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

• Published in: British journal of pharmacology Vol. 178; no. 12; pp. 2393 - 2411
• Main Authors: Peach, Chloe J., Kilpatrick, Laura E., Woolard, Jeanette, Hill, Stephen J.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.06.2021
• Subjects: Angiogenesis; BRET; Cell surface; co‐receptor; Endothelial cells; growth factor; Isoforms; Kinetics; Ligands; NanoBiT; Neuropilin; receptor tyrosine kinase; Vascular endothelial growth factor; BRET; growth factor; co-receptor; receptor tyrosine kinase; NanoBiT; kinetics
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/bph.15426

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.