DISCUSSION

NanoBiT technologies were used to quantify the real-time binding of two fluorescent VEGF-A isoforms at a defined receptor/co-receptor complex between VEGFR2 and NRP1 in living cells at 37°C. Previous work identified differences between VEGFR2 and NRP1 pharmacology in terms of their binding kinetics and localisation when expressed on their own (Peach et al., 2018a). VEGFR2 and NRP1 are, however, endogenously co-expressed together in endothelial cells and tumour cells (Whitaker et al., 2001; Prahst et al., 2008; Fantin et al., 2013; Koch et al., 2014; Lee-Montiel et al., 2015). We first demonstrated that full-length VEGFR2 and NRP1 constitutively formed a heteromeric complex in living HEK293T cells. To then probe how this specific receptor/co-receptor heteromer interacted with ligand, we established a novel approach to quantify fluorescent VEGF-A binding at a defined complex using split NanoBiT fragments (Dixon et al., 2016). VEGFR2 and NRP1 tagged at their N-terminus with HiBiT and LgBiT tags led to NanoBiT complementation with minimal luminescence when each was expressed alone. The formation of this NanoBiT complex could be prevented by increasing amounts of an unlabelled version of one of the heteromer components. As such, the BRET signal was specific to interactions between the VEGFR2/NRP1 heteromer (BRET donor) and fluorescent VEGF-A (BRET acceptor). This allowed us to monitor ligand binding to a defined RTK/co-receptor oligomeric complex. Un-complemented VEGFR2 or NRP1 that still bind to the fluorescent ligand do not, however, contribute to the BRET signal due to the lack of complemented donor luminescence and the requirement for donor and acceptor to be within 10 nm of each other.
Numerous biochemical techniques have suggested that VEGFR2 and NRP1 form heteromeric complexes, including co-immunoprecipitation studies in endothelial cells (Whitaker et al., 2001; Prahst et al., 2008; Gelfand et al., 2014) and proximity-ligation assays using antibodies in situ on tumour tissue (Koch et al., 2014). Förster Resonance Energy Transfer has also been used to demonstrate complex formation using truncated VEGFR2 and full-length NRP1 tagged with fluorophores at their C-terminus (King et al., 2018). Here, we initially used BRET between full-length VEGFR2 and NRP1 tagged at their N-terminus with NanoLuc or a fluorophore to confirm complex formation in the absence of added VEGF-A. The approach monitored complex formation that originated at the cell membrane since membrane-impermeant fluorophore-conjugated HaloTag or SnapTag substrates were used. Basal VEGFR2/NRP1 complex formation was also confirmed using both HiBiT-VEGFR2 and LgBiT-NRP1 complementation and the reverse LgBiT-VEGFR2 and HiBiT-NRP1 orientation.
Following the discovery that VEGF165a had faster binding kinetics for binding to NRP1 than to VEGFR2 when expressed on their own (Peach et al., 2018a), it was proposed that the presence of NRP1 might enhance VEGF165a binding to the heteromeric complex. The application of both NanoBiT technology and NanoBRET to monitor exclusively VEGF165a-TMR binding to VEGFR2/NRP1 complexes allowed us to test this hypothesis directly. Interestingly, the initial association kinetics (during the first 20 minutes) for VEGF165a-TMR binding to the VEGFR2/NRP1 heteromeric complex were closer to those observed at NanoLuc-VEGFR2 in isolation than to NanoLuc-NRP1. This was evident from quantification of the observed rate constant from matched experiments at a saturating concentration (10 nM) of fluorescent VEGF165a where kobs was 0.33 ± 0.04 min-1 for the VEGFR2/NRP1 NanoBit complex, 0.31 ± 0.03 min-1 for NanoLuc-VEGFR2 and 0.93 ± 0.09 min-1 for NanoLuc-NRP1. Furthermore, the removal of the binding site for VEGF165a on NRP1 by site-directed mutagenesis of residue Y297 to alanine did not alter the ability of VEGFR2 and NRP1 to form complexes or the binding of VEGF165a-TMR to the heteromeric complex. It is possible therefore that heteromerization between VEGFR2 and NRP1 masks the high affinity binding site for VEGF165a on NRP1 and just leaves the VEGFR2 binding site available.
There were some subtle differences between the kinetics of fluorescent VEGF-A isoforms at the VEGFR2/NRP1 heteromeric complex. Pro-angiogenic VEGF165a and anti-angiogenic VEGF165b are functionally distinct VEGF-A isoforms, however these isoforms only differ by six amino acid residues at their C-terminus. Despite observed physiological distinctions between VEGF-A isoforms, there were no differences observed at the level of ligand binding to NanoLuc-VEGFR2 when it was expressed alone (Peach et al., 2018a). VEGF165b is, however, selective for VEGFR2 and unable to interact with NRP1 (Peach et al., 2018a). The real-time BRET signal for VEGF165b-TMR remained elevated in intact cells at the NanoBiT complex over the full 90-minute time course. This resembled observations made with NanoLuc-VEGFR2 in membrane preparations and was quite different to the decline in BRET signal normally observed in intact HEK293T cells (Peach et al., 2019). In contrast, the profile for VEGF165a-TMR at the HiBiT complex had a small decrease at latter time points, albeit to a lesser extent than at NanoLuc-VEGFR2 in intact cells (Peach et al., 2019). This reduction in BRET signal for NanoLuc-VEGFR2 following 20 minutes has been linked to VEGF-A/VEGFR2 endocytosis leading to a change in localisation and local pH, as this decline was absent in membrane preparations and not observed for binding to NanoLuc-NRP1 (Peach et al, 2019). These data suggest that the presence of NRP1 in VEGFR2 heteromeric complexes may reduce the extent of VEGFR2 endocytosis normally seen when VEGFR2 is expressed alone.
Imaging studies exploited the compatibility of HaloTag and SnapTag technologies to label distinct receptors co-expressed by the same cell to monitor colocalisation at 37°C. Unlike immunofluorescent antibody labelling, these experiments can be performed in living cells and do not require cell fixation or cell permeabilization to access internalised receptor. These distinct tags confirmed that VEGFR2 was largely intracellular whereas NRP1 was highly localised around the plasma membrane when they were both co-expressed in the same cell. NRP1 was also localised in filopodia-like projections in HEK293T cells that resembled the filopodia of endothelial tip cells (Fantin et al., 2013, 2015). Although co-localisation studies were limited by the axial resolution limit of basic confocal microscopy, experiments monitoring receptor-receptor BRET confirmed that VEGFR2 and NRP1 were in close proximity (<10 nm). Live cell confocal imaging and bioluminescence imaging data both suggested that VEGFR2 and NRP1 were colocalised in both intracellular compartments and at the plasma membrane. VEGFR2 is subject to macropinocytosis in the absence or presence of ligand (Basagiannis and Christoforidis, 2016; Basagiannis et al., 2016). This bulk transport mechanism could therefore non-selectively engulf surrounding NRP1 in living cells. There is evidence in HUVECs for colocalisation between VEGFR2 and NRP1 both at the plasma membrane in the absence of stimulation (Lee-Montiel et al., 2015) or within intracellular sites following 20 minute VEGF165a stimulation (Muhl et al., 2017). As the NanoLuc/NanoBiT substrate furimazine is membrane-permeable, luminescence could be emitted from complexes anywhere in the cell regardless of subcellular localisation.
NanoBiT technologies take advantage of NanoLuc, a small enzyme engineered from a deep sea shrimp with bright, ATP-independent luminescence emissions (Hall et al., 2012). The small, 11 amino acid NanoBiT fragment also has mutations that confer differing intrinsic affinities for the LgBiT fragment. For example, HiBiT has a much higher intrinsic affinity for LgBiT than SmBiT (Dixon et al., 2016). Luminescence emissions from HiBiT-containing complexes were higher than for the corresponding SmBiT-containing complex, as observed previously for NanoBiT-tagged GPCRs (Botta et al., 2019). The intrinsic affinity between HiBiT and LgBiT can vary according to the expression system and protein conformation, as observed for chemokine GPCRs using the purified exogenous tag in different assay setups (White et al., 2020). While the intrinsic affinity between NanoBiT tags should be considered, luminescence emissions from both HiBiT and SmBiT complexes were displaceable by increasing amounts of competing NRP1 (Figure 3d). The kinetic parameters derived from HiBiT and SmBiT complexes were also comparable suggesting that VEGFR2-NRP1 complex formation was not being driven by the affinity of the HiBiT tag for LgBiT.
Despite its ability to upregulate VEGF-A/VEGFR2 signalling in physiological and patho-physiology, the mechanism by which NRP1 upregulates VEGFR2 signalling remains largely unknown. NRP1 can interact with a number of other growth factors (West et al., 2005; Banerjee et al., 2006; Rizzolio et al., 2012), therefore understanding how NRP1 co-expression influences RTK function has implications for other receptors contributing to cancer drug resistance. Our NanoBiT approach allowed us to isolate VEGF-A ligand binding at a defined complex of VEGFR2 and NRP1 and suggested that NRP1 did not increase the affinity or association binding kinetics of VEGF165a at VEGFR2. While NRP1 appeared to have no direct effect on ligand binding to a VEGFR2/NRP1 complex expressed within the same cell, NRP1 (which is quite often expressed endogenously at higher levels than VEGFR2) could still act as a reservoir for growth factors and create a localised gradient due to its interactions with the extracellular matrix (Shintani et al., 2006; Windwarder et al., 2016).
In summary, we have described here an approach using NanoBiT technology and NanoBRET to monitor in real time the binding of VEGF-A isoforms to defined heteromeric complexes containing both VEGFR2 and NRP1. This allowed us to determine for the first time the ligand-binding kinetics of VEGF165a-TMR and VEGF165b-TMR to the VEGFR2-NRP1 complex. We were able to use bioluminescence imaging and confocal microscopy to determine that VEGFR2-NRP1 complexes are localised in both intracellular compartments and at the plasma membrane. At the plasma membrane, the presence of NRP1 within the heteromeric complex appeared to reduce the extent of agonist-induced VEGFR2 endocytosis normally observed when it is expressed alone. The presence of NRP1 within the VEGFR2-NRP1 heteromeric complexes did not enhance VEGF165a-TMR binding, and a NRP1 binding-dead mutant (Y297A) had no effect on the binding of VEGF165a-TMR, or the formation of VEGFR2-NRP1 complexes, suggesting that the high affinity binding site for VEGF165a on NRP1 might be masked within the heteromeric complexes. In keeping with this conclusion VEGF165b-TMR, which does not bind to NRP1, had a very similar binding profile to the heteromeric complex to that observed with VEGF165a-TMR. This approach to monitor the binding profile of defined oligomeric complexes should be applicable to a wide range of receptor systems and facilitate drug discovery aimed a heteromeric complexes.
ACKNOWLEDGEMENTS
This work was funded by BBSRC (grant number BB/L019418/1) and the British Pharmacological Society. CJP held an AJ Clark studentship from the British Pharmacological Society. LEK holds an Anne McLaren Fellowship from the University of Nottingham. We thank the Centre of Membrane Proteins and Receptors (COMPARE) for financial support and Promega Corporation for synthesising VEGF165a-TMR and VEGF165b-TMR and providing HaloTag- and NanoLuc-tagged constructs. We also thank the School of Life Sciences Imaging (SLIM) team for support in imaging facilities and analysis.
AUTHOR CONTRIBUTIONS
Conceived the study: Hill, Woolard, Kilpatrick, Peach.
Participated in research design: Peach, Kilpatrick, Hill, Woolard.
Conducted experiments: Peach.
Performed data analysis: Peach, Hill.
Wrote or contributed to the writing of the manuscript: Peach, Kilpatrick, Woolard, Hill.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.