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. and Friedman-Ohana, Rachel and Alcobia, Diana C. and Riching, Kristin and Peach, Chloe J. and Wheal, Amanda J. and Briddon, Stephen J. and Robers, Matthew B. and Zimmerman, Kris and Machleidt, Thomas and Wood, Keith V. and Woolard, Jeanette and Hill, Stephen 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 . ISSN 1873-2968 (In Press)

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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.

Item Type: Article
Keywords: VEGF; VEGFR2; BRET; Ligand binding; Receptor tyrosine kinase inhibitors
Schools/Departments: University of Nottingham, UK > Faculty of Medicine and Health Sciences > School of Life Sciences
Identification Number: 10.1016/j.bcp.2017.04.006
Depositing User: Eprints, Support
Date Deposited: 21 Apr 2017 08:18
Last Modified: 21 Apr 2017 19:23
URI: http://eprints.nottingham.ac.uk/id/eprint/42120

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