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
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Alexander Faussner
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.
References
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© Walter de Gruyter
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Articles in the same Issue
- Tapasin and other chaperones: models of the MHC class I loading complex
- Expression analysis of BCL2L12, a new member of apoptosis-related genes, in colon cancer
- SR-A1, a member of the human pre-mRNA splicing factor family, and its expression in colon cancer progression
- RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter
- Kinetic characterization of phenol and aniline derivates as substrates of peroxidase
- Non-muscle α-actinin-4 interacts with plasminogen activator inhibitor type-1 (PAI-1)
- Oxidative modification of low-density lipoprotein: lipid peroxidation by myeloperoxidase in the presence of nitrite
- An apoptotic inducer, aralin, is a novel type II ribosome-inactivating protein from Aralia elata
- Inhibition of inducible TNF-α expression by oxaspirodion, a novel spiro-compound from the ascomycete Chaetomium subspirale
- 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
- Effects of short-term chemical ablation of the GIP receptor on insulin secretion, islet morphology and glucose homeostasis in mice
- Comprehensive analysis of metabolites in Corynebacterium glutamicum by gas chromatography/mass spectrometry
