RESULTS
Co-localisation between VEGFR2 and NRP1 co-expressed in
living HEK293T
cells
To investigate where VEGFR2 and NRP1 were localised when both receptors
were expressed together in HEK293T cells at 37°C, we labelled each cell
surface receptor with a distinct fluorophore. Receptors were
simultaneously labelled using different substrates containing a HaloTag
chloroalkane or SnapTag benzylguanine moiety, exploiting the fact that
the membrane-impermeant fluorophore-conjugated substrate only labels
receptors at the plasma membrane. HaloTag-VEGFR2 and SnapTag-NRP1 were
labelled with membrane-impermeant HaloTag-AlexaFluor488 and
SnapTag-AlexaFluor647 (Figure 1a). Constitutive internalisation of
HaloTag-VEGFR2 was observed (Figure 1a, green regions) whereas
SnapTag-NRP1 was largely expressed at the plasma membrane (Figure 1a,
magenta regions). Sites of spatial overlay between VEGFR2 and NRP1 were
both intracellular and at regions around the plasma membrane (Figure 1a,
white). The same cell population was stimulated with a saturating
concentration of unlabelled VEGF165b (upper panels) or
VEGF165a (lower panels) for 60 minutes (Figure 1a, right
panels). Representative images show a large proportion of NRP1 remained
at the plasma membrane independent of VEGF-A stimulation. To account for
heterogeneity between cells, regions of interest were drawn around any
cell successfully co-expressing both RTK and co-receptor to quantify
colocalisation between HaloTag-VEGFR2 and SnapTag-NRP1. Upon stimulation
with VEGFR2-selective VEGF165b, there was a reduction in
the proportion of NRP1 in VEGFR2-positive regions relative to vehicle
(Figure 1b). In contrast, there was a higher correlation between
VEGFR2/NRP1 colocalisation upon VEGF165a stimulation
compared to vehicle (Figure 1c). Both parameters indicated that VEGFR2
and NRP1 were co-localised in the absence of ligand.
BRET can also be applied to monitor proximity between receptors tagged
with a bioluminescent donor (NanoLuc) and fluorescent acceptor
(AlexaFluor488). Receptor-receptor BRET was used to monitor whether
VEGFR2 and NRP1 were in close proximity (<10 nm) when
co-expressed in HEK293T cells. This unbiased technique monitors
proximity from a whole cell population in 96-well plates. Cells were
simultaneously transfected with a constant amount of bioluminescent
donor, NanoLuc-VEGFR2, and increasing amounts of cell surface
fluorophore-labelled NRP1. In the absence of ligand, there was clear
saturation of the BRET signal with increasing amounts of fluorescent
NRP1 acceptor (Figure 2a). This was observed for both SnapTag-NRP1 and
HaloTag-NRP1, therefore independent of the fluorophore labelling
approach. Confirming that increasing amounts of HaloTag-NRP1 and
SnapTag-NRP1 were successfully transfected, there was also a saturable
BRET signal when plotted against raw fluorescence emissions (Figure 2b).
Both techniques confirmed the constitutive formation of heteromeric
complexes between VEGFR2 and NRP1 in living cells.
Complementation of NanoBiT fragments using N-terminal tagged
VEGFR2 and NRP1
We then applied a split NanoBiT approach to isolate luminescence
emissions from a defined VEGFR2/NRP1 heteromeric complex. Enzymatic
luciferase activity requires complementation between the large fragment
(LgBiT) and the short 11 amino acid tag (HiBiT or SmBiT). To determine
the optimal configuration for luminescence emissions, each NanoBiT
fragment was appended to the N-terminus of both full-length VEGFR2 or
NRP1. Luminescence emissions were higher for the combination with
LgBiT-tagged VEGFR2 and the short fragment attached to NRP1 (Figure 3a).
Emissions from the HiBiT complex were approximately ten-fold higher than
the SmBiT complex. NanoBiT-tagged receptors expressed independently
emitted minimal luminescence in the presence of furimazine relative to
the complemented NanoBiT complex (Figure 3b). Addition of purified
NanoBiT fragments to exogenously complement the NanoBiT tag confirmed
that individual constructs were appropriately expressed despite low
luminescence emissions in isolation (Figure 3c). The luminescence
signals from both HiBiT and SmBiT complexes were also prevented by
competition with increasing amounts of unlabelled HaloTag NRP1 (Figure
3d). Thus, despite the intrinsic affinity between HiBiT and LgBiT (Dixon
et al., 2016), luminescence emissions were reduced by increasing amounts
of NRP1.
A bioluminescence widefield imaging system was used to visualise where
the NanoBiT luminescence signal was localised. To determine the cellular
location of the NanoBiT complexes, cells were incubated with
membrane-permeable furimazine in the absence of ligand (Figure 4). The
NanoBiT complex between HiBiT-NRP1 and LgBiT-VEGFR2 was localised to
both intracellular sites and the plasma membrane. This spatial
distribution was comparable to the regions of white overlay between
HaloTag-VEGFR2 and SnapTag-NRP1 observed in Figure 1a.