Abstract
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. 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 VEGF165 b-TMR and non-selective VEGF165 a-TMR were monitored using BRET from this defined complex. Cell surface VEGFR2 and NRP1 were co-localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF165 b-TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 min. VEGF165 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 VEGF165 a-TMR. 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.
Highlights
Angiogenesis involves the growth of new blood vessels from existing vascular networks (Carmeliet, 2005)
NanoBiT technologies were used to quantify the real-time binding of two fluorescent VEGF-A isoforms at a defined receptor/co-receptor complex between VEGF receptor 2 (VEGFR2) and NRP1 in living cells at 37C
We first demonstrated that full-length VEGFR2 and NRP1 constitutively formed a heteromeric complex in living HEK293T cells
Summary
Angiogenesis involves the growth of new blood vessels from existing vascular networks (Carmeliet, 2005). These techniques were limited to quantifying protein–protein interactions at NanoLuc-tagged VEGFR2 or NRP1 expressed in isolation; endothelial cells and tumour cells endogenously express both VEGFR2 and NRP1 in the same cell (Fantin et al, 2013; Koch et al, 2014; Lee-Montiel et al, 2015; Prahst et al, 2008; Whitaker et al, 2001) As these receptors have distinct ligand binding dynamics and subcellular localisation, approaches are required that isolate the pharmacology of VEGF-A ligand binding to distinct complexes involving both VEGFR2 and NRP1. We have used this technology to investigate the kinetics of ligand binding of VEGF165a-TMR (Kilpatrick et al, 2017) and VEGF165b-TMR (Peach, Kilpatrick, et al, 2018) to oligomeric complexes containing both VEGFR2 and NRP1
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