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

Kilpatrick, Laura E.; Friedman-Ohana, Rachel; Alcobia, Diana C.; Riching, Kristin; Peach, Chloe J.; Wheal, Amanda J.; Briddon, Stephen J.; Robers, Matthew B.; Zimmerman, Kris; Machleidt, Thomas; Wood, Keith V.; Woolard, Jeanette; Hill, Stephen J.

Abstract

© 2017 The Authors 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.

Citation

Kilpatrick, L. E., Friedman-Ohana, R., Alcobia, D. C., Riching, K., Peach, C. J., Wheal, A. J., Briddon, S. J., Robers, M. B., Zimmerman, K., Machleidt, T., Wood, K. V., Woolard, J., & Hill, S. J. (2017). 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. Biochemical Pharmacology, 136, 62-75. https://doi.org/10.1016/j.bcp.2017.04.006

• Journal Article Type: Article
• Acceptance Date: Apr 4, 2017
• Online Publication Date: Apr 7, 2017
• Publication Date: Jul 15, 2017
• Deposit Date: Apr 21, 2017
• Publicly Available Date: Apr 21, 2017
• Journal: Biochemical Pharmacology
• Print ISSN: 0006-2952
• Electronic ISSN: 1873-2968
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 136
• Pages: 62-75
• DOI: https://doi.org/10.1016/j.bcp.2017.04.006
• Keywords: VEGF; VEGFR2; BRET; Ligand binding; Receptor tyrosine kinase inhibitors
• Public URL: https://nottingham-repository.worktribe.com/output/855061
• Publisher URL: http://www.sciencedirect.com/science/article/pii/S0006295217301958
• Additional Information: This article is maintained by: Elsevier; Article Title: 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; Journal Title: Biochemical Pharmacology; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.bcp.2017.04.006; Content Type: article; Copyright: © 2017 The Authors. Published by Elsevier Inc.
• Contract Date: Apr 21, 2017