Real-time Analysis of the Binding of Fluorescent VEGFa to VEGFR2 in Living Cells: Effect of Receptor Tyrosine Kinase Inhibitors and Fate of Internalized Agonist-receptor Complexes

Overview

Journal Biochem Pharmacol

Specialties Biochemistry
Pharmacology

Date 2017 Apr 11

PMID 28392095

Citations 27

Authors

Laura E Kilpatrick

Rachel Friedman-Ohana

Diana C Alcobia

Kristin Riching

Chloe J Peach

Amanda J Wheal

Stephen J Briddon

Matthew B Robers

Kris Zimmerman

Thomas Machleidt

Keith V Wood

Jeanette Woolard

Stephen J Hill

Affiliations

Soon will be listed here.

Abstract

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGFa-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGFa-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGFa-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane.

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