Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells

Peach, Chloe J. and Kilpatrick, Laura E. and Friedman-Ohana, Rachel and Zimmerman, Kris and Robers, Matthew B. and Wood, Keith V. and Woolard, Jeanette and Hill, Stephen J. (2018) Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells. Cell Chemical Biology . ISSN 2451-9456 (In Press)

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Abstract

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, single-site (N-terminal cysteine) labelled versions of VEGF165a, VEGF165b and VEGF121a were synthesised. 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 (4.4nM). 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 emphasise the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signalling.

Item Type: Article
RIS ID: https://nottingham-repository.worktribe.com/output/942666
Keywords: VEGFR2; Neuropilin-1; NanoBRET; Ligand binding kinetics; VEGF isoforms; Receptor mechanisms
Schools/Departments: University of Nottingham, UK > Faculty of Medicine and Health Sciences > School of Life Sciences
Depositing User: Eprints, Support
Date Deposited: 03 Jul 2018 09:02
Last Modified: 04 May 2020 19:42
URI: http://eprints.nottingham.ac.uk/id/eprint/52730

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