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.