\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,greek,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Use of NanoBiT and NanoBRET to monitor fluorescent VEGF-A binding
kinetics to VEGFR2/NRP1 heteromeric complexes in living cells.}



\author[1]{Chloe Peach}%
\author[1]{Laura Kilpatrick}%
\author[1]{Jeanette Woolard}%
\author[1]{Stephen Hill}%
\affil[1]{University of Nottingham}%


\vspace{-1em}



  
  \date{May 16, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background: Vascular Endothelial Growth Factor A (VEGF-A) is a key
mediator of angiogenesis, primarily signalling via VEGF Receptor 2
(VEGFR2). Endothelial cells also express the co-receptor Neuropilin-1
(NRP1) that potentiates VEGF-A/VEGFR2 signalling. VEGFR2 and NRP1 had
distinct real-time ligand binding kinetics when monitored using
Bioluminescence Resonance Energy Transfer (BRET). We previously
characterised fluorescent VEGF-A isoforms tagged at a single site with
tetramethylrhodamine (TMR). Here, we explore differences between VEGF-A
isoforms in living cells that co-expressed both receptors. Experimental
Approach: Receptor localisation was monitored in HEK293T cells
expressing both VEGFR2 and NRP1 using a membrane-impermeant HaloTag and
SnapTag technologies. To isolate ligand binding pharmacology at a
defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT
complementation technology whereby heteromerization is required for
luminescence emissions. Binding affinities and kinetics of
VEGFR2-selective VEGF165b-TMR and non-selective VEGF165a-TMR were
monitored using BRET from this defined complex. Key Results: Cell
surface VEGFR2 and NRP1 were co-localised and formed a constitutive
heteromeric complex. Despite being selective for VEGFR2, VEGF165b-TMR
had a distinct kinetic ligand binding profile at the complex that
largely remained elevated in cells over 90 minutes. VEGF165a-TMR bound
to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2
alone. Using a binding-dead mutant of NRP1 had no impact on the binding
kinetics or affinity of VEGF165a-TMR. Conclusions and Implications: This
NanoBiT approach enabled real-time ligand binding to be quantified in
living cells at 37\selectlanguage{ngerman}°C from a specified complex between a receptor
tyrosine kinase and its co-receptor for the first time.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\section*{INTRODUCTION}

{\label{introduction}}

Angiogenesis involves the growth of new blood vessels from existing
vascular networks (Carmeliet, 2005). This important physiological
process can also be dysregulated in numerous pathologies, such as in
tumour development (Chung and Ferrara, 2011). Vascular endothelial
growth factor A (VEGF-A) is a key mediator of angiogenesis that
primarily signals via its cognate receptor tyrosine kinase (RTK), the
VEGF Receptor 2 (VEGFR2) (Simons et al., 2016; Peach et al., 2018b).
VEGF-A binds across immunoglobulin-like domains 2 and 3 of VEGFR2 (Ruch
et al., 2007; Leppanen et al., 2010). Agonist binding results in
conformational changes throughout the VEGFR2 dimer that lead to auto-
and trans-phosphorylation of key intracellular tyrosine residues. This
triggers numerous signalling cascades that ultimately initiate
endothelial cell proliferation, migration and survival, as well as
increased vascular permeability (Koch et al., 2011).

VEGFR2 is subject to complex trafficking via clathrin-dependent and
clathrin-independent endocytosis (Ewan et al., 2006; Basagiannis and
Christoforidis, 2016; Basagiannis et al., 2016). It internalises in both
the presence and absence of VEGF-A (Ewan et al., 2006; Jopling et al.,
2009; Jopling et al., 2011). VEGF-A can also bind to the VEGFR2
co-receptor Neuropilin-1 (NRP1), a type 1 transmembrane glycoprotein
(Soker et al., 1998, 2002). VEGFR2 signalling is upregulated by NRP1
(Fantin et al., 2011; Djordjevic and Driscoll, 2013; Gelfand et al.,
2014). Endothelial cells express both VEGFR2 and NRP1 (Soker et al.,
1998; Witmer et al., 2002). NRP1 is also overexpressed in numerous
tumour subtypes (Jubb et al., 2012; Goel and Mercurio, 2013; Lee et al.,
2014) and immune cells in the tumour microenvironment (Roy et al.,
2017). VEGF-A interacts with VEGFR2 via residues encoded at the
N-terminus of VEGF-A (Leppanen et al., 2010; Brozzo et al., 2011), while
the C-terminus can interact with NRP1 (Mamluk et al., 2002; Vander Kooi
et al., 2007; Parker et al., 2012).

VEGF-A is an anti-parallel, disulphide-linked homodimer. Alternative
splicing of VEGF-A mRNA leads to a number of distinct VEGF-A isoforms
(Woolard et al., 2009; Peach et al., 2018b). VEGF-A isoforms have
different signalling properties in physiological systems with distinct
expression profiles in health and disease (Vempati et al., 2014). VEGF-A
isoforms differ in length, such as pro-angiogenic
VEGF\textsubscript{165}a or the shorter VEGF\textsubscript{121}a
isoform. A major site of splicing occurs at exon 8, where proximal
splicing results in VEGF\textsubscript{xxx}a isoforms that contain exon
8a-encoded residues (CDKPRR) and VEGF\textsubscript{xxx}b isoforms that
instead contain exon 8b-encoded residues (SLTKDD). While
VEGF\textsubscript{165}a stimulates angiogenesis as a full agonist,
VEGF\textsubscript{165}b is a partial agonist with reported
anti-angiogenic activity \emph{in vivo} (Woolard et al., 2004; C\selectlanguage{ngerman}ébe
Suarez et al., 2006; Eswarappa et al., 2014). The b1 domain of NRP1 can
interact with VEGF\textsubscript{165}a via an arginine residue encoded
by exon 8a (Mamluk et al., 2002; Vander Kooi et al., 2007; Parker et
al., 2012). In contrast, `anti-angiogenic' VEGF\textsubscript{165}b
isoforms are unable to interact with NRP1 (Cébe Suarez et al., 2006;
Kawamura et al., 2008; Delcombel et al., 2013).

Fluorescence-based technologies have been used to advance our
pharmacological understanding of G protein-coupled receptors (GPCRs),
RTKs and other classes of membrane protein (Stoddart et al., 2017). For
example, Bioluminescence Resonance Energy Transfer (BRET) is a
proximity-based assay that can quantify real-time binding at 37°C in
living cells (Stoddart et al., 2015). A receptor is tagged at the
N-terminus with a 19 kDa NanoLuciferase (NanoLuc) such that NanoLuc
emits luminescence upon oxidation of the furimazine substrate. This can
excite a nearby fluorophore in close proximity (\textless{}10 nm), such
as a compatible fluorescent ligand bound at the receptor's orthosteric
site. We previously developed fluorescent VEGF-A isoforms that were
single-site labelled with tetramethylrhodamine (TMR), to monitor ligand
binding at full-length VEGFR2 or NRP1 tagged with NanoLuc (Kilpatrick et
al., 2017; Peach et al., 2018a, 2019). Despite having a similar
nanomolar binding affinity, VEGF\textsubscript{165}a-TMR binding
kinetics were significantly faster at NRP1 than VEGFR2 (Peach et al.,
2018a). VEGFR2 and NRP1 were also subject to distinct subcellular
trafficking in the absence or presence of ligand when expressed alone.
These techniques were limited to quantifying protein-protein
interactions at NanoLuc-tagged VEGFR2 or NRP1 expressed in isolation,
however endothelial cells and tumour cells endogenously express both
VEGFR2 and NRP1 in the same cell (Whitaker et al., 2001; Prahst et al.,
2008; Fantin et al., 2013; Koch et al., 2014; Lee-Montiel et al., 2015).
Since these receptors have distinct ligand binding dynamics and
subcellular localisation, approaches are required that isolate the
pharmacology of VEGF-A ligand binding to distinct complexes involving
both VEGFR2 and NRP1.

NanoLuc Binary Technology (NanoBiT) uses a modified NanoLuc split into a
large fragment (LgBiT; 156 amino acids) and a small 11 amino acid tag
(HiBiT or SmBiT; Dixon et al., 2016). Complementation of fragments is
required for luminescence emission. Numerous variants were developed of
the small tag with different intrinsic affinities for complementation
with the LgBiT fragment, including the `higher affinity' HiBiT fragment
(K\textsubscript{d} \textasciitilde{}0.7 nM) and the lower affinity
SmBiT fragment (K\textsubscript{d} \textasciitilde{}190 \selectlanguage{greek}µ\selectlanguage{english}M). Used in
combination with a fluorescent ligand, interactions between the ligand
and a particular protein pairing can be monitored using NanoBiT and
BRET. Here we have used this technology to investigate the kinetics of
ligand binding of VEGF\textsubscript{165}a-TMR (Kilpatrick et al., 2017)
and VEGF\textsubscript{165}b-TMR (Peach et al., 2018a) to oligomeric
complexes containing both VEGFR2 and NRP1.

\par\null

\section*{METHODS}

{\label{methods}}

\subsection*{Cell Culture and
Materials}

{\label{cell-culture-and-materials}}

HEK293T cells (CCLV Cat\# CCLV-RIE 1018, RRID:CVCL\_0063) were
maintained at 37\selectlanguage{ngerman}°C/5\% CO\textsubscript{2} in Dulbecco's Modified
Eagle's Medium (DMEM; Sigma-Aldrich, USA) supplemented with 10\% Fetal
Calf Serum (FCS; Sigma-Aldrich, USA). For a consistent cell background
with functional studies performed using a reporter gene assay, all
HEK293T cells also expressed a Firefly luciferase reporter gene
(RE-Luc2P) that was inserted downstream of the NFAT promoter. Control
experiments confirmed that HEK293T-NFAT-ReLuc2P cells did not emit
luminescence in response to furimazine alone that interfered with
NanoBiT or NanoBRET assays. Cells were passaged at 70-80\% confluency
using phosphate buffered saline (PBS; Lonza, Switzerland) and trypsin
(0.25\% w/v in versene; Lonza). Fluorescent VEGF\textsubscript{165}a and
VEGF\textsubscript{165}b were labelled at a single N-terminal cysteine
residue with TMR using the HaloTag mammalian protein detection and
purification system (G6795; Promega Corporation, USA) as described
previously (Kilpatrick et al., 2017; Peach et al., 2018a). Fluorescent
ligands were characterised in terms of labelling efficiency,
dimerisation and function as described in Kilpatrick et al. (2017) and
Peach et al. (2018a). Ligands were stored at -20°C in 2.5mg/ml
protease-free bovine serum albumin (BSA; Millipore, USA). Unlabelled
recombinant human VEGF isoforms were purchased from R\&D Systems
(Abingdon, UK). Furimazine and purified NanoBiT fragments were purchased
from Promega Corporation (Madison, USA).

\subsection*{Generating Constructs}

{\label{generating-constructs}}

N-terminal NanoLuc-tagged VEGFR2 (NM\_002253) and NRP1 (NM\_003873.5)
were cloned in a pFN31K vector encoding the secretory IL\selectlanguage{english}-6 signal
peptide fused to the N\selectlanguage{english}-terminus of NanoLuc, followed by a GSSGAIA linker
before the receptor. HaloTag-VEGFR2 and HaloTag-NRP1 were cloned in a
pFN21A vector with the IL\selectlanguage{english}-6 signal peptide followed by a sequence
encoding HaloTag and an EPTTEDLYFQSDNAIA linker at the receptor
N\selectlanguage{english}-terminus. SnapTag-NRP1 was cloned into a pcDNA3.1 vector encoding a
murine 5HT3A signal sequence followed by the SnapTag and a
STSPVWWNSADIQHSGGRSSGAIA linker. The receptor-encoded sequence from
NanoLuc-NRP1 vector was used to generate SnapTag-NRP1 using the XhoI and
XbaI restriction sites. N-terminal LgBiT-VEGFR2 and LgBiT-NRP1 were
cloned in the pFN21A vector with the IL-6 signal peptide, LgBiT sequence
and a flexible GSSGGGGSGGGGSSGGAIA linker. The LgBiT tag sequence from
N198A pBiT1.1-N, available from the NanoBiT Multiple Cloning Site
Starter System (N2014, Promega Corporation), was cut using SacII and
SgfI. HiBiT-NRP1 (WT), HiBiT-NRP1 (Y297A), HiBiT-VEGFR2, SmBiT-NRP1
(WT), SmBiT-NRP1 (Y297A) and SmBiT-VEGFR2 were also cloned in a pFN21A
vector with the IL-6 signal peptide, 11 amino acid sequence and a
GSSGGSSGAIA linker. The VEGF-A binding-dead mutant of NRP1 (Y297A) was
described previously (Peach et al., 2018a). The 11 amino acid NanoBiT
tags (HiBiT: VSGWRLFKKIS; SmBIT VTGYRLFEEIL) were obtained as custom
oligonucleotide sequences from Sigma-Aldrich, annealed into double
stranded DNA and phosphorylated with T4 PNK (New England Biolabs) and
inserted using SacII and SgfI sites.

\subsection*{NFAT Luciferase Reporter Gene
Assay}

{\label{nfat-luciferase-reporter-gene-assay}}

HEK293T-NFAT-ReLuc2P cells stably expressed LgBiT-VEGFR2, HiBiT-VEGFR2
or SmBiT-VEGFR2. Cells were seeded at 25,000 cells/well in white 96-well
plates pre-coated with poly-D-lysine in DMEM containing 10\% FBS.
Following incubation for 24 hours at 37\%/5\% CO2, medium was replaced
with serum-free DMEM and cells were incubated for a further 24 hours. On
the day of experimentation, medium was replaced with serum-free DMEM
containing 0.1\% BSA. Cells were stimulated with increasing
concentrations of VEGF165a (R\&D Systems) for 5 hours at 37\%/5\% CO2.
Medium was replaced with 50 \selectlanguage{greek}μ\selectlanguage{english}l/well serum-free DMEM/0.1\% BSA and 50
\selectlanguage{greek}μ\selectlanguage{english}l/well ONE-Glo Luciferase reagent. Following a 5 minute delay to allow
reagent to react with luciferase and background luminescence to subside,
luminescence emissions were measured using a TopCount platereader
(Perkin Elmer, UK).

\subsection*{Confocal Imaging of HaloTag-VEGFR2 and
SnapTag-NRP1}

{\label{confocal-imaging-of-halotag-vegfr2-and-snaptag-nrp1}}

HEK293T-NFAT-ReLuc2P cells were plated in 8-well plates (Nunc Lab-Tek,
Thermo Fisher Scientific) pre-coated with poly-D-lysine (0.01mg/ml in
PBS) at 30,000 cells per well in DMEM/10\% FBS. Following incubation for
24 hours, cells were transfected with a mixture of HaloTag-VEGFR2 and
SnapTag-NRP1. Control wells were also transfected with a single
construct and empty vector, such as SnapTag-NRP1 and pcDNA3.1/Neo.
Transient transfections used FuGENE\selectlanguage{ngerman}® HD at a 3:1 ratio of reagent to
cDNA with a total 100 ng cDNA/well, with receptors transfected at equal
amounts of 50 ng cDNA/well. Transfection solutions were made up in
serum-free DMEM and added as 11 \selectlanguage{greek}μ\selectlanguage{english}l/well. Cells were incubated for a
further 24 hours at 37\selectlanguage{ngerman}°C/5\% CO2. Receptors were then labelled with a
solution of serum-free DMEM/0.1\% BSA containing both 0.5 \selectlanguage{greek}μ\selectlanguage{english}M membrane
impermeant HaloTag-AlexaFluor488 substrate (G1002; Promega Corporation,
USA) and 0.5 \selectlanguage{greek}μ\selectlanguage{english}M membrane impermeant SNAP-Surface AlexaFluor647 (S9136S;
New England BioLabs). These were incubated for 30 minutes (37\selectlanguage{ngerman}°C/5\%
CO2). Cells were washed twice with 200 \selectlanguage{greek}μ\selectlanguage{english}l/well HBSS/0.1\% BSA, then
replaced with a final volume of 225 \selectlanguage{greek}μ\selectlanguage{english}l/well. Cells were incubated with
vehicle, 10 nM unlabelled VEGF165b or 10 nM unlabelled VEGF165a for 60
minutes at 37\selectlanguage{ngerman}°C, adding 25 \selectlanguage{greek}μ\selectlanguage{english}l to a total volume of 250 \selectlanguage{greek}μ\selectlanguage{english}l. Cells were
imaged live using a temperature-controlled LSM710 confocal microscope
fit with a 40x water objective (Pan Apochromat objective, NA 1.2).
Wavelengths were imaged simultaneously using the 488/561/633
beamsplitter. HaloTag-VEGFR2 AlexaFluor488 was imaged using an Argon 488
nm laser (493-628 nm bandpass; 2.5\% power); SnapTag-NRP1 AlexaFluor647
was imaged with a HeNe633 nm laser (638-747 nm; 2.5\% power). All images
were taken as 12 bit images with 1024x1024 pixels per frame with 4
averages and similar gains per replicate.

\subsection*{Bioluminescence Imaging of NanoBiT
Complexes}

{\label{bioluminescence-imaging-of-nanobit-complexes}}

HEK293T-NFAT-ReLuc2P cells were plated in 6-well plates at 400,000
cells/well in DMEM/10\% FBS. On day 2, cells were transfected using
FuGENE\selectlanguage{ngerman}® HD at a 3:1 ratio of reagent to cDNA with a total 1500 ng
cDNA/well made up in serum-free DMEM. Cells were transfected with equal
amounts of LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 WT (750 ng
cDNA/well). Alternatively, NanoLuc-VEGFR2 or NanoLuc-NRP1 were
transfected at 750 ng cDNA/well with an equal amount of pcDNA3.1/Zeo
(750 ng cDNA/well). On day 3, transfected cells were transferred to a 4-
compartment 35/10 mm glass bottomed dish (CELLview, Greiner Bio-One).
Dishes were pre-coated with poly-D-lysine (0.01mg/ml in PBS) and cells
were plated at 75,000 cells/well in DMEM containing 0.1\% FBS. On the
day of experimentation (day 4), medium was replaced with HBSS/0.1\% BSA.
For cells expressing full-length NanoLuc, furimazine was added at 26 \selectlanguage{greek}μ\selectlanguage{english}M.
In contrast, cells expressing the NanoBiT complex were incubated with a
higher furimazine concentration (104 \selectlanguage{greek}μ\selectlanguage{english}M) for optimal imaging. Following
incubation for 10 minutes to allow for substrate oxidation, cells were
imaged live at 37\selectlanguage{ngerman}°C using the inverted Olympus LV200 Bioluminescence
Imaging System, fitted with a 60x oil immersion objective (super
Apochromat UPLSAPO 60xO objective; NA 1.35) with a 0.5x tube lens to
focus the image, therefore images had a final magnification of 30x.
Luminescence was collected using a Hammamatsu Image EMx2 Electron
Multiplying Charge Coupled Device (EMCCD) camera. Transmitted light
images were collected using the camera in conventional CCD mode with a
200 ms exposure time. Luminescence emissions from the full-length
NanoLuc or the NanoBiT complex were measured for 5 second exposure with
a gain of 200. Images were taken as 8 bit images with 512x512 pixels per
frame.

\subsection*{BRET Between NanoLuc-VEGFR2 and Fluorescent
NRP1}

{\label{bret-between-nanoluc-vegfr2-and-fluorescent-nrp1}}

HEK293T-NFAT-ReLuc2P cells were plated in white 96-well plates
pre-coated with poly-D-lysine (0.01mg/ml in PBS) at 25,000 cells per
well in DMEM containing 10\% FBS. Following 24 hours, cells were
transiently transfected with a total 125 ng cDNA/well using FuGENE® HD
at a 3:1 ratio of reagent to cDNA. Cells were transfected with a
constant amount of NanoLuc-VEGFR2 (25 ng cDNA/well). Cells were
simultaneously transfected with increasing concentrations of
HaloTag-NRP1 or SnapTag-NRP1 (2.5-100 ng cDNA/well). Additional wells
only contained NanoLuc-VEGFR2. These transfection solutions were made up
to equivalent to 125 ng per well using empty pcDNA3.1/Zeo vector in
serum-free DMEM. Cells were incubated for another 24 hours at 37°C/5\%
CO2. On the day of the experiment, cells were treated with 0.2 \selectlanguage{greek}μ\selectlanguage{english}M
membrane impermeant HaloTag-AlexaFluor488 substrate or 0.2 \selectlanguage{greek}μ\selectlanguage{english}M
SNAP-Surface AlexaFluor488 substrate in serum-free-DMEM/0.1\% BSA. Cells
were incubated for 30 minutes at 37\selectlanguage{ngerman}°C/5\% CO2. They were then washed
twice with 100 \selectlanguage{greek}μ\selectlanguage{english}l/well HBSS/0.1\% BSA and replaced with a final volume
of 50 \selectlanguage{greek}μ\selectlanguage{english}l/well HBSS/0.1\% BSA. At this stage, fluorescence emissions were
quantified using the PHERAstar FS platereader using filters for
excitation at 485 nm and emission at 520 nm. Cells were then incubated
with the NanoLuc substrate furimazine (10 \selectlanguage{greek}μ\selectlanguage{english}M) for 5 minutes. Emissions
were recorded using the PHERAstar FS platereader using filters
simultaneously measuring NanoLuc emissions at 475 nm (30 nm bandpass)
and AlexaFluor488 emissions at 535 nm (30 nm bandpass). BRET ratios were
calculated as fluorescence over luminescence emissions from the second
of three cycles.

\subsection*{Luminescence from NanoBiT
Complementation}

{\label{luminescence-from-nanobit-complementation}}

To characterise luminescence emissions from a NanoBiT complex,
HEK293T-NFAT-ReLuc2P cells were plated as 25,000 cells/well in white
96-well plates pre-coated with poly-D-lysine (0.01mg/ml in PBS) in DMEM
containing 10\% FBS. Following 24 hours, cells were transiently
transfected using FuGENE\selectlanguage{ngerman}® HD at a 3:1 ratio of reagent to cDNA with a
total 100 ng cDNA/well. Cells were transfected with a combination of
LgBiT-tagged (50 ng cDNA/well) and HiBiT-/SmBiT-tagged receptors (50 ng
cDNA/well). Alternatively, cells were transfected with single constructs
(50 ng cDNA/well) with empty pcDNA3.1/Zeo vector (50 ng cDNA/well).
Transfection mixtures were made up in serum-free DMEM and added as 5
\selectlanguage{greek}μ\selectlanguage{english}l/well without replacing DMEM/10\% FBS on cells. Cells were incubated
at 37\selectlanguage{ngerman}°C/5\% CO2 for a further 24 hours. Medium was replaced with
HBSS/0.1\% BSA containing 10 \selectlanguage{greek}μ\selectlanguage{english}M furimazine, in the absence or presence
of purified LgBiT protein (N401B, Promega Corporation) or HiBiT protein
(N301A, Promega Corporation). Cells were incubated at 37\selectlanguage{ngerman}°C for 10
minutes to allow NanoBiT complementation and the oxidation of
furimazine. To prevent the loss of signal through the bottom of the
plate, an adhesive plate BackSeal was added at this point. Luminescence
emissions were measured on the PHERAstar platereader using the filter
settings measuring emissions between 475-505 nm.

Additional experiments aimed to disrupt the recomplemented NanoBiT
complex using increasing concentrations of competing receptor.
HEK293T-NFAT-ReLuc2P cells were plated as 25,000 cells/well in white
96-well plates pre-coated with poly-D-lysine (0.01mg/ml in PBS) in DMEM
containing 10\% FBS. Following 24 hours, cells were transiently
transfected using FuGENE® HD at a 3:1 ratio of reagent to cDNA. Cells
were transfected with a constant amount of LgBiT-VEGFR2 (50 ng
cDNA/well) and either HiBiT-NRP1 or SmBiT-NRP1 (50 ng cDNA/well). Cells
were also transfected with increasing concentrations of HaloTag-NRP1
(25-200 ng cDNA/well). This was made up to 300 ng cDNA/well with empty
pcDNA3.1/Zeo vector. Additional wells only contained the LgBiT-VEGFR2
and HiBiT/SmBiT-NRP1 complex. Cells were incubated with transfection
solution for 24 hours at 37°C/5\% CO2. On the day of the experiment,
cells were treated with 0.2 \selectlanguage{greek}μ\selectlanguage{english}M membrane impermeant HaloTag-AlexaFluor488
substrate in serum-free-DMEM/0.1\% BSA (30 minutes, 37\selectlanguage{ngerman}°C/5\% CO2). They
were then washed twice with 100 \selectlanguage{greek}μ\selectlanguage{english}l/well HBSS/0.1\% BSA and replaced with
a final volume of 50 \selectlanguage{greek}μ\selectlanguage{english}l/well HBSS/0.1\% BSA. Fluorescence emissions were
quantified using the PHERAstar FS platereader using filters for
excitation at 485 nm and emission at 520 nm. Cells were incubated with
10 \selectlanguage{greek}μ\selectlanguage{english}M furimazine for 10 minutes, then luminescence and fluorescence
emissions were recorded using PHERAstar FS platereader. Emissions were
simultaneously measured for NanoLuc at 475 nm (30 nm bandpass) and
AlexaFluor488 at 535 nm (30 nm bandpass).

\subsection*{Fluorescent VEGF-A Binding at a VEGFR2/NRP1 NanoBiT
Complex}

{\label{fluorescent-vegf-a-binding-at-a-vegfr2nrp1-nanobit-complex}}

HEK293T-NFAT-ReLuc2P cells were plated in 6-well plates at 400,000
cells/well in DMEM containing 10\% FBS. On day 2, cells were transfected
using FuGENE\selectlanguage{ngerman}® HD at a 3:1 ratio of reagent to cDNA with a total 1500 ng
cDNA/well made up in serum-free DMEM. Cells were transfected with equal
amounts of LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 WT or Y297A
(750 ng cDNA/well), or equal amounts of LgBiT-VEGFR2 (750 ng cDNA/well)
with SmBiT-NRP1 WT (750 ng cDNA/well). For experiments monitoring
kinetics at HiBiT complexes, matched controls were performed alongside
in which cells were transfected with single receptors conjugated to
full-length NanoLuc. NanoLuc-VEGFR2 or NanoLuc-NRP1 were transfected at
750 ng cDNA/well, made up to 1500 ng cDNA/well with empty pcDNA3.1/Zeo
vector (750 ng cDNA/well). On day 3, cells were transferred from 6-well
plates. Cells were washed with 1 ml/well PBS, detached with 500 \selectlanguage{greek}μ\selectlanguage{english}l/well
trypsin and resuspended in 2 ml DMEM containing 10\% FBS. Cells were
seeded in white 96-well plates pre-coated with poly-D-lysine (0.01mg/ml
in PBS) at 30,000 cells/well. On the day of experimentation (day 4),
medium was replaced with HBSS/0.1\% BSA.

For saturation experiments, increasing concentrations of VEGF165a--TMR
or VEGF165b-TMR (0.5-20 nM) were added in the presence or absence of a
high concentration of corresponding unlabelled ligand (100nM,
\textasciitilde{}100-fold greater than the estimated Kd value).
Following incubation for 60 minutes in the dark at 37\selectlanguage{ngerman}°C, the NanoLuc
substrate furimazine (10 \selectlanguage{greek}μ\selectlanguage{english}M) was added to each well and equilibrated for
5 minutes to enable NanoLuc-mediated furimazine oxidation and resulting
luminescence emissions. Emissions were recorded using the PHERAstar FS
platereader (BMG Labtech) using a filter simultaneously measuring
NanoLuc emissions at 450 nm (30 nm bandpass) and TMR emissions using a
longpass filter at 550 nm. BRET ratios were calculated as fluorescence
over luminescence emissions from the second of three cycles.

For kinetic experiments, cells were pre-treated with furimazine (10 \selectlanguage{greek}μ\selectlanguage{english}M)
for 5 minutes to enable NanoLuc-mediated furimazine oxidation and
resulting luminescence emissions. BRET ratios were then measured per
well using the PHERAstar FS platereader using the filters above.
Following 4 initial measurements, intact cells were stimulated with
0.5-20 nM VEGF165a-TMR or VEGF165b-TMR Emissions were recorded every 30
seconds for 20 minutes or 90 minutes, using the temperature control
function of the PHERAstar FS platereader to maintain conditions at 37\selectlanguage{ngerman}°C.

\subsection*{Data Analysis}

{\label{data-analysis}}

Data were analysed using GraphPad Prism 7.02 (GraphPad Software, La
Jolla, CA, USA). Data are presented as mean ± S.E.M.. Statistical
significance was defined as \emph{P\textless{}} 0.05. Confocal images
were collected using Zen 2010 software (Zeiss, Germany). Confocal images
were processed and analysed using ImageJ Fiji 1.52 software (National
Institutes of Health, US).

For colocalization analysis, confocal images were corrected to the
background fluorescence intensity from each experimental replicate
determined using un-transfected cells in each field of view
(HaloTag-VEGFR2, 488 nm; SnapTag-NRP1, 647 nm). The mean background
intensity was calculated for each experimental replicate (n=4) and
subtracted from each image for manual thresholding. To quantify
colocalization, regions of interest (ROIs) were drawn around each cell
that co-expressed HaloTag-VEGFR2 and SnapTag-NRP1. Following subtraction
of the region outside the ROI, colocalisation was determined using
pixel-based measures between HaloTag-VEGFR2 and SnapTag-NRP1 using the
ImageJ plugin Coloc 2. Mander's Overlap Coefficients measure
co-occurrence as the proportion of SnapTag-NRP1 pixels (red) overlapping
with HaloTag-VEGFR2 (green). Pearson's Correlation Coefficients measure
whether there is a correlation between these channels. Colocalisation
parameters were calculated on a per cell basis, with a total number of
97 cells (vehicle) and 54 cells (VEGF165a stimulation) and 68 cells
(VEGF165b stimulation), pooled from 4 independent experiments.

Saturation binding curves were fitted simultaneously for total
(VEGF\textsubscript{165}a-TMR or VEGF\textsubscript{165}b-TMR alone) and
non\selectlanguage{english}-specific binding (obtained in the presence of 100 nM of unlabelled
VEGF\selectlanguage{english}-A) using the equation:

\begin{equation}
Total\ Binding=\ B_{\max}.\frac{\ [L]}{\left[L\right]+\ K_{d}}+M.\left[L\right]+C\nonumber \\
 \end{equation}

describing the nanomolar fluorescent ligand concentration, {[}L{]};
maximal binding, Bmax; the equilibrium dissociation constant of the
labelled ligand, Kd, in the same units as {[}L{]}, the slope of the
non-specific binding component, M; and the y axis intercept, c.

Kinetic studies of fluorescent ligand binding measured over time were
fitted to a mono-exponential association function:

\begin{equation}
Binding=y_{\max}\ .\ (1\ -e^{-k_{\text{obs}}\text{\ .\ t}})\nonumber \\
 \end{equation}

describing time, t, plotted on the x axis; maximum response at infinite
time, y\textsubscript{max}; and the rate constant observed for
association, k\textsubscript{obs}. Additionally, k\textsubscript{on} and
k\textsubscript{off} values were determined by simultaneously fitting
association curves at different fluorescent ligand concentrations
({[}L{]}). This utilised the following relationship with
k\textsubscript{obs}:

\begin{equation}
k_{\text{obs}}=k_{\text{on}}.\left[L\right]+\ k_{\text{off}}\nonumber \\
 \end{equation}

further describing association rate, k\textsubscript{on}, in units of
min\textsuperscript{-1} M\textsuperscript{-1}; and dissociation rate,
k\textsubscript{off}, in min\textsuperscript{-1}. These kinetic data
were also used to estimate the binding affinities, due to the
relationship between dissociation and association rates within an
equilibrium:

\begin{equation}
K_{d}=\ \frac{k_{\text{off}}}{k_{\text{on}}}\nonumber \\
 \end{equation}

Residence time was calculated as the reciprocal of k\textsubscript{off}.
Additionally, assuming a first order reaction, half life
(t\textsubscript{1/2}) was calculated for a given concentration.

\begin{equation}
t_{1/2}=\ \frac{\ln 2}{k_{\text{on}}.\left[L\right]+\ k_{\text{off}}}\nonumber \\
 \end{equation}\begin{equation}
t_{1/2}=\ \frac{0.693}{k_{\text{obs}}}\nonumber \\
 \end{equation}

\section*{RESULTS}

{\label{results}}

\subsection*{Co-localisation between VEGFR2 and NRP1 co-expressed in
living HEK293T
cells}

{\label{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\selectlanguage{ngerman}°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 VEGF\textsubscript{165}b (upper panels) or
VEGF\textsubscript{165}a (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 VEGF\textsubscript{165}b, 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 VEGF\textsubscript{165}a 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 (\textless{}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.

\subsection*{Complementation of NanoBiT fragments using N-terminal tagged
VEGFR2 and
NRP1}

{\label{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.

\textbf{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 VEGF\textsubscript{165}a 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 VEGF\textsubscript{165}a (LgBiT-VEGFR2
pEC\textsubscript{50} = 9.95 ± 0.11; HiBiT-VEGFR2 pEC\textsubscript{50}=
10.06 ± 0.12; SmBiT-VEGFR2 pEC\textsubscript{50} = 10.23 ± 0.23; n=5 for
each). These were comparable to potency values derived for
VEGF\textsubscript{165}a at wild type VEGFR2 (Kilpatrick et al., 2017).

\subsection*{Nanomolar affinity of fluorescent VEGF-A at a defined
VEGFR2/NRP1
complex}

{\label{nanomolar-affinity-of-fluorescent-vegf-a-at-a-defined-vegfr2nrp1-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
VEGF\textsubscript{165}b-TMR selectively binds to NanoLuc-VEGFR2 (and
not NRP1), whereas VEGF\textsubscript{165}a-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
VEGF\textsubscript{165}b-TMR (Figure 6b) or VEGF\textsubscript{165}a-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 (K\textsubscript{d}) in the
nanomolar range at the VEGFR2/NRP1 complex (VEGF\textsubscript{165}b-TMR
K\textsubscript{d} = 16.26 ± 3.81 nM, pK\textsubscript{d} = 7.82 ± 0.11;
VEGF\textsubscript{165}a-TMR K\textsubscript{d} = 2.53 ± 0.49,
pK\textsubscript{d} = 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).

\subsection*{Real-time kinetics of fluorescent VEGF-A isoforms at a
heteromeric VEGFR2/NRP1 NanoBiT
complex}

{\label{real-time-kinetics-of-fluorescent-vegf-a-isoforms-at-a-heteromeric-vegfr2nrp1-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 VEGF\textsubscript{165}b-TMR with that of
VEGF\textsubscript{165}a-TMR at the VEGFR2/NRP1 NanoBiT complex in
living cells. The kinetic binding profile of
VEGF\textsubscript{165}b-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 VEGF\textsubscript{165}b-TMR (Figure 7a).
Fitted to a global association curve (Table 1),
VEGF\textsubscript{165}b-TMR had a slightly slower association rate
constant (k\textsubscript{on}) for the VEGFR2/NRP1 complex (2.29 x
10\textsuperscript{6} ± 0.30 x
10\textsuperscript{6}min\textsuperscript{-1}.M\textsuperscript{-1})
compared to NanoLuc-VEGFR2 alone (7.29 x
10\textsuperscript{6}min\textsuperscript{-1}.M\textsuperscript{-1};
Peach et al., 2018a). We then directly compared the real-time binding
profile for a saturating concentration of VEGF\textsubscript{165}b-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 VEGF\textsubscript{165}b-TMR. There was no BRET detected between
VEGF\textsubscript{165}b-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
VEGF\textsubscript{165}a-TMR (Figure 7c). Unlike
VEGF\textsubscript{165}b-TMR, there was a small decline in BRET ratio
between 30-60 minutes for VEGF\textsubscript{165}a-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).
VEGF\textsubscript{165}a-TMR had a slower dissociation rate constant
(k\textsubscript{off}) at the HiBiT complex (0.046 ± 0.007
min\textsuperscript{-1}; Table 1) compared to that previously reported
for NanoLuc-NRP1 expressed alone (0.26 min\textsuperscript{-1}; Peach et
al., 2018a). As a consequence, the kinetic binding profile for 10 nM
VEGF\textsubscript{165}a-TMR was directly compared between the NanoBiT
complex and either NanoLuc-VEGFR2 or NanoLuc-NRP1 (Figure 7d).
VEGF\textsubscript{165}a-TMR association kinetics at the NanoBiT complex
in the initial 20 minutes were more comparable to NanoLuc-VEGFR2 than
NanoLuc-NRP1 (NanoBiT k\textsubscript{obs} = 0.33 ± 0.04
min\textsuperscript{-1}, NanoLuc-VEGFR2 k\textsubscript{obs} = 0.31 ±
0.03 min\textsuperscript{-1}, NanoLuc-NRP1 k\textsubscript{obs} = 0.93 ±
0.09 min\textsuperscript{-1}; n=5 per group). These observed rate
constants were significantly slower at the complex than NRP1 alone
(repeated-measures ANOVA and Holm-\selectlanguage{polish}Š\selectlanguage{english}id\selectlanguage{ngerman}ák's multiple
comparisons;\emph{P\textless{}} 0.05, n=5 for each). These data suggest
that the ligand binding profile for VEGF\textsubscript{165}a-TMR at the
NanoBiT complex reflected VEGFR2 binding kinetics, as opposed to the
faster binding observed at NRP1.

\subsection*{Fluorescent VEGF-A kinetics were similar for the SmBiT
Complex}

{\label{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 VEGF\textsubscript{165}b-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).
VEGF\textsubscript{165}b-TMR had a slower dissociation rate
(k\textsubscript{off}) from the SmBiT complex compared to the HiBiT
complex (Kruskal-Wallis test, \emph{P\textless{}} 0.05, n=5 per group).
Plotting the individual observed association rate constants
(k\textsubscript{obs}) against VEGF\textsubscript{165}b-TMR
concentration, there was a linear relationship observed at both HiBiT
and SmBiT complexes (Figure 8b). The interaction between
VEGF\textsubscript{165}b-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
VEGF\textsubscript{165}a-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
VEGF\textsubscript{165}a-TMR for the HiBiT and SmBiT complexes
(Kruskal-Wallis test and Dunn's multiple comparisons
test,\emph{P\textgreater{}} 0.05, n=5 per group). There was a linear
relationship between the derived observed association rate
(k\textsubscript{obs}) constants and VEGF\textsubscript{165}a-TMR
concentration (Figure 8d). Despite having the potential to bind to both
receptors within the complex, the interaction between
VEGF\textsubscript{165}a-TMR and the NanoBiT complex could also be
defined by a first order reaction.

\subsection*{Similar complex pharmacology using a binding-dead mutant of
NRP1}

{\label{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
VEGF\textsubscript{165}a-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,
VEGF\textsubscript{165}a-TMR exhibited saturable binding at the NanoBiT
complex (Figure 9c). This was displaced by a high concentration of
unlabelled VEGF\textsubscript{165}a, 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
(VEGF\textsubscript{165}a-TMR/NanoBiT Y297A K\textsubscript{d} = 1.55 ±
0.38; pK\textsubscript{d} 8.84 ± 0.11; n=3). Binding kinetics at the
mutant NanoBiT complex were then monitored using four concentrations of
VEGF\textsubscript{165}a-TMR (Figure 9d). This had an identical profile
compared to VEGF\textsubscript{165}a-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 (k\textsubscript{on} =
3.71x10\textsuperscript{7} ±
0.21x10\textsuperscript{7}min\textsuperscript{-1}M\textsuperscript{-1};
k\textsubscript{off} = 0.054 ± 0.008 min\textsuperscript{-1}; kinetic
pK\textsubscript{d} = 8.85 ± 0.04; n=5). These data suggest that
VEGF\textsubscript{165}a-TMR bound the NanoBiT complex with similar
kinetics, regardless of the ability to simultaneously engage NRP1.

\section*{DISCUSSION}

{\label{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 \emph{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 VEGF\textsubscript{165}a 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 VEGF\textsubscript{165}a binding to the heteromeric complex. The
application of both NanoBiT technology and NanoBRET to monitor
exclusively VEGF\textsubscript{165}a-TMR binding to VEGFR2/NRP1
complexes allowed us to test this hypothesis directly. Interestingly,
the initial association kinetics (during the first 20 minutes) for
VEGF\textsubscript{165}a-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 VEGF\textsubscript{165}a where
k\textsubscript{obs} was 0.33 ± 0.04 min\textsuperscript{-1} for the
VEGFR2/NRP1 NanoBit complex, 0.31 ± 0.03 min\textsuperscript{-1} for
NanoLuc-VEGFR2 and 0.93 ± 0.09 min\textsuperscript{-1} for NanoLuc-NRP1.
Furthermore, the removal of the binding site for
VEGF\textsubscript{165}a 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 VEGF\textsubscript{165}a-TMR to the
heteromeric complex. It is possible therefore that heteromerization
between VEGFR2 and NRP1 masks the high affinity binding site for
VEGF\textsubscript{165}a 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
VEGF\textsubscript{165}a and anti-angiogenic VEGF\textsubscript{165}b
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).
VEGF\textsubscript{165}b is, however, selective for VEGFR2 and unable to
interact with NRP1 (Peach et al., 2018a). The real-time BRET signal for
VEGF\textsubscript{165}b-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
VEGF\textsubscript{165}a-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 (\textless{}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
VEGF\textsubscript{165}a 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 VEGF\textsubscript{165}a 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 VEGF\textsubscript{165}a-TMR and VEGF\textsubscript{165}b-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
VEGF\textsubscript{165}a-TMR binding, and a NRP1 binding-dead mutant
(Y297A) had no effect on the binding of VEGF\textsubscript{165}a-TMR, or
the formation of VEGFR2-NRP1 complexes, suggesting that the high
affinity binding site for VEGF\textsubscript{165}a on NRP1 might be
masked within the heteromeric complexes. In keeping with this conclusion
VEGF\textsubscript{165}b-TMR, which does not bind to NRP1, had a very
similar binding profile to the heteromeric complex to that observed with
VEGF\textsubscript{165}a-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 VEGF\textsubscript{165}a-TMR and
VEGF\textsubscript{165}b-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.

\section*{REFERENCES}

{\label{references}}

Ballmer-Hofer, K., Andersson, A.E., Ratcliffe, L.E., and Berger, P.
(2011). Neuropilin-1 promotes VEGFR-2 trafficking through Rab11 vesicles
thereby specifying signal output. \emph{Blood} \emph{118} : 816--26.

Banerjee, S., Sengupta, K., Dhar, K., Mehta, S., D'Amore, P., Dhar, G.,
et al. (2006). Breast Cancer Cells Secreted Platelet-Derived Growth
Factor-Induced Motility of Vascular Smooth Muscle Cells Is Mediated
Through Neuropilin-1. \emph{Mol. Carcinog.} \emph{45} : 871--80.

Basagiannis, D., and Christoforidis, S. (2016). Constitutive endocytosis
of VEGFR2 protects the receptor against shedding. \emph{J. Biol. Chem.
291} : 16892--903.

Basagiannis, D., Zografou, S., Murphy, C., Fotsis, T., Morbidelli, L.,
Ziche, M., et al. (2016). VEGF induces signalling and angiogenesis by
directing VEGFR2 internalisation via macropinocytosis. \emph{J. Cell
Sci. 129} : 4091--104.

Botta, J., Bibic, L., Killoran, P., McCormick, P., and Howell, L.
(2019). Design and development of stapled transmembrane peptides that
disrupt the activity of G-protein coupled receptor oligomers. \emph{J.
Biol. Chem. 294} : 16587--603.

Brozzo, M.S., Bjelic, S., Kisko, K., Schleier, T., Leppánen, V.-M.,
Alitalo, K., et al. (2011). Thermodynamic and structural description of
allosterically regulated VEGF receptor 2 dimerization. \emph{Blood119} :
1781--88.

Carmeliet, P. (2005). Angiogenesis in life, disease and
medicine.\emph{Nature} \emph{438} : 932--36.

Cébe Suarez, S., Pieren, M., Cariolato, L., Arn, S., Hoffman, U.,
Bogucki, A., et al. (2006). A VEGF-A splice variant defective for
heparan sulfate and neuropilin-1 binding shows attenuated signaling
through VEGFR-2. Cell. Mol. \emph{Life Sci.} \emph{63} : 2067--77.

Chung, A.S., and Ferrara, N. (2011). Developmental and pathological
angiogenesis. \emph{Annu. Rev. Cell Dev. Biol.} \emph{27} : 563--84.

Delcombel, R., Janssen, L., Vassy, R., Gammons, M., Haddad, O., Richard,
B., et al. (2013). New prospects in the roles of the C-terminal domains
of VEGF-A and their cooperation for ligand binding, cellular signaling
and vessels formation. \emph{Angiogenesis} \emph{16} : 353--71.

Dixon, A.S., Schwinn, M.K., Hall, M.P., Zimmerman, K., Otto, P., Lubben,
T.H., et al. (2016). NanoLuc Complementation Reporter Optimized for
Accurate Measurement of Protein Interactions in Cells. \emph{ACS Chem.
Biol.} \emph{11} : 400--08.

Djordjevic, S., and Driscoll, P.C. (2013). Targeting VEGF signalling via
the neuropilin co-receptor. \emph{Drug Discov. Today} \emph{18} :
447--45.

Eswarappa, S.M., Potdar, A.A., Koch, W.J., Fan, Y., Vasu, K., Lindner,
D., et al. (2014). Programmed translational readthrough generates
antiangiogenic VEGF-Ax. \emph{Cell} \emph{157} : 1605--18.

Ewan, L.C., Jopling, H.M., Jia, H., Mittar, S., Bagherzadeh, A., Howell,
G.J., et al. (2006). Intrinsic tyrosine kinase activity is required for
vascular endothelial growth factor receptor 2 ubiquitination, sorting
and degradation in endothelial cells. \emph{Traffic} \emph{7} :
1270--82.

Fantin, A., Herzog, B., Mahmoud, M., Yamaji, M., Plein, A., Denti, L.,
et al. (2014). Neuropilin 1 (NRP1) hypomorphism combined with defective
VEGF-A binding reveals novel roles for NRP1 in developmental and
pathological angiogenesis. \emph{Development} \emph{141} : 556--62.

Fantin, A., Lampropoulou, A., Gestri, G., Raimondi, C., Senatore, V.,
Zachary, I., et al. (2015). NRP1 Regulates CDC42 Activation to Promote
Filopodia Formation in Endothelial Tip Cells. \emph{Cell Rep.11} :
1577--90.

Fantin, A., Schwarz, Q., Davidson, K., Normando, E.M., Denti, L., and
Ruhrberg, C. (2011). The cytoplasmic domain of neuropilin 1 is
dispensable for angiogenesis, but promotes the spatial separation of
retinal arteries and veins. \emph{Development} \emph{138} : 4185--91.

Fantin, A., Vieira, J.M., Plein, A., Denti, L., Fruttiger, M., Pollard,
J.W., et al. (2013). NRP1 acts cell autonomously in endothelium to
promote tip cell function during sprouting angiogenesis. \emph{Blood121}
: 2352--62.

Gelfand, M. V, Hagan, N., Tata, A., Oh, W.-J., Lacoste, B., Kang, K.-T.,
et al. (2014). Neuropilin-1 functions as a VEGFR2 co-receptor to guide
developmental angiogenesis independent of ligand binding. \emph{eLife3}
: e03720. doi: 10.7554/eLife.03720.

Goel, H.L., and Mercurio, A.M. (2013). VEGF targets the tumour
cell.\emph{Nat. Rev. Cancer} \emph{13} : 871--82.

Hall, M.P., Unch, J., Binkowski, B.F., Valley, M.P., Butler, B.L., Wood,
M.G., et al. (2012). Engineered luciferase reporter from a deep sea
shrimp utilizing a novel imidazopyrazinone substrate. \emph{ACS Chem.
Biol.} \emph{7} : 1848--57.

Herzog, B., Pellet-Many, C., Britton, G., Hartzoulakis, B., and Zachary,
I.C. (2011). VEGF binding to NRP1 is essential for VEGF stimulation of
endothelial cell migration, complex formation between NRP1 and VEGFR2,
and signaling via FAK Tyr407 phosphorylation. \emph{Mol. Biol. Cell22} :
2766--76.

Jubb, A.M., Strickland, L.A., Liu, S.D., Mak, J., Schmidt, M., and
Koeppen, H. (2012). Neuropilin-1 expression in cancer and
development.\emph{J. Pathol.} \emph{226} : 50--60.

Kawamura, H., Li, X., Harper, S.J., Bates, D., and Claesson-Welsh, L.
(2008). Vascular Endothelial Growth Factor (VEGF)-A165b Is A Weak In
vitro Agonist for VEGF Receptor-2 Due to Lack of Coreceptor Binding and
Deficient Regulation of Kinase Activity. \emph{Cancer Res.} \emph{68} :
4683--92.

Kilpatrick, L.E., Friedman-Ohana, R., Alcobia, D.C., Riching, K., Peach,
C.J., Wheal, A., et al. (2017). Real-time analysis of the binding of
fluorescent VEGF\textsubscript{165}a to VEGFR2 in living cells: Effect
of receptor tyrosine kinase inhibitors and fate of internalized
agonist-receptor complexes. \emph{Biochem. Pharmacol. 136} : 62--75.

King, C., Wirth, D., Workman, S., and Hristova, K. (2018). Interactions
between NRP1 and VEGFR2 molecules in the plasma membrane. \emph{Biochim.
Biophys. Acta - Biomembr.} \emph{1860} : 2118--25.

Koch, S., Meeteren, L.A. Van, Morin, E., Testini, C., Weström, S.,
Björkelund, H., et al. (2014). NRP1 Presented in trans to the
endothelium arrests VEGFR2 endocytosis, preventing angiogenic signaling
and tumor initiation. \emph{Dev. Cell} \emph{28} : 633--46.

Koch, S., Tugues, S., Li, X., Gualandi, L., and Claesson-Welsh, L.
(2011). Signal transduction by vascular endothelial growth factor
receptors. \emph{Biochem. J. 437} : 169--83.

Lee-Montiel, F.T., Li, P., and Imoukhuede, P.I. (2015). Quantum dot
multiplexing for the profiling of cellular receptors. Nanoscale
18504--18514.

Lee, S.W., Lee, J.E., Yoo, C.Y., Ko, M.S., Park, C.S., and Yang, S.H.
(2014). NRP-1 expression is strongly associated with the progression of
pituitary adenomas. Oncol. Rep. \emph{32} : 1537--1542.

Leppanen, V.M., Prota, A.E., Jeltsch, M., Anisimov, A., Kalkkinen, N.,
Strandin, T., et al. (2010). Structural determinants of growth factor
binding and specificity by VEGF receptor 2. \emph{Proc. Natl. Acad. Sci.
U.S.A.} \emph{107} : 2425--30.

Mamluk, R., Gechtman, Z., Kutcher, M.E., Gasiunas, N., Gallagher, J.,
and Klagsbrun, M. (2002). Neuropilin-1 binds vascular endothelial growth
factor 165, placenta growth factor-2, and heparin via its b1b2
domain.\emph{J. Biol. Chem.} \emph{277} : 24818--25.

Muhl, L., Folestad, E.B., Gladh, H., Wang, Y., Moessinger, C.,
Jakobsson, L., et al. (2017). Neuropilin 1 binds platelet-derived growth
factor (PDGF)-D and is a co-receptor in PDGF-D/PDGF receptor \selectlanguage{greek}β
\selectlanguage{english}signaling. \emph{J. Cell Sci.} \emph{130} : 1365--78.

Parker, M.W., Xu, P., Li, X., and Vander Kooi, C.W. (2012). Structural
basis for selective vascular endothelial growth factor-A (VEGF-A)
binding to neuropilin-1. \emph{J. Biol. Chem.} \emph{287} : 11082--89.

Peach, C.J., Kilpatrick, L.E., Friedman-Ohana, R., Zimmerman, K.,
Robers, M.B., Wood, K. V., et al. (2018a). Real-Time Ligand Binding of
Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in
Living Cells. \emph{Cell Chem. Biol.} \emph{25} : 1208--18.

Peach, C.J., Mignone, V.W., Arruda, M.A., Hill, S.J., Kilpatrick, L.E.,
and Woolard, J. (2018b). Molecular Pharmacology of VEGF-A Isoforms:
Binding and Signalling at VEGFR2. \emph{Int. J. Mol. Sci.} \emph{19} :
1--27.

Peach, C.J., Kilpatrick, L.E., Woolard, J., and Hill, S.J. (2019).
Comparison of the ligand binding properties of fluorescent VEGF-A
isoforms to VEGFR2 in living cells and membrane preparations using
NanoBRET. \emph{Br. J. Pharmacol.} \emph{176} : 3220--35.

Prahst, C., H\selectlanguage{ngerman}éroult, M., Lanahan, A.A., Uziel, N., Kessler, O.,
Shraga-Heled, N., et al. (2008). Neuropilin-1-VEGFR-2 complexing
requires the PDZ-binding domain of neuropilin-1. \emph{J. Biol.
Chem.283} : 25110--14.

Rizzolio, S., Rabinowicz, N., Rainero, E., Lanzetti, L., Serini, G.,
Norman, J., et al. (2012). Neuropilin-1-dependent regulation of
EGF-receptor Signaling. \emph{Cancer Res.} \emph{72} : 5801--11.

Roy, S., Bag, A.K., Singh, R.K., Talmadge, J.E., Batra, S.K., and Datta,
K. (2017). Multifaceted role of neuropilins in the immune system:
Potential targets for immunotherapy. \emph{Front. Immunol.} \emph{8} :
1--27.

Ruch, C., Skiniotis, G., Steinmetz, M.O., Walz, T., and Ballmer-Hofer,
K. (2007). Structure of a VEGF--VEGF receptor complex determined by
electron microscopy. \emph{Nat. Struct. Mol. Biol.} \emph{14} : 249--50.

Shintani, Y., Takashima, S., Asano, Y., Kato, H., Liao, Y., Yamazaki,
S., et al. (2006). Glycosaminoglycan modification of neuropilin-1
modulates VEGFR2 signaling. \emph{EMBO J.} \emph{25} : 3045--55.

Simons, M., Gordon, E., and Claesson-Welsh, L. (2016). Mechanisms and
regulation of endothelial VEGF receptor signalling. \emph{Nat. Rev. Mol.
Cell Biol.} \emph{17} : 611--25.

Soker, S., Miao, H.Q., Nomi, M., Takashima, S., and Klagsbrun, M.
(2002). VEGF165 mediates formation of complexes containing VEGFR-2 and
neuropilin-1 that enhance VEGF165-receptor binding. \emph{J. Cell.
Biochem.} \emph{85} : 357--68.

Soker, S., Takashima, S., Hua, Miao, Q., Neufeld, G., and Klagsbrun, M.
(1998). Neuropilin-1 Is Expressed by Endothelial and Tumor Cells as an
Isoform-Specific Receptor for Vascular Endothelial Growth
Factor.\emph{Cell} \emph{92} : 735--45.

Stoddart, L., Johnstone, E.K.M., Wheal, A.J., Goulding, J., Robers,
M.B., Machleidt, T., et al. (2015). Application of BRET to monitor
ligand binding to GPCRs. \emph{Nat. Methods} \emph{12} : 661--63.

Stoddart, L.A., Kilpatrick, L.E., and Hill, S.J. (2017). NanoBRET
Approaches to Study Ligand Binding to GPCRs and RTKs. Trends Pharmacol.
Sci. \emph{39} : 136--47.

Vander Kooi, C.W., Jusino, M.A., Perman, B., Neau, D.B., Bellamy, H.D.,
and Leahy, D.J. (2007). Structural basis for ligand and heparin binding
to neuropilin B domains. \emph{Proc. Natl. Acad. Sci. U.S.A.104} :
6152--57.

Vempati, P., Popel, A.S., and Mac Gabhann, F. (2014). Extracellular
regulation of VEGF: Isoforms, proteolysis, and vascular
patterning.\emph{Cytokine Growth Factor Rev} . \emph{25} : 1--19.

West, D.C., Rees, C.G., Duchesne, L., Patey, S.J., Terry, C.J.,
Turnbull, J.E., et al. (2005). Interactions of multiple heparin binding
growth factors with neuropilin-1 and potentiation of the activity of
fibroblast growth factor-2. \emph{J. Biol. Chem.} \emph{280} :
13457--64.

Whitaker, G.B., Limberg, B.J., and Rosenbaum, J.S. (2001). Vascular
Endothelial Growth Factor Receptor-2 and Neuropilin-1 Form a Receptor
Complex that is Responsible for the Differential Signaling Potency of
VEGF165 and VEGF121. \emph{J. Biol. Chem.} \emph{276} : 25520--31.

White, C.W., Caspar, B., Vanyai, H.K., Pfleger, K.D.G., and Hill, S.J.
(2020). CRISPR-Mediated Protein Tagging with Nanoluciferase to
Investigate Native Chemokine Receptor Function and Conformational
Changes. \emph{Cell Chem. Biol. 27:} 499--510.

Windwarder, M., Yelland, T., Djordjevic, S., and Altmann, F. (2016).
Detailed characterization of the O-linked glycosylation of the
neuropilin-1 c/MAM-domain. \emph{Glycoconj. J.} \emph{33} : 387--97.

Witmer, A.N., Dai, J., Weich, H.A., Vrensen, G.F.J.M., and Schlingemann,
R.O. (2002). Expression of vascular endothelial growth factor receptors
1, 2, and 3 in quiescent endothelia. \emph{J. Histochem. Cytochem.50} :
767--77.

Woolard, J., Bevan, H.S., Harper, S.J., and Bates, D. (2009). Molecular
diversity of VEGF-A as a regulator of its biological
activity.\emph{Microcirculation} \emph{16} : 572--92.

Woolard, J., Wang, W., Bevan, H.S., Qiu, Y., Morbidelli, L.,
Pritchard-jones, R.O., et al. (2004). VEGF 165b, an Inhibitory Vascular
Endothelial Growth Factor Splice Variant: Mechanism of Action , In vivo
Effect On Angiogenesis and Endogenous Protein Expression. \emph{Cancer
Res.} \emph{64} : 7822--35.

\section*{FIGURE LEGENDS}

{\label{figure-legends}}

Figure 1. \textbf{Localisation of VEGFR2 and NRP1 co-expressed in living
HEK293T cells} . (a) HEK293T cells expressing HaloTag-VEGFR2 and
SnapTag-NRP1 were simultaneously labelled with membrane-impermeant 0.5
\selectlanguage{greek}μ\selectlanguage{english}M HaloTag-AlexaFluor488 and 0.5 \selectlanguage{greek}μ\selectlanguage{english}M SnapTag-AlexaFluor647 for 30 minutes
(37\selectlanguage{ngerman}°C). Cells were washed twice in HEPES Buffered Saline Solution (HBSS)
containing 0.1\% Bovine Serum Albumin (BSA) and incubated at 37°C. Cells
were imaged on the LSM710 Confocal Microscope (40X objective). The same
cell population were imaged in the presence of vehicle or following
treatment with 10 nM unlabelled VEGF\textsubscript{165}b or
VEGF\textsubscript{165}a for 60 minutes (37°C). Images show
HaloTag-VEGFR2 (green) and SnapTag-NRP1 (magenta), showing regions of
spatial overlay in white. Images are representative from 4 independent
experiments. (b,c) ImageJ/Fiji software was used to analyse images with
channels corresponding to HaloTag-VEGFR2 or SnapTag-NRP1.
Co-localisation was quantified based on regions of interest drawn around
cells co-expressing both receptors. Mander's Overlap Coefficients
represent the proportion of SnapTag-NRP1 in HaloTag-VEGFR2+ regions (b),
whereas Pearson's Correlation Coefficients compare the relationship
between the intensity of VEGFR2 and NRP1 pixels (c). All coefficient
values were pooled from 4 independent experiments, with a total of 97
cells (vehicle), 68 cells (VEGF\textsubscript{165}b) or 54 cells
(VEGF\textsubscript{165}a). Coefficients were compared between
conditions using a Kruskal-Wallis test and post-Hoc Dunn's multiple
comparisons test between vehicle, VEGF\textsubscript{165}b or
VEGF\textsubscript{165}a stimulation (* \emph{P\textless{}} 0.05).

Figure 2. \textbf{Oligomer formation between VEGFR2 and NRP1.} (a,b)
HEK293T cells were transiently transfected with a fixed concentration of
NanoLuc-VEGFR2 (25 ng cDNA/well) and increasing concentrations of
fluorescent acceptor (HaloTag-NRP1 or SnapTag-NRP1, 0-100 ng cDNA/well).
All wells were transfected with 125 ng cDNA/well total with empty
pcDNA3.1/Zeo vector. NRP1 was labelled with 0.2 \selectlanguage{greek}µ\selectlanguage{english}M HaloTag-AlexaFluor488
substrate or 0.2 \selectlanguage{greek}μ\selectlanguage{english}M SNAP-Surface AlexaFluor488 substrate for 30 minutes
(37\selectlanguage{ngerman}°C). Cells were washed twice with HBSS/0.1\% BSA then incubated in 10
\selectlanguage{greek}µ\selectlanguage{english}M furimazine for 5 minutes (37\selectlanguage{ngerman}°C). Emissions from the luminescent donor
and fluorescent acceptor receptor were simultaneously monitored by the
PHERAstar FS platereader. Data are expressed as (a) mean ± S.E.M. from 5
independent experiments with duplicate wells or (b) individual data
points from a representative experiment plotting BRET ratio values
against fluorescence emissions (485-520 nm).

Figure 3. \textbf{Complementation of a VEGFR2/NRP1 NanoBiT complex} .
(a) To determine the optimal orientation of labelling with NanoLuc
Binary Technology (NanoBiT) fragments, each receptor was tagged with the
18 kDa fragment (LgBiT) and a smaller 11 amino acid fragment. HiBiT has
a higher intrinsic affinity to complement with LgBiT compared to SmBiT
(Dixon et al., 2016). HEK293T cells were transiently transfected in
96-well plates with equal amounts of LgBiT-tagged receptor (50 ng
cDNA/well) and HiBiT- or SmBiT-tagged receptor (50 ng cDNA/well). Cells
were incubated with 10 \selectlanguage{greek}µ\selectlanguage{english}M furimazine in HBSS/0.1\% BSA for 10 minutes
(37\selectlanguage{ngerman}°C). Data were normalised to un-transfected cells (0\%) and
HiBiT-NRP1/LgBiT-VEGFR2 (100\%) per experiment. Data are expressed as
mean ± S.E.M. from 5 independent experiments (LgBiT-VEGFR2) or 3
independent experiments (LgBiT-NRP1), each with triplicate wells. (b) To
compare emissions from individual NanoBiT-tagged receptors relative to a
complemented NanoBiT complex, HEK293T cells were transiently transfected
in 96-well plates with LgBiT-VEGFR2, HiBiT-NRP1 or SmBiT-NRP1 (50 ng
cDNA/well). Dual expression cells expressed a complemented NanoBiT
complex (filled bars) whereas single constructs (empty bars) were
transfected with 50 ng cDNA/well empty pcDNA3.1/Zeo vector for 100 ng
total cDNA/well. Experiments were repeated in 5 independent experiments.
Raw emissions were plotted from a representative experiment as mean ±
S.E.M. from triplicate wells. (c) Cells expressed a single
NanoBiT-tagged construct, in the absence (open bars) or presence (filled
bars) of 20 nM purified HiBiT or LgBiT. Data are representative from 5
independent experiments from the same experiment as (b). Raw emissions
were plotted as mean ± S.E.M. from triplicate wells. (d) Prevention of
NanoBiT complex formation by co-expression of increasing amounts of
competing VEGFR2 or NRP1. HEK293T cells were transfected with equal
amounts of LgBiT-VEGFR2 (50 ng cDNA/well) and either HiBiT-NRP1 (lined
bars) or SmBiT-NRP1 (solid bars) at 50 ng cDNA/well. Cells were also
transfected with increasing amounts of HaloTag-NRP1 (0-200 ng
cDNA/well), as well as with pcDNA3.1/Zeo empty vector (for 300 ng total
cDNA/well). Data were normalised to un-transfected cells (0\%) and the
complemented NanoBiT complex in the absence of competing receptor
(100\%) per experiment. Data are expressed as mean ± S.E.M. from 3
independent experiments, each with triplicate wells. (a-d) Cells were
incubated with furimazine (10 \selectlanguage{greek}µ\selectlanguage{english}M) in HBSS/0.1\% BSA for 10 minutes
(37\selectlanguage{ngerman}°C). Luminescence emissions (475-505 nm) were measured by the
PHERAstar FS platereader.

Figure 4. \textbf{Bioluminescence imaging of NanoLuc-VEGFR2,
NanoLuc-NRP1 or NanoBiT-complemented VEGFR2-NRP1 complexes.} HEK293T
cells were transfected with LgBiT-VEGFR2 (750 ng cDNA/well) and
HiBiT-NRP1 (750 ng cDNA/well). Following 24 hours, transfected cells
were seeded into 35 mm\textsuperscript{2} glass-bottomed dishes. Cells
were incubated with furimazine for 10 minutes at 37°C (26 \selectlanguage{greek}μ\selectlanguage{english}M for
full-length NanoLuc; 104 \selectlanguage{greek}μ\selectlanguage{english}M for NanoBiT complex). Cells were imaged live
using a widefield Olympus LV200 Bioluminescence Imaging System as
described under Methods. Images are representative from 3 independent
experiments.

Figure 5. \textbf{Characterisation of NFAT signalling from VEGFR2 tagged
with LgBiT, HiBiT or SmBiT moieties.} HEK293T cells stably expressed
both NFAT-ReLuc2P and either LgBiT-VEGFR2, HiBiT-VEGFR2 or SmBiT-VEGFR2.
(a) Cells were serum-starved for 24 hours. On the day of
experimentation, cells were stimulated with increasing concentrations of
VEGF\textsubscript{165}a for 5 hours and 37\selectlanguage{ngerman}°C/5\% CO\textsubscript{2}.
Data were normalised to mean vehicle (0\%) or 10 nM unlabelled
VEGF\textsubscript{165}a (100\%) per experiment. Data are expressed as
mean ± S.E.M. from 5 independent experiments with duplicate wells per
experiment.

Figure 6. \textbf{Saturation binding of VEGF\textsubscript{165}b-TMR and
VEGF\textsubscript{165}a-TMR at a HiBiT complex of VEGFR2 and NRP1} .
(a) Fluorescent VEGF-A ligand binding was monitored at a defined complex
of LgBiT-VEGFR2 and HiBiT-NRP1. In the presence of furimazine,
individual receptors do not emit luminescence in isolation. Upon NanoBiT
complementation, luminescence emissions can excite the
tetramethylrhodamine (TMR) in close proximity. NanoBiT therefore only
acts as a luminescent donor when VEGFR2 and NRP1 are in complex. (b,c)
HEK293T cells were transfected in 6-well plates with equal amounts of
LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 (750 ng cDNA/well).
Following 24 hours, transfected cells were seeded in 96-well plates. On
the day of experimentation, cells were incubated with increasing
concentrations of VEGF\textsubscript{165}b-TMR (b) or
VEGF\textsubscript{165}a-TMR (c). This was performed in the presence or
absence of 100 nM VEGF\textsubscript{165}b (b) or
VEGF\textsubscript{165}a (c) to determine non-specific binding.
Following 60 minutes at 37°C, 10 \selectlanguage{greek}μ\selectlanguage{english}M furimazine was added for 10 minutes
(37\selectlanguage{ngerman}°C). Emissions were measured on the PHERAstar platereader. BRET
ratios are expressed as mean ± S.E.M. from 3 independent experiments
with duplicate wells.

Figure 7. \textbf{Real-time binding of fluorescent VEGF-A isoforms at
the NanoBiT complex compared to isolated receptors.} (a) HEK293T cells
were transfected in 6-well plates with equal amounts of LgBiT-VEGFR2
(750 ng cDNA/well) and HiBiT-NRP1 (750 ng cDNA/well). Alternatively,
cells were transfected with equal amounts of NanoLuc-VEGFR2 or
NanoLuc-NRP1 (750 ng cDNA/well) and empty pcDNA3.1/Zeo vector (750 ng
cDNA/well). Following 24 hours, transfected cells were seeded in 96-well
plates. On the day of experimentation, cells were pre-treated with
furimazine (10 \selectlanguage{greek}µ\selectlanguage{english}M) and left to equilibrate at 37\selectlanguage{ngerman}°C for 10 minutes. (a)
Cells expressing the NanoBiT complex (LgBiT-VEGFR2/HiBiT-NRP1) were
stimulated with 4 concentrations of VEGF\textsubscript{165}b-TMR added
at x=0. Kinetic data were fitted to a global association model with an
unconstrained k\textsubscript{on} from the 90 minute time course. (b) On
the same plate, the real-time binding profile of 20 nM
VEGF\textsubscript{165}b-TMR was monitored in cells only expressing
either NanoLuc-VEGFR2 or NanoLuc-NRP1 (left y axis, grey symbols). This
was directly compared to binding of the same concentration of
VEGF\textsubscript{165}b-TMR at the LgBiT-VEGFR2/HiBiT-NRP1 NanoBiT
Complex (right y axis, red circular symbols). (c) Cells expressing the
HiBiT complex were stimulated with 4 concentrations of
VEGF\textsubscript{165}a-TMR. Kinetic data were fitted to a global
association model without a constrained k\textsubscript{on} from the
initial 20 minutes due to the latter decline in BRET ratio. (d) The
real-time binding profile of a saturating concentration of
VEGF\textsubscript{165}a-TMR (10 nM) was compared between cells
expressing LgBiT-VEGFR2/HiBiT-NRP1 (right y axis, blue circular symbols)
to cells only expressing NanoLuc-VEGFR2 or NanoLuc-NRP1 (left y axis,
open symbols). For each experiment, emissions were simultaneously
measured on the PHERAstar FS platereader every 30 seconds for 90 minutes
at 37°C. BRET ratios were baseline-corrected to vehicle at each time
point per experimental replicate. In (b) and (d), the x axis was split
to highlight the initial association (20 minutes) and long-term BRET
signal (90 minutes). Data represent mean ± S.E.M. from 5 independent
experiments with duplicate wells. Derived k\textsubscript{on},
k\textsubscript{off} and kinetic pK\textsubscript{d} parameters are in
Table 1.

Figure 8\textbf{. Fluorescent VEGF-A binding kinetics at a NanoBiT
VEGFR2/NRP1 complex using split tags with lower intrinsic affinity} .
HEK293T cells were transfected in 6-well plates with equal amounts of
LgBiT-VEGFR2 (750 ng cDNA/well) and SmBiT-NRP1 (750 ng cDNA/well).
Following 24 hours, transfected cells were seeded in 96-well plates.
Cells were pre-treated with furimazine (10 \selectlanguage{greek}µ\selectlanguage{english}M) and left to equilibrate
at 37\selectlanguage{ngerman}°C for 10 minutes. (a) Cells were stimulated with 4 concentrations
of VEGF\textsubscript{165}b-TMR added at x=0. Kinetic data were fitted
to a global association model without a constrained
k\textsubscript{on}from the 90 minute time course. For clarity, the 10
nM data set has not been included in the figure. (b) The derived rate
constant, k\textsubscript{obs}, was obtained from exponential
association curves fitted for each of the four fluorescent ligand
concentrations. These were plotted against VEGF\textsubscript{165}b-TMR
concentration and fitted against a linear regression (HiBiT Complex y =
0.0023x + 0.034, R\textsuperscript{2} = 0.46; SmBiT Complex y = 0.0026x
+ 0.01, R\textsuperscript{2} = 0.65). (c) Cells were stimulated with 4
concentrations of VEGF\textsubscript{165}a-TMR. Kinetic data were fitted
to a global association model without a constrained
k\textsubscript{on}from the initial 20 minutes due to the latter decline
in BRET ratio. For clarity, the 5 nM data set has not been included in
the figure. (d) The derived k\textsubscript{obs} for each fluorescent
ligand at all four concentrations were plotted against each
VEGF\textsubscript{165}a-TMR concentration and fit with a linear
regression (HiBiT Complex y = 0.0024x + 0.10, R\textsuperscript{2} =
0.72; SmBiT Complex y = 0.0025x + 0.09, R\textsuperscript{2} = 0.62).
Emissions were simultaneously measured on the PHERAstar FS platereader
every 30 seconds for 90 minutes at 37°C. BRET ratios were
baseline-corrected to vehicle at each time point per replicate. Data
represent mean ± S.E.M. from 5 independent experiments with duplicate
wells in each independent experiment. Derived k\textsubscript{on},
k\textsubscript{off} and kinetic pK\textsubscript{d} parameters are in
Table 1.

Figure 9. \textbf{Ligand binding of VEGF\textsubscript{165}a-TMR at a
NanoBiT complex with a binding-dead NRP1 mutant} . (a)
VEGF\textsubscript{165}a-TMR ligand binding was monitored at a defined
NanoBiT complex between LgBiT-VEGFR2 and a HiBiT-NRP1 VEGF-A
binding-dead mutant in the b1 domain. (b) HEK293T cells were transiently
transfected in 96-well plates with LgBiT-VEGFR2, HiBiT-NRP1 Y297A or
SmBiT-NRP1 Y297A (50 ng cDNA/well). Dual expression cells expressed a
complemented NanoBiT complex with the HiBiT or SmBiT tag. Cells also
expressed single constructs (empty bars) were transfected with 50 ng
cDNA/well empty pcDNA3.1/Zeo vector. Cells were incubated with 10 \selectlanguage{greek}µ\selectlanguage{english}M
furimazine in HBSS/0.1\% BSA for 10 minutes (37\selectlanguage{ngerman}°C). Luminescence
emissions (475-505 nm) were measured by the PHERAstar FS platereader.
Data were normalised to un-transfected cells (0\%) and HiBiT-NRP1
Y297A/LgBiT-VEGFR2 (100\%) per experiment. Data are expressed as mean ±
S.E.M. from 5 independent experiments, each with triplicate wells. (c,d)
HEK293T cells were transfected in 6-well plates with equal amounts of
LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 Y297A (750 ng cDNA/well).
Following 24 hours, transfected cells were seeded in 96-well plates. (c)
On the day of experimentation, cells were incubated with increasing
concentrations of VEGF\textsubscript{165}a-TMR in the presence or
absence of 100 nM VEGF\textsubscript{165}a to determine non-specific
binding. Following 60 minutes at 37°C, 10 \selectlanguage{greek}μ\selectlanguage{english}M furimazine was added for 10
minutes (37\selectlanguage{ngerman}°C). Emissions were measured on the PHERAstar platereader
(550-LP/460-480 nm). BRET ratios are expressed as mean ± S.E.M. from 3
independent experiments with duplicate wells. Derived equilibrium
dissociation constants (pK\textsubscript{d}) are in the text. (d) Cells
were pre-treated with furimazine (10 \selectlanguage{greek}µ\selectlanguage{english}M) and left to equilibrate at 37\selectlanguage{ngerman}°C
for 10 minutes. Cells were incubated with 4 concentrations of
VEGF\textsubscript{165}a-TMR. Kinetic data were fitted to a global
association model without a constrained k\textsubscript{on} from the
initial 20 minutes. Emissions were simultaneously measured on the
PHERAstar FS platereader every 30 seconds for 90 minutes at 37°C. BRET
ratios were baseline-corrected to vehicle at each time point per
experimental replicate. Data represent mean ± S.E.M. from 5 independent
experiments with duplicate wells. Derived k\textsubscript{on},
k\textsubscript{off} and kinetic pK\textsubscript{d} parameters are
noted in the text.

\section*{TABLE}

{\label{table}}

Table 1. \textbf{Summary of binding parameters derived at the NanoBiT
complex for VEGF\textsubscript{165}b-TMR and
VEGF\textsubscript{165}a-TMR, compared to published values from
receptors expressed alone} .\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& Kinetic pK\textsubscript{d} & k\textsubscript{on}
(min\textsuperscript{-1}M\textsuperscript{-1}) & k\textsubscript{off}
(min\textsuperscript{-1})\tabularnewline
\midrule
\endhead
VEGF\textsubscript{165}b-TMR & VEGF\textsubscript{165}b-TMR &
VEGF\textsubscript{165}b-TMR &
VEGF\textsubscript{165}b-TMR\tabularnewline
HiBiT Complex & 7.81 \selectlanguage{ngerman}± 0.10 (5) & 2.29x10\textsuperscript{6} \selectlanguage{ngerman}±
0.30x10\textsuperscript{6} (5) & 0.037 \selectlanguage{ngerman}± 0.007 (5)\tabularnewline
SmBiT Complex & 8.43 \selectlanguage{ngerman}± 0.17 (5) & 2.94x10\textsuperscript{6} \selectlanguage{ngerman}±
0.55x10\textsuperscript{6} (5) & 0.012 \selectlanguage{ngerman}± 0.003 (5)\tabularnewline
VEGF\textsubscript{165}a-TMR & & &\tabularnewline
HiBiT Complex & 8.83 \selectlanguage{ngerman}± 0.12 (5) & 3.12x10\textsuperscript{7} \selectlanguage{ngerman}±
0.43x10\textsuperscript{7} (5) & 0.046 \selectlanguage{ngerman}± 0.007 (5)\tabularnewline
SmBiT Complex & 8.83 \selectlanguage{ngerman}± 0.31 (5) & 2.83x10\textsuperscript{7} \selectlanguage{ngerman}±
0.69x10\textsuperscript{7} (5) & 0.046 \selectlanguage{ngerman}± 0.020 (5)\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Parentheses represent the number of independent experiments, each with
duplicate wells. Kinetic parameters were derived from a global
association fit from the full 90 minute time course
(VEGF\textsubscript{165}b-TMR) or the initial 20 minutes
(VEGF\textsubscript{165}a-TMR). Experiments with LgBiT-VEGFR2 and
HiBiT-NRP1 (HiBiT Complex) are shown in Figure 7a and c. Kinetic
experiments with LgBiT-VEGFR2 and SmBiT-NRP1 (SmBiT Complex) are shown
in Figure 8a and c.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1v2/Figure-1v2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2v2/Figure-2v2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3v2/Figure-3v2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4v2/Figure-4v2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5v2/Figure-5v2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6v2/Figure-6v2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-7v2/Figure-7v2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-8v2/Figure-8v2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-9v2/Figure-9v2}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

