Quantifying VEGF binding at VEGFR2 and Neuropilin-1 using NanoBRET

[Speaker] Chloe Peach:1
[Co-author] Laura Kilpatrick:1, Rachel Friedman-Ohana:2, Matt Robers:2, Jeanette Woolard:1, Steve Hill:1
1:Cell Signalling Research Group, University of Nottingham, UK, 2:Promega Corporation, Madison, USA

Background: Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis. VEGF primarily signals via VEGF receptor 2 (VEGFR2), however signalling can be selectively potentiated by its co-receptor Neuropilin-1 (NRP1) (1). Despite approved anti-cancer therapeutics targeting VEGF signalling (2), limited information is available on the ligand-binding characteristics of the full-length VEGFR2 or NRP1. To quantify ligand binding using bioluminescence resonance energy transfer (BRET), we monitored interactions between fluorescent VEGF165a (single-site labelled with tetramethylrhodamine (TMR)) and N-terminal NanoLuc-tagged VEGFR2 or NRP1 expressed in living cells.
Method: HEK293 cells stably expressing either NanoLuc-VEGFR2 or NanoLuc-NRP1 were seeded 24 hours prior to experimentation in 96-well plates. For saturation experiments, increasing concentrations of fluorescent VEGF165a-TMR was added in the presence or absence of 100nM unlabelled VEGF165a in Hanks buffered saline solution/0.1% bovine serum albumin (HBSS/BSA; pH 7.4). Following 60min stimulation at 37C, the NanoLuc substrate furimazine (10uM) was added and BRET ratios were recorded using a BMG Pherastar. For kinetic experiments, fluorescent VEGF (1-20nM) was added following 5min furimazine incubation and BRET ratios were measured every 30 seconds for 20 minutes at 37C. Data are expressed as mean +/- SEM.
Results: Saturable binding was observed for VEGF165a-TMR at both NanoLuc-VEGFR2 and NanoLuc-NRP1, for which comparable binding affinities were derived (NanoLuc-VEGFR2 Kd=1.21+/-0.17; NanoLuc-NRP1 Kd=2.57+/-0.29; n=3). Minimal non-specific binding was observed for both VEGFR2 and NRP1. Real-time binding kinetics showed VEGF165a-TMR reached maximal binding within 5 minutes at NanoLuc-NRP1 compared to 20 minutes at NanoLuc-VEGFR2 (n=5). Fitted to a simple association model, similar nanomolar affinities were also derived despite faster Kon and slower Koff rates at NanoLuc-NRP1 relative to NanoLuc-VEGFR2.
Conclusions: NanoBRET with fluorescently labelled VEGF quantified high affinity (nM) ligand binding of VEGF165a to NanoLuc-tagged VEGFR2 and NRP1 in HEK293 cells. Despite comparable binding affinities, VEGF165a-TMR had faster binding kinetics at NRP1 compared to VEGFR2. Measuring NanoBRET in real-time has enabled the ligand binding characteristics of VEGFR2 and its co-receptor NRP1 to be determined separately in living cells at 37C.
References: (1) Woolard et al. (2009) Microcirculation 16:572-592; (2) Ferrara and Adamis (2016) Nat.Rev.Drug Discov. 15:385-403; (3) Kilpatrick et al. (2017) Biochem.Pharmacol. 136:62-75.
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