Comparison of the ligand-binding properties of fluorescent VEGF-A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET.
Binding Sites
/ drug effects
Bioluminescence Resonance Energy Transfer Techniques
Cell Membrane
/ drug effects
Cells, Cultured
Dose-Response Relationship, Drug
Endosomes
/ drug effects
Fluorescence
HEK293 Cells
Humans
Ligands
Protein Isoforms
/ drug effects
Protein Kinase Inhibitors
/ chemistry
Quinazolines
/ chemistry
Structure-Activity Relationship
Vascular Endothelial Growth Factor A
/ antagonists & inhibitors
Vascular Endothelial Growth Factor Receptor-2
/ antagonists & inhibitors
Journal
British journal of pharmacology
ISSN: 1476-5381
Titre abrégé: Br J Pharmacol
Pays: England
ID NLM: 7502536
Informations de publication
Date de publication:
09 2019
09 2019
Historique:
received:
08
12
2018
revised:
01
05
2019
accepted:
21
05
2019
pubmed:
5
6
2019
medline:
29
8
2020
entrez:
5
6
2019
Statut:
ppublish
Résumé
Vascular endothelial growth factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF Ligand-binding kinetics were monitored in both intact HEK293T cells and membranes (expressing nanoluciferase-tagged VEGFR2) using BRET between tagged receptor and fluorescent analogues of VEGF Quantitative analysis of the effect of fluorescent VEGF-A isoforms on VEGFR2 endocytosis in cells demonstrated that they produce a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand-binding association curves were maintained for the duration of the 90-min experiment. Measurement of the k These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/k
Sections du résumé
BACKGROUND AND PURPOSE
Vascular endothelial growth factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF
EXPERIMENTAL APPROACH
Ligand-binding kinetics were monitored in both intact HEK293T cells and membranes (expressing nanoluciferase-tagged VEGFR2) using BRET between tagged receptor and fluorescent analogues of VEGF
KEY RESULTS
Quantitative analysis of the effect of fluorescent VEGF-A isoforms on VEGFR2 endocytosis in cells demonstrated that they produce a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand-binding association curves were maintained for the duration of the 90-min experiment. Measurement of the k
CONCLUSIONS AND IMPLICATIONS
These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/k
Identifiants
pubmed: 31162634
doi: 10.1111/bph.14755
pmc: PMC6692582
mid: EMS84031
doi:
Substances chimiques
Ligands
0
Protein Isoforms
0
Protein Kinase Inhibitors
0
Quinazolines
0
VEGFA protein, human
0
Vascular Endothelial Growth Factor A
0
KDR protein, human
EC 2.7.10.1
Vascular Endothelial Growth Factor Receptor-2
EC 2.7.10.1
cediranib
NQU9IPY4K9
Types de publication
Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
3220-3235Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/L019418/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/N020081/1
Pays : United Kingdom
Informations de copyright
© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.
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