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

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 VEGF(165)b-TMR and non-selective VEGF(165)a-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, VEGF(165)b-TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 min. VEGF(165)a-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 VEGF(165)a-TMR. Conclusion and Implications This NanoBiT approach enabled real-time ligand binding to be quantified in living cells at 37 degrees C from a specified complex between a receptor TK and its co-receptor for the first time.

Automated summary

VEGF-A is an important mediator of angiogenesis that signals primarily through VEGF receptor 2 (VEGFR2). In this study, researchers investigated the differences in ligand binding kinetics of VEGF-A isoforms in living cells expressing both VEGFR2 and neuropilin-1 (NRP1). They found that despite the selectivity for VEGFR2, one isoform had distinct binding kinetics when interacting with the VEGFR2/NRP1 complex, while the other isoform showed kinetics similar to VEGFR2 alone.