Comparison of the ligand‐binding properties of fluorescent VEGF‐A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET

Peach, Chloe J.; Kilpatrick, Laura E.; Woolard, Jeanette; Hill, Stephen J.

Abstract

Background and Purpose: Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF165a to NanoLuciferase-tagged VEGF Receptor 2 (VEGFR2) in living cells is that the bioluminescence resonance energy transfer (BRET) signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here we have compared the binding of three fluorescent VEGF-A isoforms to VEGFR2 in cells and isolated membrane preparations.

Experimental Approach: Ligand binding kinetics were monitored in both intact HEK293T cells and membranes (expressing NanoLuciferase tagged VEGFR2) using BRET between the tagged receptor and fluorescent analogues of VEGF165a, VEGF165b and VEGF121a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand-associated receptors in intracellular endosomes using automated quantitative imaging.

Key Results: Quantitiative analysis of the effect of fluorescent VEGF-A isoforms on
VEGFR2 endocytosis in cells demonstrated that they produced a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand binding association curves were maintained for the duration of the 90 minute experiment. Measurement of koff at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4.

Conclusions and Implications: These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/koff) as VEGFR2 moves from the plasma membrane to intracellular endosomes.

Citation

Peach, C. J., Kilpatrick, L. E., Woolard, J., & Hill, S. J. (2019). Comparison of the ligand‐binding properties of fluorescent VEGF‐A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET. British Journal of Pharmacology, 176(17), 3220-3235. https://doi.org/10.1111/bph.14755

• Journal Article Type: Article
• Acceptance Date: May 21, 2019
• Online Publication Date: Jun 4, 2019
• Publication Date: 2019-09
• Deposit Date: Jun 7, 2019
• Publicly Available Date: Aug 9, 2019
• Journal: British Journal of Pharmacology
• Print ISSN: 0007-1188
• Electronic ISSN: 1476-5381
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 176
• Issue: 17
• Pages: 3220-3235
• DOI: https://doi.org/10.1111/bph.14755
• Keywords: BRET, Receptor tyrosine kinase, VEGFR2, Fluorescent VEGF-A isoforms, Receptor endocytosis, Ligand binding kinetics, VEGFR2 phosphorylation
• Public URL: https://nottingham-repository.worktribe.com/output/2158485
• Publisher URL: https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14755