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.