 20, D-80336 München, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Cornelia Seidl
  • Ludwig-Maximilians-Universität München, Abteilung für Klinische Chemie und Klinische Biochemie, Nussbaumstrasse 20, D-80336 München, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Marianne Jochum
  • Ludwig-Maximilians-Universität München, Abteilung für Klinische Chemie und Klinische Biochemie, Nussbaumstrasse 20, D-80336 München, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2005-06-01 | DOI: https://doi.org/10.1515/BC.2004.109

Abstract

Depending on their interaction with intracellular proteins, G protein-coupled receptors (GPCR) often display different affinities for agonists at 37°C. Determining the affinity at that temperature is often difficult in intact cells as most GPCRs are internalized after activation. When sequestration of the B2 bradykinin receptor (B2R) was inhibited by either 0.5 M sucrose or phenylarsine oxide (PAO), a shift in the affinity was detected when the incubation temperature was raised from 4°C to 37°C or lowered from 37°C to 4°C. In contrast, binding of the antagonist [3H]NPC 17731 was temperature-independent. B2R mutants displayed different affinity shifts allowing conclusions on the role of the involved amino acids. By inhibiting receptor sequestration it was possible to determine also dissociation of [3H]BK and of [3H]NPC 17731 from intact cells at 37°C. Surprisingly, both dissociation rates were markedly enhanced by the addition of unlabeled ligand, most likely via prevention of reassociation of dissociated [3H]ligand. This suggests that dissociated [3H]ligand cannot move freely away from the receptor.

In summary, our data demonstrate that inhibition of receptor internalization either by PAO or sucrose provides an excellent method to study receptor function and the effects of mutations in intact cells.

Keywords: G protein-coupled receptor; internalization; kinin; NPXXY

About the article

Received: June 2, 2004

Accepted: July 13, 2004

Published Online: 2005-06-01

Published in Print: 2004-09-01


Citation Information: Biological Chemistry, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2004.109.

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[1]
A Faussner, S Schüssler, J Feierler, M Bermudez, J Pfeifer, K Schnatbaum, T Tradler, M Jochum, G Wolber, and C Gibson
British Journal of Pharmacology, 2012, Volume 167, Number 4, Page 839
[2]
Irina Kalatskaya, Steffen Schüssler, Cornelia Seidl, Marianne Jochum, and Alexander Faussner
Biological Chemistry, 2006, Volume 387, Number 5
[3]
Alexander Faussner, Alexandra Bauer, Irina Kalatskaya, Steffen Schüssler, Cornelia Seidl, David Proud, and Marianne Jochum
FEBS Journal, 2004, Volume 272, Number 1, Page 129
[4]
Alexander Faussner, Goeran Wennerberg, Steffen Schüssler, Jens Feierler, Cornelia Seidl, Marianne Jochum, and David Proud
FEBS Journal, 2009, Volume 276, Number 13, Page 3491

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