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Use of NanoBiT and NanoBRET to monitor fluorescent VEGF-A binding kinetics to VEGFR2/NRP1 heteromeric complexes in living cells.
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  • Chloe Peach,
  • Laura Kilpatrick,
  • Jeanette Woolard,
  • Stephen Hill
Chloe Peach
University of Nottingham

Corresponding Author:mbxcp7@exmail.nottingham.ac.uk

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Laura Kilpatrick
University of Nottingham
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Jeanette Woolard
University of Nottingham
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Stephen Hill
University of Nottingham
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Abstract

Background: Vascular Endothelial Growth Factor A (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 Bioluminescence Resonance Energy Transfer (BRET). We previously characterised fluorescent VEGF-A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explore 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 a 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 heteromerization 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 minutes. VEGF165a-TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding-dead mutant of NRP1 had no impact on the binding kinetics or affinity of VEGF165a-TMR. Conclusions 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 tyrosine kinase and its co-receptor for the first time.
21 Aug 2020Submitted to British Journal of Pharmacology
24 Aug 2020Submission Checks Completed
24 Aug 2020Assigned to Editor
29 Aug 2020Reviewer(s) Assigned
03 Nov 2020Review(s) Completed, Editorial Evaluation Pending
06 Nov 2020Editorial Decision: Revise Minor
06 Feb 20211st Revision Received
07 Feb 2021Submission Checks Completed
07 Feb 2021Assigned to Editor
07 Feb 2021Reviewer(s) Assigned
19 Feb 2021Review(s) Completed, Editorial Evaluation Pending
23 Feb 2021Editorial Decision: Accept
Jun 2021Published in British Journal of Pharmacology volume 178 issue 12 on pages 2393-2411. 10.1111/bph.15426