You are here: Home :: Area NEM :: Life sciences :: Biochemistry

Alexander Faussner, Steffen Schuessler, Cornelia Seidl, Marianne Jochum

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

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

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.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 385, 09/2004
Pages: 835 - 843

Show full article (external site)

Show all available items of this journal

 

Area NEM