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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.

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



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.

Journal Article Type Article
Publication Date Jul 15, 2017
Journal Biochemical Pharmacology
Print ISSN 0006-2952
Electronic ISSN 1873-2968
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 135
Pages 62-75
APA6 Citation Kilpatrick, L. E., Friedman-Ohana, R., Alcobia, D. C., Riching, K., Peach, C. J., Wheal, A. 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, 135, (62-75). doi:10.1016/j.bcp.2017.04.006. ISSN 0006-2952
DOI https://doi.org/10.1016/j.bcp.2017.04.006
Keywords VEGF; VEGFR2; BRET; Ligand binding; Receptor tyrosine kinase inhibitors
Publisher URL http://www.sciencedirect.com/science/article/pii/S0006295217301958
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0
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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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