Objective
Background: Vascular
endothelial growth factor (VEGF) is a key mediator of angiogenesis and vascular
permeability. 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 quantitative information
is available on the binding characteristics of VEGF to full-length VEGFR2 or
NRP1. To monitor ligand binding in real-time at 37°C, bioluminescence resonance
energy transfer (BRET) was used to quantify interactions between fluorescent VEGF
165a
single-site labelled with tetramethylrhodamine (TMR) (3), and N-terminal
NanoLuc-tagged VEGFR2 or NRP1 expressed in living cells.
Methods: 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 60 minute
stimulation at 37°C, the NanoLuc substrate furimazine (10μM) was added and BRET
ratios were recorded using a BMG Pherastar. For kinetic experiments,
fluorescent VEGF (1-20nM) was added following 5 minute furimazine incubation and
BRET ratios were measured every 30 seconds for 20 minutes at 37°C. Data are
expressed as mean ± S.E.M.
Results: Saturable
binding was observed for VEGF165a-TMR at both NanoLuc-VEGFR2 and
NanoLuc-NRP1, for which comparable binding affinities were derived
(n=5; Table 1). Minimal non-specific binding was observed for both VEGFR2 and NRP1. Real-time
binding kinetics then 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 (Table 1).
Conclusions: NanoBRET
with fluorescently labelled VEGF quantified high affinity (nM) ligand binding
of VEGF165a to NanoLuc-tagged VEGFR2 and NRP1 in living cells. Despite
comparable binding affinities, VEGF165a-TMR had faster binding
kinetics at NRP1 compared to VEGFR2. Measuring NanoBRET in real-time showed
marked differences in the binding profiles at VEGFR2 and its co-receptor NRP1
for the first time, emphasizing the importance of ligand binding kinetics for the
overall dynamics of VEGF signalling.
References: (1) Peach
et al. (2018) Intl. J. Mol. Sci. 19:1-27; (2) Ferrara and Adamis (2016) Nat. Rev. Drug Discov. 15:385-403; (3) Kilpatrick et al. (2017) Biochem. Pharmacol. 136:62-75.
