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Use of NanoBiT and NanoBRET to monitor fluorescent VEGF-A binding kinetics to VEGFR2/NRP1 heteromeric complexes in living cells

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

Use of NanoBiT and NanoBRET to monitor fluorescent VEGF-A binding kinetics to VEGFR2/NRP1 heteromeric complexes in living cells Thumbnail


Authors

Chloe J. Peach

JEANETTE WOOLARD Jeanette.Woolard@nottingham.ac.uk
Professor of Cardiovascular Physiology and Pharmacology

STEPHEN HILL STEVE.HILL@NOTTINGHAM.AC.UK
Professor of Molecular Pharmacology



Abstract

Background and Purpose:
VEGF‐A is a key mediator of angiogenesis, primarily signalling via VEGF receptor 2 (VEGFR2). Endothelial cells also express the co‐receptor neuropilin‐1 (NRP1) that potentiates VEGF‐A/VEGFR2 signalling. VEGFR2 and NRP1 had distinct real‐time ligand binding kinetics when monitored using BRET. We previously characterised fluorescent VEGF‐A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explored differences between VEGF‐A isoforms in living cells that co‐expressed both receptors.


Experimental Approach:
Receptor localisation was monitored in HEK293T cells expressing both VEGFR2 and NRP1 using membrane‐impermeant HaloTag and SnapTag technologies. To isolate ligand binding pharmacology at a defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT complementation technology whereby heteromerisation is required for luminescence emissions. Binding affinities and kinetics of VEGFR2‐selective VEGF165b‐TMR and non‐selective VEGF165a‐TMR were monitored using BRET from this defined complex.

Key Results:
Cell surface VEGFR2 and NRP1 were co‐localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF165b‐TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 min. VEGF165a‐TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding‐dead mutant of NRP1 did not affect the binding kinetics or affinity of VEGF165a‐TMR.

Conclusion and Implications:
This NanoBiT approach enabled real‐time ligand binding to be quantified in living cells at 37°C from a specified complex between a receptor TK and its co‐receptor for the first time.

Journal Article Type Article
Acceptance Date Feb 23, 2021
Online Publication Date Apr 8, 2021
Publication Date May 24, 2021
Deposit Date Mar 5, 2021
Publicly Available Date Apr 8, 2021
Journal British Journal of Pharmacology
Print ISSN 0007-1188
Electronic ISSN 1476-5381
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 178
Issue 12
Pages 2393-2411
DOI https://doi.org/10.1111/bph.15426
Keywords Pharmacology
Public URL https://nottingham-repository.worktribe.com/output/5368354
Publisher URL https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15426

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