Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells

Peach, Chloe J.; Kilpatrick, Laura E.; Friedman-Ohana, Rachel; Zimmerman, Kris; Robers, Matthew B.; Wood, Keith V.; Woolard, Jeanette; Hill, Stephen J.

Abstract

© 2018 The Author(s) Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165a, VEGF165b, and VEGF121a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a, and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling. Peach et al. have used fluorescent VEGF-A isoforms to demonstrate that they can discriminate between VEGFR2 and its co-receptor NRP1 in real-time ligand binding studies in live cells. This precision chemical biology approach showed that fluorescent VEGF165a binds more rapidly to NRP1 than VEGFR2.

Citation

Peach, C. J., Kilpatrick, L. E., Friedman-Ohana, R., Zimmerman, K., Robers, M. B., Wood, K. V., Woolard, J., & Hill, S. J. (2018). Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells. Cell Chemical Biology, 25(10), 1208-1218.e5. https://doi.org/10.1016/j.chembiol.2018.06.012

• Journal Article Type: Article
• Acceptance Date: Jun 29, 2018
• Online Publication Date: Jul 26, 2018
• Publication Date: Oct 18, 2018
• Deposit Date: Jul 3, 2018
• Publicly Available Date: Jul 27, 2019
• Journal: Cell Chemical Biology
• Electronic ISSN: 2451-9456
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 25
• Issue: 10
• Pages: 1208-1218.e5
• DOI: https://doi.org/10.1016/j.chembiol.2018.06.012
• Keywords: VEGFR2; Neuropilin-1; NanoBRET; Ligand binding kinetics; VEGF isoforms; Receptor mechanisms
• Public URL: https://nottingham-repository.worktribe.com/output/942666
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S2451945618302265
• Additional Information: This article is maintained by: Elsevier; Article Title: Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells; Journal Title: Cell Chemical Biology; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.chembiol.2018.06.012; Content Type: article; Copyright: © 2018 The Author(s). Published by Elsevier Ltd.
• Contract Date: Jul 3, 2018