Real-time analysis of the binding of fluorescent VEGF165a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes

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

doi:10.1016/j.bcp.2017.04.006

Laura E Kilpatrick, Rachel Friedman-Ohana + Show 11 more

Open Access

https://doi.org/10.1016/j.bcp.2017.04.006

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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 (VEGF165a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF165a-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 VEGF165a-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.

Highlights

Vascular endothelial growth factor A (VEGF-A) is an important mediator of tumour-induced angiogenesis [1,2,3,4]
Cys-VEGF165a was released from the beads by proteolytic cleavage using HaloTEV protease, while HaloTag and HaloTEV remained permanently attached to the beads eliminating the need for post cleavage removal
This study has provided for the first time quantitative information on the real time binding of VEGF-A isoforms to full length VEGFR2 in living cells

Summary

Introduction

Vascular endothelial growth factor A (VEGF-A) is an important mediator of tumour-induced angiogenesis [1,2,3,4]. The binding of VEGF to VEGFRs leads to a change in conformation of their intracellular domains and auto- or trans-phosphorylation of specific tyrosine residues of the receptor dimer resulting in the activation of different intracellular signalling cascades [11,12,13,14]. These signalling pathways are relatively well understood with tyrosine residues Y1175 and Y1214 in the human VEGFR2 being two important autophosphorylation sites activated by VEGF binding and tyrosine kinase activation. Phosphorylation of these specific residues creates binding sites for key intracellular signalling proteins such as

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