Vascular endothelial growth factor (VEGF) is a
dimeric hormone that controls much of vascular development through
binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are
located near the poles of the dimer and straddle the interface between
subunits. Deletion experiments in KDR indicate that of the seven
IgG-like domains in the extracellular domain, only domains 2-3 are
needed for tight binding of VEGF. Monomeric forms of the extracellular
domain of KDR bind ~100 times weaker than dimeric forms showing a
strong avidity component for binding of VEGF to predimerized forms of
the receptor. Based upon these structure-function studies and a
mechanism in which receptor dimerization is critical for signaling, we
constructed a receptor antagonist in the form of a heterodimer of VEGF
that contained one functional and one non-functional site. These
studies establish a functional foundation for the design of VEGF
analogs, mimics, and antagonists.
 |
INTRODUCTION |
Formation of the vasculature is one of the most intriguing and
physiologically important processes in human biology. Induction of new
blood vessels is clinically relevant following stroke or heart attack,
whereas inhibition of vascularization is likely to curtail the growth
of tumors or retinopathy disease (1, 2). Vascular endothelial growth
factor (VEGF)1 regulates
vascularization by acting as an endothelial cell mitogen and a vascular
permeability factor (3). VEGF can bind to two different
single-transmembrane receptors, kinase domain receptor (KDR) and
fms-like tyrosine kinase-1 (Flt-1). Gene disruption experiments in mice
show that both receptors are necessary for embryonic development;
however, KDR is the primary mitogenic receptor for endothelial cells
(4, 5).
VEGF is a member of the cystine-knot family of growth factors which
includes PDGF, tissue growth factor-
, nerve growth factor, and
others (6). The hormone is a dimer that is held together by two
intermolecular disulfide bonds. Alternatively spliced forms of VEGF are
found in vivo that range in length from 121 to 206 residues
(7, 8). The NH2-terminal 110 residues of the most prevalent
form of VEGF, VEGF1-165, codes for the receptor-binding domain, whereas the next 55 residues code for a heparin-binding domain.
We have recently solved the x-ray crystal structure of the
receptor-binding domain of VEGF (VEGF8-109 which contains residues 8 to 109) and provided a high resolution functional map of the
binding site for KDR by alanine-scanning mutagenesis (9) (Fig. 1).
Here, we characterize the functional requirements for binding and
signaling between VEGF1-109 and KDR. Mutational and biophysical studies show that two molecules of KDR dimerize across the
subunit interface of the VEGF and initiate signaling. Deletion experiments in KDR show that IgG-like domains 2-3 are sufficient for
tight binding and domains 4-7 are not essential for signaling. Based
upon these studies we have produced a VEGF analog that antagonizes the
action of native VEGF. These studies provide a basis for developing antagonists and mimics of VEGF and should apply to other members of the
cystine-knot family of hormones and their receptors.
 |
EXPERIMENTAL PROCEDURES |
The Expression and Purification of Deletion Variants of KDR-IgG
Fusions and KDR Monomers--
The plasmid pHEBO23 containing the
cDNA coding for the KDR extracellular domain was fused to cDNA
encoding amino acids 216-443 of the human heavy chain IgG (10). A
Genenase 1 site (Ala-Ala-His-Tyr) was put in between the KDR and the
IgG by Kunkle mutagenesis (11). To generate KDR deletion mutants, a
KpnI restriction site was put at the junction of each domain
by Kunkle mutagenesis. Various deletion mutants were constructed by
cutting at a unique ClaI site outside of the 5' end of the
gene and the KpnI at each junction. Qiagen columns were used
to purify DNA to transiently transfect into the human kidney 293 cell
line. KDR-IgG mutants were expressed in 4-day conditioned serum-free
media in a quantity ranging from 100 to 400 µg/15-cm diameter dish.
Proteins were purified by protein A affinity chromatography and
quantified by their UV absorption at 280 nm and total amino acid
hydrolysis. Constructs containing KDR 1-5, 1-4, 1-3, 1-2, and 2-3
correspond to amino acids 1-552, 1-424, 1-335, 1-222, and 1-335
(with 24-116 deleted), respectively. Residues 1-19 constitute the
signal sequence based on NH2-terminal analysis of the
secreted protein, KDR 1-7.
To generate monomers of KDR domain 1-7 or KDR 1-3, the corresponding
IgG fusion was incubated with 20:1 molar ratio of fusion protein to
Genenase 1 in 20 mM Tris-HCl (pH 8) and 2 M
NaCl for 4 h at room temperature (12). The mixture was then passed
through a protein A column to capture the digested IgG portion and any incompletely digested fusion protein. The digested protein was further
purified on a gel filtration column, Superdex 200 (Pharmacia), to
remove Genenase 1 and some protein aggregates. For large scale production of KDR monomer, the KDR 1-7 and 1-3 IgG fusion with Genenase cut site were cloned into plasmid for stable dihydrofolate reductase-Chinese Hamster cells expression (13).
Stoichiometry as Determined by Gel Filtration
Chromatography--
For the stoichiometry studies, proteins were
characterized for purity by SDS-polyacrylamide gel electrophoresis and
gel filtration chromatography and quantified by total amino acids acid
hydrolysis. Different ratios of KDR with VEGF8-109 or the
corresponding heterodimer containing one intact binding site (hV-1) was
incubated at room temperature for 2 h before injecting onto the
Superdex 200 column. For KDR 1-3, one column was used. For KDR 1-7,
two columns were used in tandem.
Binding Assays--
KDR-IgG fusions or KDR monomers were
incubated with 125I-VEGF1-165 (ICN, DuPont)
and increasing concentrations of VEGF variants for 18 h at room
temperature in 100 µl of binding buffer containing 0.5% bovine serum
albumin, 0.05% Tween 20, 0.15 N NaCl, and 20 mM Tris-HCl (pH 7.5). The mixture was transferred to a 96-well plate coated with anti-Fc antibody for KDR-IgG assays or MAKD5
for KDR monomer assays and allowed 15 min to capture the complex. The
plate was then washed and counted in Topcount Microplate Scintillation
counter (Packard, Downers Grove, IL). Biotinylated
VEGF1-109 was used as tracer for some assays and
horseradish peroxidase-conjugated strepavidin was added at the end. The
concentration of receptor and 125I-VEGF1-165
or biotinylated VEGF1-109 were adjusted so that they were
at least a factor of four below the estimated Kd.
For mAb binding assays, KDR-IgG was captured on the rabbit anti-human
Fc antibody Fab (Jackson ImmunoResearch Laboratory Inc., West Grove,
PA) coated 96-well plate. Serial dilutions of mAbs were put in the
plate and allowed to bind for 2 h and the plates washed thoroughly
with incubation buffer. Horseradish peroxidase-conjugated rabbit
anti-mouse Fab antibody was added which had been preabsorbed with human
Fc.
Purification and Refolding of the VEGF Variants--
The
purification and refolding of VEGF1-109 and
VEGF8-109 was performed as described in Muller et
al. (9). The refolding of the VEGF1-109 heterodimer
was performed essentially as described by Potgens and co-workers (14).
VEGF with the C51S/I46A/I83A mutations and VEGF1-109 with
C60S/F17A/E64A mutations were purified separately from
Escherichia coli. The variants were mixed and unfolded with
6 M guanidine HCl plus 1 mM oxidized
glutathione at pH 6, and dialyzed against 10 volumes of 2 M
urea with 2 mM reduced glutathione and 0.5 mM
of oxidized glutathione in 20 mM Tris-HCl at pH 8 for
18 h. Urea was removed by dialyzing slowly against 20 volumes of
20 mM Tris-HCl (pH 8) overnight at 4 °C. The covalently
linked heterodimer was finally purified by FPLC (Pharmacia) on a Mono Q
anion exchange column and the identity confirmed by SDS-PAGE and mass
spectrometry.
KDR Transfected 3T3 Cell Lines--
The KDR 1-7 or KDR 1-3 was
fused to residue 768 of Flt-4 (1-1363) so that the fusion contains the
transmembrane (residues 776-800) and full intracellular domain of
Flt-4 in a similar fashion as the CSF-1R-Flt-4 fusion described by
Pajusola et al. (20). NIH 3T3 cells were transfected with
the purified plasmid containing the fusion and one-tenth molar of NEO
(G418)-resistant plasmid by the calcium phosphate precipitation method
(15) and selected with G418 (Life Technologies, Inc.) first 200 µg/ml, later 500 µg/ml. The NEO control cells were given the NEO
plasmid. The cells were cloned by limited dilution and selected for
their response to VEGF in the [3H]thymidine incorporation
assay. The positive clones were maintained with Dulbecco's modified
minimal essential medium/F-12 media with 10% fetal bovine serum and
400 µg/ml G418.
[3H]Thymidine Incorporation Assay--
For 3T3
cells transfected with KDR 1-7 or KDR 1-3 fused to the Flt-4
transmembrane and intracellular domains, 2000 cells were plated in each
well of the 96-well dishes and fasted with Dulbecco's modified minimal
essential F-12 media supplemented with 1% dialyzed fetal bovine serum
for 48 h. Cells were then treated with VEGF8-109 or
VEGF variants for 18 h and pulsed with 0.5 µCi/well of
[3H]thymidine (Amersham) for 6 h and harvested and
counted with Topcount Microplate Scintillation counter (Packard). For
assays with HuVEC cells (purchased from Cell System, Kirkland, WA),
cells were passed from 2-5 times. Cells were maintained in complete growth media (Cell System) on dishes coated with attachment factors (Cell System). Cells were seeded in coated 96-well plates (4000 cells
per well) and fasted in Dulbecco's modified minimal essential F-12
media with 1% dialyzed fetal bovine serum for 24 h. VEGF and
variants were added in fresh fasting media and incubated for 18 h.
Cells were pulsed with [3H]thymidine (0.5 µCi/well) for
6 h, harvested, and counted with Topcount.
 |
RESULTS |
KDR Binds at the Subunit-Subunit Interface of VEGF--
Single
alanine mutations that disrupt binding to VEGF1-109 map to
the poles of the dimer (Fig. 1). From
these mutagenesis data alone one cannot distinguish if KDR binds across
the subunit-subunit interface or if each KDR binds entirely to one
subunit (Fig. 2A). To evaluate
these models for binding we produced two mutated forms of VEGF: a
monomeric form and a heterodimer in which one pole was mutated to
obliterate receptor binding. Monomeric VEGF1-109 was
produced by substituting with arginine the two cysteine residues (Cys-51 and Cys-60) that are responsible for the intermolecular disulfides. The C51R/C60R double mutant was purified and shown to be
monomeric by native gel filtration and SDS-PAGE under nonreducing conditions (data not shown). The fact that the monomeric VEGF bound
equally well as the wild type VEGF to an anti-VEGF monoclonal antibody
suggests that its structure was largely intact; mutational studies show
this epitope is discontinuous and therefore requires proper folding of
VEGF for binding to occur.2
The monomer was unable to compete with wild type VEGF1-109 (Kd ~ 5 nM) for binding to KDR at
concentrations approaching 1 µM (Fig. 2B).
However, at much higher concentrations, monomeric VEGF could displace
wild-type VEGF with an approximate IC50 of ~20
µM (Table II). Thus, although the monomer appears to be
properly folded its binding affinity for KDR is reduced
>1000-fold.

View larger version (79K):
[in this window]
[in a new window]
|
Fig. 1.
Epitopes for binding of KDR to the VEGF
dimer. A space-filling rendition of the two VEGF subunits are
shown in white and light gray (left).
All residues that were alanine-scanned are colored: light
blue (<1.0 kcal/mol impact on binding free energy),
blue (1.0-2.0 kcal/mol), yellow (2.0-3.0
kcal/mol), and red (>3.0 kcal/mol). An end-on view
generated by rotating the molecule up by 90° (right). The
figure is reproduced with permission (9).
|
|

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2.
Panel A, inter-subunit and intra-subunit
models for binding two molecules of KDR receptor to dimeric VEGF. The
inter-subunit disulfide bonds in VEGF are indicated. Panel
B, displacement of biotinylated VEGF1-109 from
binding to monomeric KDR 1-7 by a monomeric form of
VEGF1-109 containing the double mutant C51R/C60R, a
heterodimeric form of VEGF1-109 that possesses either both
binding sites (hV-2), or a single site at one pole of the hormone
(hV-1) and the wild-type VEGF1-109 with IC50
values 500, 10, 8.9, and 4.6 nM, respectively.
Dilutions of these VEGF variants were added with fixed amounts of
biotinylated VEGF (1 nM) to KDR (0.5 nM) and
incubated for 18 h. The complex was captured with a mAb to KDR
(MAKD5) as described under "Experimental Procedures."
|
|
Variants of VEGF containing a single intermolecular disulfide bond were
produced using a strategy reported by Potgens and co-workers (14). A
C51S mutation was introduced into one subunit, and a C60S variant was
produced in the other. C51S and C60S mutants were purified separately
from E. coli and refolded together (see "Experimental
Procedures") and a heterodimer of VEGF containing two functional
receptor binding sites (hV-2) was generated. The hV-2 heterodimer bound
to KDR with virtually the same affinity as the wild-type dimer (Fig.
2B) and was fully active in cell-based assays (data not
shown). Thus, the heterodimer containing only one intersubunit
disulfide bonds is functional and properly folded. The individual
cysteine mutants taken through the same refolding procedure do not bind
KDR (up to 10 µM in concentration (data not shown),
presumably because they cannot form a proper homodimer.
Next we produced single binding site heterodimers (hV-1) in which one
pole on VEGF1-109 was mutated at residues shown by alanine
scanning to be most important for binding KDR (Fig. 1). One subunit
contained the C51S mutation plus I46A and I83A, and the other subunit
had C60S along with F17A and E64A. The hV-1 heterodimer was generated
as hV-2 and confirmed by mass spectrometry. The hV-1 bound to the KDR
monomer only 2-fold weaker than VEGF8-109 (Fig.
2B), indicating that it can still bind the receptor with one
pole intact. These data combined with the fact that monomeric VEGF
binds much weaker to KDR strongly support a model where each KDR binds
across the subunit-subunit interface and not exclusively to one of the
subunits (Fig. 2A).
The IgG-like Domains 2-3 in KDR Are Sufficient for High Affinity
Binding--
To determine the minimal IgG-like domain requirements for
binding of KDR to VEGF1-109, a series of deletions were
produced in which each of the seven IgG-like domains were deleted from the carboxyl terminus of the extracellular domain. The deletion variants were expressed initially as dimeric proteins by fusion to the
CH2-CH3 domain of an antibody (KDR-IgG). This was done to facilitate
purification on a Protein A affinity column (12) and to compare their
affinities to monomeric forms of KDR.
The choice of deletion junction was based on homology to other members
of the IgG superfamily (16, 17). Systematic carboxyl-terminal domain
deletions had virtually no effect on affinity for VEGF until IgG-like
domain 3 was deleted (Table I); KDR 1-2
had an affinity that was >1000-fold reduced relative to KDR 1-3 but
did show specific binding at concentrations above 2 µM
(data not shown). A variant of KDR missing the first
NH2-terminal domain, KDR 2-3, bound nearly as well as the
full-length KDR (Table I). These data suggest that domains 2-3 are
most important for high affinity binding.
To determine if these deletions had caused misfolding of the molecules,
we analyzed their binding to three different anti-KDR monoclonal
antibodies (Table I), one of which (MAKD6) blocks binding of VEGF. The
antibodies bound to nearly all the deletion variants with affinities
virtually identical to the full-length KDR 1-7 (EC50 ~1
nM). Deletion of domain 1 caused complete disruption for
binding of the non-neutralizing antibodies (MAKD1 and MAKD5) but not
the neutralizing antibody (MAKD6). Thus, the deletions do not grossly
disrupt the structure of the molecules and locate the epitopes for
MAKD1 and MAKD5 to domain 1 and for MAKD6 to domain 2. The fact that
the antibody MAKD6, which blocks binding of VEGF, binds to domain 2 further supports the importance of domain 2 for binding VEGF.
To facilitate preparation of monomeric forms of KDR, a Genenase 1 protease cleavage site (18) was engineered at the junction of the last
KDR IgG domain and the CH2 domain (19). The cleaved KDR was shown to be
monomeric based on its mobility in nonreducing SDS-PAGE and gel
filtration. Both the KDR 1-7 and KDR 1-3 monomers bound all three
mAbs and equally well to VEGF (Table I). These results show the first
three IgG-like domains are sufficient for binding of VEGF whether in
monomeric or dimeric forms.
One VEGF Dimer Binds Two Molecules of the Extracellular Domain of
KDR--
To determine the stoichiometry of binding of
VEGF1-109 to the extracellular domain of KDR, we
systematically varied the ratio of VEGF to KDR and determined the
apparent size of the complexes by gel filtration. The glycosylated
monomeric KDR 1-7 migrated as a single peak by gel filtration
chromatography with an apparent molecular mass of ~250 kDa (Fig.
3A). By comparison, the
dimeric KDR 1-7-IgG migrated as a 600-kDa peak (data not shown). Upon
addition of one equivalent of VEGF (dimer) per two equivalents of KDR
(monomer), a single complex peak was formed of apparent molecular mass
~400 kDa. A minor shoulder was seen that might represent the slight
excess KDR monomer in the mixture. The fact that the 2:1 complex is
smaller by gel filtration than expected from the sum of the component
molecular masses (520 kDa) may be that VEGF aligns the receptor
subunits in a more compact fashion.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Panel A, gel filtration chromatography
of various ratios of KDR 1-7 monomer and VEGF dimer (upper six
chromatograms) or VEGF heterodimer (hV-1) containing a single
functional binding site (lower two chromatograms). The
concentration of KDR 1-7 monomer was 1 µM except at
ratios of 3:1 and 4:1, where the concentration of KDR 1-7 was 1.5 and
2 µM, respectively. The quantitation of protein was
determined by amino acids hydrolysis and absorbance at 280 nM. Panel B, gel filtration chromatography of
various ratios of KDR 1-3 monomer and VEGF dimer (six
chromatograms on the left) or hV-1 (five
chromatograms on the right). The concentration of KDR
1-3 monomer was held constant at 1 µM except at ratios
of 3:1 and 4:1, where KDR 1-3 monomer was 1.5 and 2 µM,
respectively.
|
|
Further additions of 2 and 3 equivalents of VEGF did not change the
position of the high molecular weight peak, and excess VEGF accumulated
as the free dimeric hormone (Fig. 3A). The height of the
free VEGF peak was small because VEGF contains no tryptophan residues
and therefore has a small molar absorbance at 280 nm. When the ratio of
KDR to VEGF exceeded 2:1, free KDR 1-7 accumulated as a shoulder. The
hV-1 heterodimeric variant of VEGF forms a 1:1 complex with monomeric
KDR. This complex migrated at a position that was intermediate between
the free KDR 1-7 and the 2:1 KDR·VEGF complex. When the ratio of the
hV-1 to KDR 1-7 exceeded unity, the free heterodimer accumulated in
the chromatogram.
Parallel experiments were carried out with the monomeric form of KDR
1-3 (Fig. 3B). When no VEGF was present, KDR 1-3 migrated as a single peak of apparent molecular mass of ~70 kDa. Addition of 1 equivalent of VEGF dimer to 2 equivalent of KDR 1-3 resulted in
forming a peak with apparent molecular mass of ~160 kDa. Addition of
2 and 3 equivalents of VEGF did not change the position of the complex
peak, but free VEGF accumulated. Increasing additions of KDR 1-3 in
excess of the 2:1 ratio to VEGF dimer showed increasing appearance of
free KDR 1-3. A similar set of experiments with the hV-1 showed it
maximally formed a 1:1 complex (Fig. 3B); when the ratio of
either the variant or KDR 1-3 was skewed from unity, the free excess
component accumulated. These experiments explicitly show that two
molecules of KDR bind to one VEGF dimer, and that form of KDR lacking
IgG-like domains 4-7 are capable of producing the 2:1 complex in
solution. When VEGF is engineered to have only one functional binding
site (hV-1) it cannot dimerize the receptor in vitro.
VEGF Binds Avidly to Dimeric versus Monomeric Forms of
KDR--
Given the fact that the VEGF dimer binds two molecules of
receptor we wished to determine to what extent predimerization of the
receptor influenced affinity. This can be readily seen by comparing the
binding constants for the monomeric and dimeric forms of KDR (Table I).
KDR-IgG fusions containing domains 1-7 or 1-3 bound 50-100-fold
stronger than their monomeric counterparts. The affinity of the
single-site heterodimeric VEGF for binding to the dimeric KDR IgG
fusion was about 200-fold weaker than wild-type VEGF (Fig.
4). In contrast, binding to monomeric KDR
for the heterodimer was only 2-fold weaker than native VEGF (Fig.
2B). These data, summarized in Table
II, show that binding of dimeric VEGF to
predimerized KDR is ~100-fold stronger than when either the hormone
or receptor contains a single binding site.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
VEGF containing two sites binds much stronger
to predimerized forms of KDR. Displacement of
125I-VEGF1-165 from the dimeric KDR 1-7-IgG
is shown for wild-type VEGF1-165, VEGF8-109,
the single disulfide heterodimer containing both binding sites (hV-2)
or single binding site (hV-1), and monomeric VEGF1-109,
C51R/C60R. The IC50 values from here and Fig. 2B
are summarized in Table II.
|
|
VEGF Binds Virtually the Same Way to Monomeric and Dimeric
KDR--
Given the strong avidity component to binding of VEGF to its
receptor, we wished to determine if VEGF binds the same way to monomeric and dimeric forms of KDR 1-7. We have previously reported the alanine scan of VEGF for binding to KDR-IgG (9). Here we analyze
the binding of these same alanine mutants to monomeric KDR (Table
III). The data show that the same set of
alanine mutants that are most disruptive for binding to KDR-IgG are
also strongly disruptive to binding monomeric KDR.
View this table:
[in this window]
[in a new window]
|
Table III
The comparison of the effect alanine mutants of VEGF1-109 on
binding to KDR 1-7-IgG or KDR1-7 monomer
The relative binding affinity was expressed as the fold difference of
alanine mutants with VEGF1-109 in the competitive binding
assay as described under "Experimental Procedures." Standard
deviations in these measurements averaged ±25% of the value shown.
Residues are shown in two groups (e.g. F17A or I43A') to
indicate that they are presented in the same epitope but from different
subunits. Each mutant is present twice in the dimer.
|
|
There are some subtle and systematic differences in the way the alanine
mutants bind the monomeric versus dimeric KDR. For example
M18A, I43A, I46A, E64A, and I83A were more disruptive to affinity (by
factors ranging from 2-8-fold) when tested against the monomeric
versus dimeric KDR. Only F17A was more disruptive to the
dimer than the monomer (by ~2-fold). The biased suppression of the
disruptive effects of the alanine mutations when binding to the dimer
is likely caused from avidity in binding. We conclude there are no
gross differences in the way monomeric and dimeric forms of KDR bind to
VEGF.
KDR Domains 1-3 Are Sufficient for Signaling in Cells--
NIH
3T3 fibroblast cells that contain the extracellular domain of
colony-stimulating factor receptor fused to the transmembrane and
intracellular domain of the Flt-4 receptor incorporate
[3H]thymidine and proliferate when treated with the
colony-stimulating factor (20). To produce a VEGF responsive cell line,
we made a similar chimera in which the seven IgG-like domains of KDR
were linked to the transmembrane and intracellular kinase domain of Flt-4. At low concentrations of VEGF this cell line incorporated [3H]thymidine with an EC50 of ~100
pM (Fig. 5A); high
concentrations of VEGF (>1 µM) showed inhibition. Such a
bell-shaped dose-response curve is anticipated for a two-site hormone
dimerizing two identical receptors (21). The hV-1 was inactive (Fig.
5A).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
The agonistic effects of VEGF variants on DNA
synthesis of 3T3 cells stably expressing KDR 1-7 (ECD)/Flt4 (ICD)
(Panel A), KDR 1-3 (ECD)/Flt-4 (ICD) (Panel
B), or HuVEC (Panel C). Cells were
treated for 18 h with either VEGF9-109 (open
squares) or the VEGF1-109 heterodimer with single
binding site, hV-1 (closed circles). Cells were pulsed with
[3H]thymidine for 6 h and analyzed as described
under "Experimental Procedures."
|
|
A similar construct was produced in which only domains 1-3 of KDR were
linked to Flt-4. These cells also incorporated
[3H]thymidine in response to VEGF (Fig. 5B)
but did so with a higher EC50 (~10 nM) and
lower maximal response. We did not go to high enough concentrations to
see inhibition by VEGF. The hV-1 was virtually inactive. Primary human
umbilical vein endothelial cells (HuVEC) showed a bell-shaped
dose-response curve (Fig. 5C). We resist making quantitative
comparisons between HuVEC and KDR expressed NIH 3T3 cells given the
fact that the HuVEC contain both KDR and Flt-1 receptors (22).
The difference in EC50 values and maximal response for the
KDR 1-7 and KDR 1-3 cell lines likely resulted from the fact that the
number of functional receptors on the KDR 1-3 cell line was at least
10-fold lower based upon binding of 125I-VEGF (data not
shown). To explore the effect of receptor number on signaling directly
we isolated three different clones of cells that varied over a range of
12-fold in the amount of the KDR 1-7 that specifically bound
125I-VEGF (Fig.
6A). The maximal levels of
[3H]thymidine incorporation correlated with the number of
receptors expressed on these cells and the EC50 values
correlated inversely with the number of receptors (Fig. 6B).
It is interesting that the basal levels of [3H]thymidine
incorporation correlated with the receptor number as well, suggesting
that receptors can preassociate and signal weakly in the absence of
exogenous VEGF. All of the transfectants containing the KDR 1-3
construct expressed much lower levels of receptors which may suggest
that domains 4-7 are important for high level expression and display
of the receptor.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 6.
3T3 cells stably expressing varying amounts
of KDR 1-7 (ECD)/Flt-4 (ICD) receptor respond to
VEGF9-109 with different values of EC50 and
maximal response. Panel A, three different 3T3 cell clones
expressing varying amounts of the KDR 1-7 (ECD)/Flt-4 (ICD) were
isolated and ranked according to the amount of functional binding sites
for VEGF as determined by specific binding of
125I-VEGF1-165. The same number of 3T3 cells
from transfected clones 1, 2, and 3 or HuVEC were plated on the 24-well
plate. The 125I-VEGF1-165 (0.1 nM)
was added with (open bar) or without (filled bar)
a 200-fold molar excess of cold VEGF1-165 for 2 h and
cells were washed and counted. Panel B, these same cell
lines were treated with increasing concentrations of
VEGF8-109 and [3H]thymidine incorporation
was measured. The three clones of 3T3 cells expressing KDR 1-7/Flt-4,
NEO transfected control cells, and HuVEC were fasted and treated with
serial dilutions of VEGF8-109 for 18 h. Cells were
pulsed with [3H]thymidine for 6 h before harvesting
as described under "Experimental Procedures."
|
|
Antagonism of VEGF Receptors by the Single-site Heterodimer of
VEGF--
Given the ability of hV-1 to bind but not dimerize and
activate KDR (Fig. 5), we studied its ability to antagonize signaling of KDR. Indeed the heterodimer antagonizes [3H]thymidine
incorporation in the 3T3 cells transfected with the chimeric KDR
(1-7)-Flt-4 receptor and HuVEC with an IC50 of ~300 and
~20 nM, respectively (Fig.
7). The fact that the heterodimer is less
effective on the 3T3-transfected cells versus HuVEC likely reflects the fact that the former expresses much higher levels of
receptors (Fig. 6A).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7.
The antagonistic effects of VEGF single site
heterodimer (hV-1) on DNA synthesis of 3T3 cells stably expressing KDR
1-7 (ECD)/Flt-4 (ICD) (Panel A) or HuVEC (Panel
B). Cells were incubated with either 0.1 nM
VEGF8-109 (for 3T3 cells) or 1 nM
VEGF8-109 (HuVEC) to induce 90% maximal incorporation of
[3H]thymidine together with increasing concentrations of
the hV-1.
|
|
 |
DISCUSSION |
KDR Binds Across the VEGF Dimer Interface--
The data here
combined with previous mutational analysis (9) suggest that binding
occurs across the VEGF dimer interface (Fig. 2A). It may be
a general feature of the cystine-knot hormones that the receptor binds
at the interface between hormone subunits. A structure of domain 2 of
the Flt-1 receptor bound to VEGF shows it binds across the dimer
interface (23). A heterodimer containing one molecule of VEGF linked to
its homolog, PLGF, is only 20-50-fold reduced as a mitogen on HuVEC
cells (EC50 ~ 50 nM) whereas the PLGF
homodimers are inactive (24). The fact the VEGF/PLGF heterodimer shows
any activity can be rationalized from our mutational analysis (Table
III). Some of the critical binding determinants (Phe-17, Glu-64,
Gln-79, and Ile-83) are conserved in PLGF and others are reasonably
conservative substitutions (M18Q, I43V, Y45H and I46M). These later
substitutions would likely have a much more dramatic effect when
present in both subunits, thus accounting for the absence of
significant mitogenic activity for the PLGF homodimer up to the
concentrations that were tested (~1 µM). Similar
observations have been made for homodimers and heterodimers of PDGF
(isoforms AA, AB, and BB) for binding the PDGF-
and -
receptors
(25). Mutational analysis of nerve growth factor, another member of the
cystine-knot family of dimeric hormones, shows a broad patch of
residues involved in binding receptor that spans the interface between
subunits (26).
Requirements and Consequences for Receptor
Dimerization--
Hormone-induced receptor dimerization is a general
mechanism for activation of tyrosine kinase receptors (27). All
receptors that bind cystine-knot hormone dimers are presumed to be
activated by receptor dimerization (6). Here, gel filtration
experiments provide in vitro evidence that VEGF binds two
molecules of the extracellular domain of KDR. The dimerized complex
appears to be very stable since excess VEGF is unable to dissociate the
complex to 1:1 complex. This dimerization reaction is critical for
signaling because the VEGF heterodimer, hV-1, with only one functional
site is inactive in cell-based assays and antagonizes the action of wild-type VEGF. Receptor dimerization is also supported by the observation that cell-based assays show a bell-shaped dose-response curve with respect to VEGF. PDGF isoforms have been shown to induce dimerization of the extracellular domains of the PDGF-
and -
receptors in vitro (28). Binding of the dimeric hormone,
SCF, to the extracellular domain of the Kit receptor, a tyrosine kinase receptor of the IgG class, causes dimerization in vitro
(29-31), and induces a bell-shaped dose-response curve in
vivo (30).
Predimerized forms of KDR bind VEGF 100-fold more tightly than
monomeric forms of KDR showing a strong avidity component in binding.
Dimeric receptor fusion proteins, such as IgG fusions, or receptors
bound to monoclonal antibodies are often used as convenient assay
reagents for hormones and their variants. The data presented here show
that there is a significant avidity component to binding in these
fusions that affects the affinity constants. The avidity effect
observed here is not the result of an alternate way that VEGF binds the
dimeric KDR because alanine mutations in VEGF that are most disruptive
to binding monomeric KDR are also the ones that most affect binding to
dimeric KDR (Table III).
We observed that wild-type VEGF1-109 binds about 100-fold
more tightly than the single-site heterodimer to cells expressing KDR
1-7 (not shown). This suggests that receptors on cells may be loosely
associated. Moreover, NIH 3T3 cells expressing larger numbers of VEGF
receptors showed a higher basal level of [3H]thymidine
incorporation in the absence of VEGF (Fig. 6B), suggesting that receptors on cells have an intrinsic ability to dimerize in the
absence of ligand. Similar observations have been made for cells
overexpressing various tyrosine kinase receptors, such as variants of
the EGF receptor (27). Overexpression of the PDGF receptors can induce
receptor autophosphorylation in the absence of ligand, and it is even
possible to cross-link small amounts of the extracellular domains in
the absence of PDGF (28). The fact we did not see evidence for
dimerization by gel filtration of the ecodomains of KDR 1-7 or 1-3 in
the absence of VEGF may only reflect the sensitivity of the method and
that the receptors have a much higher effective concentration on
cells than in our solution experiments (~1 µM).
Deletion experiments showed that domains 2-3 of KDR are sufficient and
necessary for high affinity binding of VEGF (Table I). Cells can signal
when transfected with KDR domain 1-3 linked to Flt-4, even with low
receptor expression, suggesting that domains 4-7 are not essential for
signaling. We cannot rule out other roles for these domains; they may
stabilize the signal transduction complex and/or provide for better
display and expression of the receptor. Systematic deletion experiments
have been conducted on at least four other tyrosine kinase receptors of
the IgG class, and generally show that binding is dominated by IgG-like
domains 2-3. Deletion experiments showed the first three of the five
IgG-like domains in the Kit receptor are required for binding of SCF,
but there is uncertainty regarding the role of domain 4 in signaling (30, 31). An antibody directed toward domain 4 blocked signaling in
cells transfected with Kit, and deletion of domain 4 blocked signaling
but not stem cell factor binding (30). In contrast, biophysical
experiments (31) showed that Kit 1-3 can dimerize in solution with
stem cell factor and both the enthalpy and free energy of binding were
indistinguishable from Kit 1-5. In either case, both groups agree that
the ligand-binding site for stem cell factor is contained in the first
three IgG-like domains.
Deletion analysis of PDGF-
receptor, which contains five IgG-like
domains, has shown that domains 2-3 are sufficient for binding PDGF
isoforms although the presence of domain 1 has a small differential
effect on binding PDGF-AA versus PDGF-BB (32). Deletion
analysis of the fibroblast growth factor receptor, which contains three
IgG-like domains, showed that domains 2-3 are sufficient for high
affinity binding of fibroblast growth factor (33). Deletion experiments
in Flt-1, which like KDR contains seven IgG-like domains, have shown
that the VEGF-binding site is located among the first three IgG-like
domains (34-36) and domain 2 of Flt-1 alone can bind VEGF tightly
(23). Thus, domain 2 may plays a dominant role in all five of these
tyrosine kinase receptors that have IgG-like domains and may be general
to the other members of this class.
Mechanism-based Antagonsits of VEGF--
Antagonists to VEGF may
be very useful in preventing tumor angiogenesis and retinopathy
diseases. Here, we have elucidated the functional requirements for
receptor binding and activation and designed an antagonist, hV-1, for
the proliferation of HuVEC cells based on this knowledge. The fact the
IC50 of hV-1 for inhibiting VEGF in HuVEC (~20
nM) is ~100-fold higher than the EC50 of VEGF stimulating growth (~0.2 nM) likely reflects the avidity
effect described above. We believe the hV-1 antagonize VEGF stimulation of HuVEC by blocking the dimerization of KDR since KDR is more important for signaling mitogensis. However, the hV-1 does bind Flt-1
with near wild-type affinity and we are currently looking at its
ability to activate Flt-1. Alanine scanning of both receptor-binding sites on VEGF suggests that the binding sites for KDR and Flt-1 overlap
and are not identical (9, 23, 37). Based on these results it should
also be possible to design receptor specific antagonists and further
elucidate the functions of the two receptors. Overall, these studies
provide a basis from which we can produce new analogs of VEGF to both
probe its biology and generate new and potent therapeutics.
We thank Toni Klassen and Jin Kim for mAbs to
KDR, Hans Christinger for providing purified VEGF, Napoleone Ferrara
for providing the plasmid pHEBO23-KDR-IgG and pHEBO23-Flt-1-IgG,
Jennifer Singh, Richard DeMarco, Michael Clasen for technical support,
the DNA Synthesis Group at Genentech for oligonucleotides, and David
Wood for the graphics.