Influence of NanoBiT tags on VEGFR2-mediated signalling.
We confirmed that the NanoBiT fragments did not interfere with VEGFR2
signalling using an NFAT reporter gene assay (Kilpatrick et al., 2017).
Concentration-response curves for VEGF165a were compared
between cells stably expressing VEGFR2 tagged at the N-terminus with
LgBiT, HiBiT or SmBiT (Figure 5). Each receptor exhibited a
concentration-dependent increase in NFAT gene transcription in response
to increasing concentrations of VEGF165a. Each cell line had a similar
potency derived for VEGF165a (LgBiT-VEGFR2
pEC50 = 9.95 ± 0.11; HiBiT-VEGFR2 pEC50= 10.06 ± 0.12; SmBiT-VEGFR2 pEC50 = 10.23 ± 0.23; n=5
for each). These were comparable to potency values derived for
VEGF165a at wild type VEGFR2 (Kilpatrick et al., 2017).
Nanomolar affinity of fluorescent VEGF-A at a defined
VEGFR2/NRP1
complex
Fluorescent VEGF-A ligand binding was monitored at full-length VEGFR2
and NRP1 tagged at their N-terminus with LgBiT and HiBiT, respectively.
Since uncomplemented receptors cannot oxidise furimazine, luminescence
was confined to proteins where complementation from a defined
heteromeric VEGFR2/NRP1 NanoBiT complex had occurred (Figure 6a). BRET
therefore only derived from the receptor/co-receptor complex and the
fluorescent VEGF-A acceptor. We have previously demonstrated that
VEGF165b-TMR selectively binds to NanoLuc-VEGFR2 (and
not NRP1), whereas VEGF165a-TMR can bind to both
NanoLuc-VEGFR2 or NanoLuc-NRP1 with nanomolar affinity (Peach et al.,
2018a). At the complemented HiBiT complex, there was saturable binding
in the presence of increasing concentrations of
VEGF165b-TMR (Figure 6b) or VEGF165a-TMR
(Figure 6c). This was displaced by a high concentration of unlabelled
ligand, demonstrating low non-specific binding. Both fluorescent ligands
had equilibrium dissociation constants (Kd) in the
nanomolar range at the VEGFR2/NRP1 complex (VEGF165b-TMR
Kd = 16.26 ± 3.81 nM, pKd = 7.82 ± 0.11;
VEGF165a-TMR Kd = 2.53 ± 0.49,
pKd = 8.61 ± 0.09; n=3 for both). Estimated ligand
binding affinities were similar to those derived at isolated receptors
tagged with full-length NanoLuc (Peach et al., 2018a).
Real-time kinetics of fluorescent VEGF-A isoforms at a
heteromeric VEGFR2/NRP1 NanoBiT
complex
Taking advantage of the NanoBiT approach to monitor real-time ligand
binding at 37°C to a complex, we compared the kinetics of ligand binding
of VEGF165b-TMR with that of
VEGF165a-TMR at the VEGFR2/NRP1 NanoBiT complex in
living cells. The kinetic binding profile of
VEGF165b-TMR (which should only bind to VEGFR2; Peach et
al., 2018a) continued to increase over the full 90 minute time course in
intact cells, producing a classic ligand binding association maintained
for each concentration of VEGF165b-TMR (Figure 7a).
Fitted to a global association curve (Table 1),
VEGF165b-TMR had a slightly slower association rate
constant (kon) for the VEGFR2/NRP1 complex (2.29 x
106 ± 0.30 x 106min-1.M-1) compared to
NanoLuc-VEGFR2 alone (7.29 x 106min-1.M-1; Peach et al., 2018a). We
then directly compared the real-time binding profile for a saturating
concentration of VEGF165b-TMR between the NanoBiT
complex and cells expressing NanoLuc-tagged receptors alone in matched
time course experiments (Figure 7b). Compared to NanoLuc-VEGFR2, the
small decline in BRET signal after a peak at 20 minutes in intact cells
was absent when monitored at the NanoBiT complex for
VEGF165b-TMR. There was no BRET detected between
VEGF165b-TMR and NanoLuc-NRP1, however this selective
ligand had a distinct long-term kinetic profile at the VEGFR2/NRP1
complex compared to VEGFR2 alone (Figure 7b).
Kinetic experiments were repeated with four concentrations of
VEGF165a-TMR (Figure 7c). Unlike
VEGF165b-TMR, there was a small decline in BRET ratio
between 30-60 minutes for VEGF165a-TMR at the HiBiT
complex (Figure 7c). Association binding curves were globally fitted to
kinetic data from the initial 20 minutes due to this decline (Table 1).
VEGF165a-TMR had a slower dissociation rate constant
(koff) at the HiBiT complex (0.046 ± 0.007
min-1; Table 1) compared to that previously reported
for NanoLuc-NRP1 expressed alone (0.26 min-1; Peach et
al., 2018a). As a consequence, the kinetic binding profile for 10 nM
VEGF165a-TMR was directly compared between the NanoBiT
complex and either NanoLuc-VEGFR2 or NanoLuc-NRP1 (Figure 7d).
VEGF165a-TMR association kinetics at the NanoBiT complex
in the initial 20 minutes were more comparable to NanoLuc-VEGFR2 than
NanoLuc-NRP1 (NanoBiT kobs = 0.33 ± 0.04
min-1, NanoLuc-VEGFR2 kobs = 0.31 ±
0.03 min-1, NanoLuc-NRP1 kobs = 0.93 ±
0.09 min-1; n=5 per group). These observed rate
constants were significantly slower at the complex than NRP1 alone
(repeated-measures ANOVA and Holm-Šidák’s multiple comparisons;P< 0.05, n=5 for each). These data suggest that the
ligand binding profile for VEGF165a-TMR at the NanoBiT
complex reflected VEGFR2 binding kinetics, as opposed to the faster
binding observed at NRP1.
Fluorescent VEGF-A kinetics were similar for the SmBiT
Complex
Considering the distinct kinetic observations at the HiBiT complex, we
further probed ligand binding kinetics at the SmBiT complex to explore
possible influences of the NanoBiT tag characteristics (Dixon et al.,
2016). Using four concentrations of VEGF165b-TMR,
binding was monitored over 90 minutes (Figure 8a). The binding profile
remained elevated throughout the time course with similarities to
kinetics observed with the HiBiT complex. Kinetic data were globally
fitted to a simple exponential association (Table 1).
VEGF165b-TMR had a slower dissociation rate
(koff) from the SmBiT complex compared to the HiBiT
complex (Kruskal-Wallis test, P< 0.05, n=5 per group).
Plotting the individual observed association rate constants
(kobs) against VEGF165b-TMR
concentration, there was a linear relationship observed at both HiBiT
and SmBiT complexes (Figure 8b). The interaction between
VEGF165b-TMR and the NanoBiT complex can therefore be
defined as a first order reaction. Binding kinetics were also monitored
at the SmBiT complex using four concentrations of
VEGF165a-TMR (Figure 8c). Fitted data from the initial
20 minute period using a global fit, there were no differences between
the association kinetic parameters derived for
VEGF165a-TMR for the HiBiT and SmBiT complexes
(Kruskal-Wallis test and Dunn’s multiple comparisons test,P> 0.05, n=5 per group). There was a linear
relationship between the derived observed association rate
(kobs) constants and VEGF165a-TMR
concentration (Figure 8d). Despite having the potential to bind to both
receptors within the complex, the interaction between
VEGF165a-TMR and the NanoBiT complex could also be
defined by a first order reaction.
Similar complex pharmacology using a binding-dead mutant of
NRP1
In addition to comparing binding between selective and non-selective
fluorescent VEGF-A isoforms, site-directed mutagenesis was an
alternative approach to probe the contribution of NRP1 engagement to the
pharmacological characteristics of the VEGFR2/NRP1 complex. Using a
previously characterised binding-dead NRP1 mutant (Y297A; Herzog et al.,
2011; Fantin et al., 2014; Peach et al., 2018a), comparisons were made
using the same ligand in the absence of interactions between
VEGF165a-TMR and NRP1 within the heteromeric NanoBiT
complex (Figure 9a). Upon co-expression of LgBiT-VEGFR2 and either
HiBiT- or SmBiT-NRP1 (Y297A), there were high luminescence emissions
resulting from NanoBiT complementation (Figure 9b). Luminescence
emissions from this NanoBiT complex were comparable to wild type NRP1,
therefore this amino acid residue was not required for constitutive
VEGFR2/NRP1 complex formation. NanoBiT constructs expressed in isolation
from their complementary fragment also had minimal luminescence
emissions in the presence of furimazine (Figure 9b). Isolating ligand
binding from this VEGFR2/NRP1 Y297A complex,
VEGF165a-TMR exhibited saturable binding at the NanoBiT
complex (Figure 9c). This was displaced by a high concentration of
unlabelled VEGF165a, confirming that there was low
non-specific binding. Derived equilibrium dissociation constants were in
the nanomolar range and similar to the wild type NanoBiT complex
(VEGF165a-TMR/NanoBiT Y297A Kd = 1.55 ±
0.38; pKd 8.84 ± 0.11; n=3). Binding kinetics at the
mutant NanoBiT complex were then monitored using four concentrations of
VEGF165a-TMR (Figure 9d). This had an identical profile
compared to VEGF165a-TMR at the wild type HiBiT complex
(Figure 7c), whereby there was a small decline in BRET ratio following
30-60 minutes. Association kinetics were derived from the initial 20
minutes using a global fit (kon =
3.71x107 ± 0.21x107min-1M-1; koff =
0.054 ± 0.008 min-1; kinetic pKd =
8.85 ± 0.04; n=5). These data suggest that VEGF165a-TMR
bound the NanoBiT complex with similar kinetics, regardless of the
ability to simultaneously engage
NRP1.