From the Department of Cardiovascular Research, Procter & Gamble Pharmaceuticals, Health Care Research Center, Mason, Ohio 45040
Received for publication, March 14, 2001, and in revised form, April 26, 2001
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ABSTRACT |
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The two most abundant secreted isoforms of
vascular endothelial growth factor A (VEGF165 and
VEGF121) are formed as a result of differential splicing of
the VEGF-A gene. VEGF165 and VEGF121 share
similar affinities at the isolated VEGF receptor (VEGFR)-2 but have
been previously demonstrated to have differential ability to activate
VEGFR-2-mediated effects on endothelial cells. Herein we investigate
whether the recently described VEGF165 isoform-specific receptor neuropilin-1 (Npn-1) is responsible for the difference in
potency observed for these ligands. We demonstrate that although VEGFR-2 and Npn-1 form a complex, this complex does not result in an
increase in VEGF165 binding affinity. Therefore, the
differential activity of VEGF165 and VEGF121
cannot be explained by a differential binding affinity for the complex.
Using an antagonist that competes for VEGF165 binding at
the VEGFR-2·Npn-1 complex, we observe specific antagonism of
VEGF165-meditated phosphorylation of VEGFR-2 without affecting the VEGF121 response. These data indicate that
the formation of the complex is responsible for the increased potency
of VEGF165 versus VEGF121. Taken
together, these data suggest a receptor-clustering role for Npn-1, as
opposed to Npn-1 behaving as an affinity-converting subunit.
The product of the
VEGF-A1 gene is required for
formation of the embryonic vasculature, because haploinsufficiency
leads to embryonic lethality due to a failure in both angiogenesis and blood island formation (1). The effects of VEGF-A on development of the
embryonic vasculature are mediated by an interaction with the VEGF
receptor (VEGFR)-1 and -2 receptor tyrosine kinases (2-4), whereas
effects on angiogenesis that occur in the adult animal appear to be
mediated largely through VEGFR-2 (5-8), with VEGFR-1 playing a
modulatory role (9-12).
VEGF-A exists in multiple protein isoforms with different heparin
proteoglycan and extracellular matrix binding properties. These
isoforms (VEGF165, VEGF121,
VEGF145, VEGF183, VEGF189, and VEGF206) arise from alternate splicing of the VEGF-A gene.
VEGF165 is the predominant isoform and has heparin binding
activity, whereas the VEGF121 isoform is freely soluble and
is devoid of heparin binding activity (13). Similarly, another VEGF
superfamily member, placenta growth factor (PlGF), exists in three
different isoforms that also exhibit differential heparin binding
ability (14). Although VEGF165 and VEGF121 bind
to VEGFR-2 with equal affinity, their activity in biochemical assays
that rely on activation of VEGFR-2 is not equivalent (15-17), implying
that their ability to activate VEGFR-2 is not solely dependent on
VEGFR-2 binding affinity.
Recently, neuropilin (Npn)-1 was identified as a receptor that binds
VEGF165 (18, 19), VEGF-E (20), PlGF152 (21), and VEGF-B (22), whereas Npn-2 has been identified as a receptor that
binds PlGF152, VEGF165, and VEGF145
(23). Neither Npn binds VEGF121, and Npn-1 will not bind
the non-heparin-binding form of PlGF, PlGF139 (21, 23).
Npn-1 and Npn-2 also bind various semaphorin ligands to mediate
repulsive guidance activity in certain neuronal populations (24). The
cytoplasmic domains of Npn-1 and Npn-2 are not required for semaphorin
signaling (25), and they do not contain sequences predictive of
enzymatic activity nor sequences predicted to be involved in coupling
to intracellular signaling molecules. Nevertheless, the Npn-1 and Npn-2
proteins are obligate binding subunits of a semaphorin signaling
receptor complex. Members of the plexin family appear to serve as the
signaling subunits of the semaphorin receptor complex (26, 27).
Potential signals that lie downstream of the neuropilin-plexin receptor complex include activation of a pertussis toxin-sensitive G-protein (28) and activation of Rac1, a small molecular mass G-protein involved in cytoskeletal rearrangement (26, 29). The carboxyl terminus
of Npn-1 has been shown to bind to a PSD-95/Dlg/ZO domain containing a
regulator of G-protein signaling protein (30, 31), although the role of
this protein in semaphorin signaling has not been established.
Although the role of Npn-1 in mediating the neuronal effects of the
semaphorins is being elucidated, much less is known about the role of
Npn-1 in VEGF165-mediated angiogenesis. Targeted disruption of the Npn-1 gene in mice results in embryonic lethality, with the
embryos exhibiting defects in the formation of brachial arch-derived vessels, impaired neural vascularization, and defects in vascular development in the yolk sac that occur in addition to expected defects
in neuronal patterning (32, 33). Transgenic overexpression of Npn-1
results in embryonic lethality associated with excessive vascular
formation, dilation, and hemorrhaging, as well as defects in skeletal
morphogenesis and neuronal guidance (34, 35). Because the semaphorin
III knockout mouse also exhibits cardiovascular abnormalities (36), it
is not currently known whether the vascular effects attributed to the
absence or the overexpression of Npn-1 are due to signaling alterations
in the semaphorin or the VEGF systems. Because VEGF121 does
not bind to Npn-1 (18, 23) and because mice engineered to express only
the VEGF121 isoform do not exhibit deficiencies in the
development of the embryonic vasculature (37), it is possible that
Npn-1 might not be required for the development of the vasculature in
response to VEGF-A. However, these same mice exhibit defects in cardiac
vascularization and suffer from ischemic cardiomyopathy, implying that
VEGF121 cannot substitute for VEGF165 in
development of the cardiac vasculature during the period of postnatal
cardiac development (37). These data suggest that the inability of
VEGF121 to interact with Npn-1 may result in insufficient
signaling to support the maturation of the cardiac vasculature.
The ability of VEGF165 to bind to VEGFR-2 and Npn-1 through
distinct regions of the VEGF165 molecule led to the
assertion that VEGFR-2 and Npn-1 may signal as a co-receptor complex
(18, 35). We demonstrate here that VEGFR-2 does indeed form a complex with Npn-1 in both heterologous systems as well as in cultured endothelial cells. We further demonstrate that, although the complex does not bind VEGF165 with an affinity that is greater than
that exhibited at either VEGFR-2 or Npn-1 alone, formation of this complex can explain the differential potencies that are observed in
cultured endothelial cells for VEGF165- and
VEGF121-mediated stimulation of VEGFR-2 activation.
Materials--
Carrier-free recombinant VEGF165 was
purchased from R & D Systems Inc. (Minneapolis, MN).
Na125I was purchased from Amersham Pharmacia Biotech.
Heparin sulfate (catalog number 9399) and bovine serum albumin (BSA)
(catalog number A3294) were purchased from Sigma. PD-10 columns and
protein G-Sepharose beads were purchased from Amersham Pharmacia
Biotech. Donor calf serum, LipofectAMINE-2000, and Optimem I were
purchased from Life Technologies, Inc. Fetal bovine serum was purchased from Hyclone Laboratories (Logan, UT). pJFE14 vector and
hVEGFR-2/pJFE14 construct were obtained from Regeneron Pharmaceuticals
(Tarrytown, NY). pLTRMCSIRES-GFP and pLTR2hFLK1(full)IRES-GFP
retroviral constructs were obtained from Regeneron Pharmaceuticals. The
pBMN-Z-I-Neo vector used to construct hNpn-1 retroviral
construct was obtained from Dr. Gary P. Nolan (Stanford
University). The PlGF152 Exon 6 peptide (amino acids
142-157) was synthesized by SynPep (Dublin, CA). All of the other
reagents were obtained from commercial sources.
Cell Culture--
Human umbilical vein endothelial cells (HUVEC)
obtained from Clonetics (Walkersville, MD) were cultured in endothelial
cell growth medium (Clonetics) and were used up to passage 3. Balb/c 3T3 A31 cells obtained from the American Type Culture Collection (Manassas, VA) were cultured in Balb/c growth medium (Dulbecco's modified Eagle's medium, 10% donor calf serum, 1%
L-glutamine, 1% antimycotics, 1% nonessential amino
acids). COS-1 cells obtained from American Type Culture Collection were
cultured in COS-1 cell growth medium (Dulbecco's modified Eagle's
medium supplemented with 1% L-glutamine, 1% nonessential
amino acids, 1% antimycotics, and 10% fetal bovine serum). QMXE
packaging cells were cultured in COS-1 cell growth medium. All of the
cells were grown at 37 °C in 5% CO2.
Plasmid Construction--
For transient transfection in COS-1
cells, the hVEGFR-2 and hNpn-1 cDNAs were cloned into the mammalian
expression plasmid pJFE14 containing the SR Transient Expression of Receptors--
COS-1 cells were
transiently transfected with pJFE14 (7.0 µg), hVEGFR-2 (5.0 µg of
hVEGFR-2 + 2.0 µg of pJFE14), hVEGFR-2 (5.0 µg) + hNpn-1(2.0 µg),
or hNpn-1 (2.0 µg of hNpn-1 + 5.0 µg of pJFE14) constructs using
LipofectAMINE-2000 in Optimem I according to the manufacturer's
instructions. The cells were plated 20 h after transfection.
Creation of Stable Cells--
Retrovirus was generated by
transfecting QMXE packaging cells with the appropriate retroviral
construct (VEGFR-2, Npn-1, or Mock) using the same medium and
methodology established for the COS-1 cells. Virus was generated
overnight (33° C, 5% CO2). Balb/c 3T3 A31 cells
(~1 × 106 cells/100-mm dish) were infected with
viral supernatants containing 6 µg/ml of polybrene. Pools of infected
Balb/c 3T3 A31 cells expressing hVEGFR-2 or GFP alone (Mock) were
selected twice by fluorescence-activated cell sorting using the
hVEGFR-2 specific monoclonal antibody J5F4A2 for the
hVEGFR-2-containing cells or by GFP emission for the Mock cells. The
hVEGFR-2 clone (D7R2) was generated by subjecting the hVEGFR-2 cell
pool to limiting dilution and identified via Western analysis. The Mock cells (PGBMGH) were used as a pool. The
hNeuropilin-1 clonal cell line (Npn-1(2)) was generated by infecting
Balb/c 3T3 A31 cells with hNpn-1 virus and selecting in the presence of
1.5-2.0 mg/ml Geneticin. hVEGFR-2/hNpn-1 stable cells
(D7R2·Npn-1(4)) were created by infecting the stable VEGFR-2 clonal
cell line (D7R2) with hNpn-1 virus and selected in the presence of
1.5-2.0 mg/ml Geneticin.
Polyclonal Antibody Production--
The Npn-1 rabbit polyclonal
antibody (NP1ECD4) was raised against the purified peptide sequence
(Ac-DLDKKNPEIKIDETGSTC-amide (amino acids 814-830)) and affinity
purified by QCB, a division of BIOSOURCE
International (Hopkinton, MA). The sequence used for generation of the
NP1ECD4 antibody is from the extracellular juxtamembrane region of
Npn-1, and this antibody is therefore not predicted to recognize
soluble Npn-1 (39), which has been demonstrated to bind
VEGF165 alone (40) or in Npn-1·VEGFR complexes (41). The
VEGFR-2 rabbit polyclonal antibody (R2.2) was raised against the
purified peptide sequence Ac-SKRKSRPVSVKTFEDIPLEEPC-amide (amino acids
1225-1246), unique to the carboxyl-terminal domain of VEGFR-2, and
affinity purified by QCB. The Npn-1 number 30 polyclonal antibody was
the kind gift of Dr. David Ginty (Johns Hopkins University). This
antibody was generated against amino acids 583-856 of rat neuropilin-1
(42). These residues correspond to the MAM domain and the
extracellular juxtamembrane region and thus recognize only full-length
Npn-1 protein. This antibody was used exclusively for Western blotting.
The C-19 antibody (catalog number SC-7239) for neuropilin-1 was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and will
only recognize the full-length Npn-1 protein. This antibody was
sometimes substituted for the NP1ECD4 antibody for immunoprecipitations
(as indicated in the figure legends).
Monoclonal Antibody Production--
For generation of the
immunogen for the J5F4A2 monoclonal antibody, the entire coding region
of the VEGFR-2 extracellular domain (amino acids 1-760) was expressed
and purified from Sf9 cells as an Fc fusion protein (Regeneron
Pharmaceuticals). The amino-terminal sequence of the purified protein
was determined to be a single sequence (ASVGL). This protein was
designated FLK-1(Ig 1-7):Fc. Unique clones were tested by Western blot
using hybridoma tissue culture supernatants and/or purified antibody.
The VEGFR-2 monoclonal antibody (J5F4A2) showed the optimal reactivity
to FLK-1(Ig 1-7):Fc and does not cross-react with VEGFR-1 (data not shown). Antibodies were purified from tissue culture supernatant by
protein G column.
Iodination of VEGF165--
Iodination was performed
using a modification of methods described previously (15, 43). Briefly,
5 µg of carrier-free human recombinant VEGF165 was
suspended in 90 µl of Dulbecco's phosphate-buffered saline. To the
reaction tube, 1 mCi of Na125I was added, followed by 40 µl of chloromine T (1 µg/µl in 0.5 M sodium phosphate
buffer, pH 7.5), and incubated for 1 min. 50 µl of sodium
metabisulfite (2 µg/µl in sodium phosphate buffer, pH 7.5) was
added to stop the reaction. 500 µl of column elution buffer (0.5%
BSA, 0.01% Tween 20 in Dulbecco's phosphate-buffered saline) was
added to the reaction and transferred to pre-equilibrated PD-10 column
for separation from unreacted iodine. The specific activity was
corrected for column recovery and varied from 4,000 to 15,000 Ci/mmol.
Radioimmunoprecipitation--
3 × 106
cells/100-mm dish were plated 24 h prior to the experiment. The
cells were incubated with 300-700 pM
[125I]VEGF165 for 4 h in 4 °C binding
buffer (minimum essential medium, 25 mM HEPES, 0.2%
BSA) containing 1 µg/ml heparin sulfate and protease inhibitors (10 µg/ml leupeptin, 10 µg/ml antipain, 50 µg/ml benzamidine, 100 µg/ml soy bean trypsin inhibitor, 10 µg/ml bestatin, 10 µg/ml pepstatin, 0.3 mM phenylmethylsufonyl fluoride). Unbound ligand was removed by washing with ice-cold BSA-free binding buffer (minimum essential medium, 25 mM HEPES). Affinity
labeling of the receptors was achieved by cross-linking with 15 mM disuccinimidyl glutarate in BSA-free binding buffer (15 min, 4 °C). Excess disuccinimidyl glutarate was removed by washing
with ice-cold Dulbecco's phosphate-buffered saline. The cells were
lysed with RIP buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM
NaI, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 1% BSA). Lysate
was immunoprecipitated overnight at 4 °C with 10 µg of antibody
(R2.2, NP1ECD4, or C-19), 0.1% SDS, and protein G-Sepharose
beads. The samples were separated on 6% Novex SDS-polyacrylimide gel
electrophoresis (PAGE) gels, and the images were analyzed on the Storm
system (Molecular Dynamics, Sunnyvale, CA).
Competition Binding Assay--
2 × 105
cells/well in a 12-well plate were plated 24 h prior to the
experiment. Depending upon the equilibrium dissociation constant
(Kd) of the receptor being studied, 100-400 pM [125I]VEGF165 was incubated (4 h, 4 °C) in the presence of increasing concentrations of unlabeled
ligands in binding buffer containing 1 µg/ml heparin sulfate and
protease inhibitors. Unbound ligand was removed by washing with
ice-cold BSA-free binding buffer. The cells were lysed with 250 µl of
RIP buffer containing 0.1% SDS and counted using a Saturation Analysis and Nonlinear Curve Fitting--
For a
direct measurement of receptor number and affinity, the saturation
analysis was performed as described above except that increasing
concentrations of 0.5-3000 pM
[125I]VEGF165 were added in the presence or
the absence of 30 nM unlabeled VEGF165 to
estimate nonspecific binding. This concentration range had previously
been determined to discriminate a heterogeneous population of
[125I]VEGF165 binding sites in some
immortalized endothelial cell lines (data not shown). The maximum
number of binding sites (Bmax) and the
Kd values were obtained using the Prism software package, which performs a statistical assessment of goodness-of-fit to
a one-binding site versus a two-binding site model. This
methodology is preferred over the Scatchard analysis because it does
not require a transformation and linearization of the data, which is
known to distort the experimental errors associated with radioligand binding data (44). In all cases described herein, the optimal fit of
the data was to a one-binding site model (Table
I and data not shown). The data points
in all figures represent the averages of triplicate determinations.
Western Blot Analysis--
Cells were solubilized with ice-cold
RIPA buffer (20 mM Tris-HCl, pH 7.6, 150 mM
NaCl, 50 mM NaF, 1 mM sodium orthovanadate, 5 mM benzamidine, 0.5% IGEPAL CA-630, and protease
inhibitors). The protein was separated by SDS-PAGE and transferred onto
polyvinylidene difluoride membranes. The membranes were probed for
VEGFR-2 (blocked with 5% BSA in Tris-buffered saline, 0.1% Tween 20 (0.1% TBST)) or Npn-1 (blocked in 0.5% Blotto in 0.1% TBST) using
rabbit R2.2 (0.55 µg/ml) or rabbit anti-Npn-1 number 30 (1:5,000) and
detected by ECL.
Anti-phosphotyrosine Assay--
HUVEC cells were plated (1 × 106 cells/flask) in T-75 flasks 1 week prior to
stimulation. The cells were rinsed with Dulbecco's modified Eagle's
medium, serum-starved (1-2 h, 37 °C), and then stimulated by
incubation with increasing concentrations of VEGF in binding buffer
containing 1 µg/ml heparin sulfate for 5 min at 37 °C. Cells were
lysed with RIPA buffer containing protease inhibitors and passed
through a 23-gauge needle. The samples were immunoprecipitated with
R2.2 antibody (10 µg) overnight at 4 °C, washed with RIPA,
separated by SDS-PAGE, and transferred onto polyvinylidene difluoride
membranes. The membranes were probed with 0.5 µg/ml 4G10
anti-phosphotyrosine antibody (Upstate Biotechnology Inc., Lake Placid,
NY; catalog number 05-321) and detected by ECL. Membranes were stripped
(200 mM glycine, pH 2.8) and processed for VEGFR-2 receptor
normalization using R2.2 (0.55 µg/ml). The bands were quantitated
using ImageQuant (Molecular Dynamics), and the EC50 values
were determined from analysis of the dose-response curves generated
with GraphPad Prism.
VEGF165 and VEGF121 Exhibit Differential
Potency in Stimulating VEGFR-2 Phosphorylation Despite Similar Affinity
at VEGFR-2--
Although VEGF165 and VEGF121
have the same affinity for the isolated purified VEGFR-2 receptor (15),
these two ligands exhibit a differential ability to activate the
VEGFR-2 receptor in vitro (15-17). In native HUVEC, this
differential activity manifests as an increased potency of
VEGF165 relative to VEGF121. This difference in
potency is characterized by an EC50 (concentration of
agonist that provokes a response halfway between the base-line and
maximal responses) for VEGF165 of 7.76 × 10 VEGFR-2 and Npn-1 Form a Co-receptor Complex--
To demonstrate
the potential for the VEGFR-2·Npn-1 complex to form, we utilized an
affinity labeling reciprocal immunoprecipitation experimental design.
In this design, cells expressing each of the receptors in isolation or
co-expressing both receptors are affinity-labeled with a high
concentration of [125I]VEGF165, chemically
cross-linked, and immunoprecipitated with antibodies specific for one
member of the suspected receptor complex. If the complex forms, the
receptor-specific antibody should more effectively precipitate the
receptor against which it was generated, and additionally precipitate a
band of the appropriate size corresponding to the alternate receptor in
the complex. Fig. 3A
demonstrates that the complex can indeed form in the presence of ligand
in the COS-1 overexpression system. In cells expressing only VEGFR-2 (lane 1) a single band of ~240 kDa is observed. This band
represents [125I]VEGF165 cross-linked to
VEGFR-2. Using our VEGFR-2 specific antibody, in COS-1 cells
co-expressing VEGFR-2 and Npn-1 we are able to immunoprecipitate not
only affinity-labeled VEGFR-2, but an additional doublet of bands that
runs at the predicted size (~140 kDa) for Npn-1 cross-linked to
[125I]VEGF165 (Fig. 3A, left
panel, lane 2). In cells overexpressing only Npn-1
(lane 3) the VEGFR-2 receptor is not present for the complex
to form, and therefore we are unable to substantially detect the Npn-1
doublet. The data in lane 3 also indicate that the VEGFR-2
antibody does not cross-react with Npn-1, and the weak doublet detected
in lane 3 may indicate formation of a complex between
endogenous VEGFR-2 and the exogenous Npn-1. Interestingly, a weak
doublet that corresponds to the size of the Npn-1 bands is also
detected with the VEGFR-2 receptor-specific antibody in cells
overexpressing VEGFR-2 (Fig. 3A, lane 1), which
may indicate formation of a complex between endogenous Npn-1 (see Npn-1
Western data; Fig. 3B, right panel) in the
presence of the exogenous VEGFR-2. The inability to detect these
affinity-labeled bands in the COS-1 cells expressing empty vector (Fig.
3A, Mock, lane 4) provides a further
indication that these bands are not nonspecific. The identity of the
lower doublet is confirmed as being Npn-1 by the immunoprecipitation
with the Npn-1-specific antibody (Fig. 3A, lane
7). The inability to detect bands in VEGFR-2 cells alone (lane 5) demonstrates that when Npn-1 is not overexpressed,
the complex with endogenous Npn-1 and overexpressed VEGFR-2 is not detected because no affinity-labeled VEGFR-2 receptor is detected in
cells immunoprecipitated by the Npn-1 specific receptor antibody. Lane 5 also demonstrates that the Npn-1 receptor-specific
antibody does not cross-react with the VEGFR-2 receptor. When both
receptors are co-expressed (Fig. 3A, lane 6) and
immunoprecipitated using the Npn-1 receptor-specific antibody, a band
corresponding to the size of affinity-labeled VEGFR-2 is apparent, in
addition to the affinity-labeled Npn-1 doublet. COS-1 cells expressing Npn-1 alone immunoprecipitated with the Npn-1 antibody confirm the
identity of the doublet as being due to
[125I]VEGF165 binding to Npn-1 (lane
7). The inability to detect the doublet in COS-1 cells expressing
empty vector (Fig. 3A, lane 8) again demonstrates
the specificity of the immunoprecipitated bands. Finally, we observe
high molecular mass bands either in cells expressing VEGFR-2 alone
(Fig. 3A, lane 1), in cells expressing Npn-1
alone (lane 7), or in cells co-expressing these receptors (lanes 2 and 6). Because it is difficult to
estimate the molecular masses of the bands in this region of the gel,
and the individual receptor homodimers are of comparable molecular
masses, we cannot identify the precise nature of these bands in cells
co-expressing both VEGFR-2 and Npn-1.
It is noteworthy that more intense labeling of VEGFR-2 is observed upon
co-expression with Npn-1 (Fig. 3A, lane 2 versus
lane 1) and that more intense labeling of Npn-1 is observed
upon co-expression with VEGFR-2 (Fig. 3A, lane 6 versus lane 7). This is because co-expression results
in increased expression of both VEGFR-2 and Npn-1 protein
versus that obtained when either receptor is expressed alone
(Fig. 3B) and is therefore not an indication of increased
binding affinity at the receptor complex (see also Table I).
Fig. 4 demonstrates the ability of the
VEGFR-2·Npn-1 complex to form in endogenous cells, as similar results
are found when the reciprocal immunoprecipitations, described above,
are performed in HUVEC cells. The VEGFR-2-specific antibody
immunoprecipitates affinity-labeled VEGFR-2 along with a
triplet of bands (~120-140 kDa), of which two bands
correspond in size to the doublet that is immunoprecipitated by
the Npn-1 antibody in affinity-labeled COS-1 cells (Fig. 3A,
lanes 6 and 7). The third band (Fig. 4, left
panel, asterisk, and Fig. 7A, lane
1, asterisk) of the triplet observed in the HUVEC cells
might represent an endogenous soluble form of Npn-1 because soluble
Npn-1 is competent to bind [125I]VEGF165 (40)
and may also form a complex with VEGFR-2 (41). This band is not
observed in the COS-1 system (Fig. 3) because the Npn-1 cDNA used
for these experiments would only produce the full-length receptor. In
the anti-Npn-1 immunoprecipitates from HUVEC cells (Fig. 4, right
panel, and Fig. 7A, lane 4), a doublet of
the size seen in the COS-1 cells (Fig. 3A) is once again
observed. The triplet seen in the anti-VEGFR-2 immunoprecipitates is
not observed here because the Npn-1 antibody used for these experiments would not be predicted to immunoprecipitate the soluble form of Npn-1
(see "Experimental Procedures"). Similar to Fig. 3, high molecular
mass bands are also observed with both antibodies, which might suggest
the formation of a heteromeric receptor complex.
To confirm the identity of the bands in the affinity labeling
experiments and to examine the possibility of ligand-independent complex formation, we utilized a reciprocal immunoprecipitation Western
blot analysis experimental design, in either the presence or the
absence of VEGF165, in both the HUVEC cells and COS-1
overexpression systems. As observed in the affinity labeling
experiments (Fig. 3A), Fig.
5A demonstrates that we are
able to immunoprecipitate Npn-1 with our VEGFR-2-specific antibody only
in COS-1 cells that co-express VEGFR-2 in concert with Npn-1 (Fig.
5A, left panels). Conversely, we are also able to
immunoprecipitate VEGFR-2 with the Npn-1-specific antibody only in
COS-1 cells co-expressing VEGFR-2 with Npn-1 (Fig. 5A,
right panels). The ability to detect the co-receptor complex
in COS-1 cells appears to be independent of the presence of
VEGF165 ligand.
Western blot analysis of COS-1 and HUVEC cells demonstrates that the
expression levels of VEGFR-2 and Npn-1 are substantially lower in the
HUVEC cells relative to the COS-1 cells (Fig. 5B). The lower
expression levels of the receptors, coupled with the limitation imposed
by the efficiency of the receptor-specific antibodies, precludes
detection of the receptor complex using the reciprocal
immunoprecipitation Western technique in HUVEC cells. Detection of the
co-receptor complex in the endogenous HUVEC system is only possible in
the presence of ligand because of the sensitivity afforded by the use
of radioiodinated ligand (compare Fig. 5A with Fig. 4). For
this reason, we are unable to determine whether a ligand-independent
complex exists in HUVEC cells. Although the COS-1 system indicates a
potential for a ligand-independent complex to form in endogenous
systems, we cannot exclude the possibility that the ligand-independent
complex observed in the COS-1 cells is driven by the higher receptor
expression levels obtained in the COS-1 cell system.
Formation of the VEGFR-2·Npn-1 Complex Does Not Result in an
Increase in Ligand Binding Affinity--
With the formation of the
co-receptor complex demonstrated, we investigated the mechanism by
which the complex increased the signaling potency of
VEGF165. The simplest explanation would be that Npn-1 acts
as an affinity converter and that the affinity of VEGF165
for VEGFR-2 is increased in the presence of Npn-1. Although the COS-1
system is an effective tool to determine whether the complex can form,
a transient expression system does not ensure that co-expression of
both receptors occurs in every cell. To avoid this caveat, we produced
stable cell lines overexpressing VEGFR-2 on an Npn-1 background. We
chose the Balb/c 3T3 A31 cell line in which to produce our stable cell
lines because these cells are reported to have endothelial cell
characteristics (45). Fig. 6A
illustrates that the endogenous Balb/c 3T3 A31 cells contain detectable
amounts of VEGF binding sites (vector control, PGBMGH) that can be
explained by the expression of detectable amounts of Npn-1 (Fig.
6B). Not surprisingly, overexpression of human Npn-1 on this
background increases the number of binding sites relative to vector
control (p = 0.050, Bmax = 143,306 ± 37,419, n = 5, in Npn-1(2)
versus 48,825 ± 16,118, n = 4 in
PGBMGH) without a change in binding affinity (Table I). When VEGFR-2 is
overexpressed on the Npn-1 background, there is a small but
statistically insignificant increase in binding site number with no
change in binding affinity (Fig. 6A and Table I, D7R2). The
lack of a statistically significant increase in
Bmax when VEGFR-2 is overexpressed on the Npn-1
background (Table I and Fig. 6A) can be attributed to the
formation of the VEGFR-2·Npn-1 complex, which the VEGF165
ligand perceives as indistinguishable from free VEGFR-2 or Npn-1. We
attempted to further increase Npn-1 expression in the D7R2 cells but
achieved only marginal success, as evidenced by the Western analysis in
Fig. 6B and a lack of statistically significant increase in
Bmax versus that in the parent D7R2
cells (Table I). Nevertheless, these data clearly indicate that binding
affinity to Npn-1 is not increased in the presence of VEGFR-2, because
we could not discern a subpopulation of higher affinity binding sites
when the data were fit to a one-site versus two-site binding
model (data not shown). Furthermore, the inability to detect a high
affinity subpopulation is independent of the ratio of VEGFR-2 to Npn-1
because these ratios are quite different in the COS-1 and Balb/c cells
(compare Western analyses in Figs. 3B, 5B, and
6B).
The limitation to this analysis is that the Balb/c 3T3 A31 cells do not
allow us to compare the binding affinity at VEGFR-2 to that of Npn-1
when each are expressed alone. However, the binding to VEGFR-2
expressed alone in the COS-1 cells (3.39 × 10 Access to the VEGFR-2·Npn-1 Complex Can Explain the Increased
Potency of VEGF165 versus VEGF121 for
Activation of VEGFR-2--
To demonstrate that the enhanced potency of
VEGF165 could be explained by enhanced signaling through
the VEGFR-2·Npn-1 complex versus VEGFR-2 alone, we chose
to specifically antagonize the binding of VEGF165 to Npn-1
to see whether the potency of VEGF165 in the
anti-phosphotyrosine assay would be reduced to match that of
VEGF121. A potential Npn-1 antagonist has been identified
by Migdal et al. (21), who examined the effects of peptides
generated from the Exon 6 and Exon 7 portions of PlGF152 on
the binding of [125I]VEGF165 in HUVEC cells.
These authors concluded that a peptide consisting of the first 16 amino
acid residues from Exon 6 of PlGF152 was sufficient to
block the binding of [125I]VEGF165 to a
120-kDa band whose characteristics were consistent with that of Npn-1.
Fig. 7A demonstrates the
ability of this peptide to compete specifically for
[125I]VEGF165 binding in HUVEC cells. With no
competitor (lane 1) [125I]VEGF165
binding to the VEGFR-2 band and the Npn-1 triplet in HUVEC cells is
detectable by immunoprecipitation with a VEGFR-2-specific antibody. An
excess of cold VEGF165 (30 nM) completely
abrogates the labeling of both the VEGFR-2 band and the Npn-1 triplet
(lane 2), and the PlGF152 Exon 6 peptide
competes for binding to the Npn-1 triplet with a proportional effect on
the labeling of the VEGFR-2 band (lane 3). Furthermore, when
HUVEC cells are affinity-labeled with
[125I]VEGF165 and immunoprecipitated with an
Npn-1-specific antibody, the PlGF152 Exon 6 peptide
completely abrogates the labeling of the Npn-1 triplet (lane
6). These data are consistent with the PlGF152 Exon 6 peptide being able to compete for binding of VEGF165 to
Npn-1 and to the VEGFR-2·Npn-1 complex in the native HUVEC cells. The
ability of the PlGF152 Exon 6 peptide to bind
preferentially to Npn-1 is demonstrated in Fig. 7B where the
peptide is able to completely compete for
[125I]VEGF165 binding in COS-1 cells
expressing Npn-1 but does not compete effectively for binding to
VEGFR-2 when it is expressed in the absence of Npn-1.
Because the PlGF152 Exon 6 peptide is able to compete for
VEGF165 binding to Npn-1, we hypothesized that if Npn-1 is
responsible for the increased potency of VEGF165,
preventing the formation of the VEGFR-2·Npn-1 complex by blocking the
binding of VEGF165 to Npn-1 should reduce the signaling
potency of VEGF165. Furthermore, because
VEGF121 does not have access to the Npn-1 receptor but activates VEGFR-2, the PlGF152 Exon 6 peptide should have
no effect on the signaling potency of VEGF121. Fig.
8 demonstrates that in the absence of the
PlGF152 Exon 6 peptide, VEGF165 and
VEGF121 demonstrate significantly different signaling
potencies in native HUVEC (VEGF165 EC50 = 283 pM; VEGF121 EC50 = 1.84 nM). However, in the presence of 100 µM
PlGF152 Exon 6 peptide, the signaling potency of
VEGF165 is dramatically reduced (VEGF165
+PlGF152 Exon 6 peptide EC50 = 1.06 nM), whereas the signaling potency of a ligand that does
not have access to Npn-1 and that signals solely through VEGFR-2
(VEGF121) is unaffected (VEGF121
+PlGF152 Exon 6 peptide EC50 = 2.38 nM). From these data, we conclude that the ability of
VEGF165 to bind to the VEGFR-2·Npn-1 complex is
responsible for the differential potency of VEGF165
relative to VEGF121 in stimulating activation of
VEGFR-2.
Neuropilin-1 has been identified as an isoform-specific receptor
for VEGF165 (18), the VEGF-E isoform VEGForfNZ2
(20), PlGF152 (21), and both splice isoforms of VEGF-B
(22). In the case of VEGF165, it was hypothesized that
Npn-1 acts as a potentiator of ligand binding to VEGFR-2, thereby
explaining the differential activity of VEGF165
versus VEGF121 in various endothelial cell signaling
assays (18). However, this conclusion was based solely on affinity
labeling data generated in cells engineered to co-express Npn-1 with
VEGFR-2. Affinity labeling experiments are not accurate indicators of
receptor-ligand binding affinity because the results can be biased by
changes in cross-linking efficiency of the ligand to the receptor that
result from conformational alterations upon ligand binding to the
receptor component within the complex versus that observed
when the receptor component is expressed alone. Additionally, the
amount of radioligand observed to cross-link a receptor component can
be influenced by alterations in receptor expression between cell lines
that express the receptor alone versus those that co-express
multiple receptor components. Indeed, we observe a similar increase in
binding to VEGFR-2 in COS-1 cells transiently expressing VEGFR-2 in
concert with Npn-1 versus that observed in COS-1 cells that
express VEGFR-2 alone, but this increased binding to VEGFR-2 can be
explained by increased expression of VEGFR-2 in the presence of Npn-1
that often occurs in this system (Fig. 3).
Further support for the contention that formation of the
VEGFR-2·Npn-1 receptor complex does not result in an
alteration in VEGF165 binding affinity comes from a
detailed analysis of the saturation isotherms in cells expressing Npn-1
alone or in concert with VEGFR-2 (Fig. 6 and Table I). The binding
affinity of [125I]VEGF165 is similar in cells
expressing Npn-1 alone or VEGFR-2 alone, and there is no increase in
affinity observed upon co-expression of VEGFR-2 on an Npn-1 background
(Fig. 6 and Table I). These data demonstrate that formation of the
VEGFR-2·Npn-1 co-receptor complex in the presence of ligand (Figs. 3,
4, and 7) does not result in formation of a subpopulation of high
affinity binding sites. In this respect, the VEGFR-2·Npn-1 receptor
complex is similar to that observed for the glial cell line-derived
neurotrophic factor receptor Despite a lack of effect on ligand binding affinity, Npn-1 appears to
be responsible for the discrimination of signaling efficiency elicited
by VEGFR-2 in response to VEGF165 versus
VEGF121. Previous attempts at blocking Npn-1 involvement in
VEGF165 signaling utilized a recombinant version of the
Npn-1-binding domain of VEGF (16, 18). This glutathione
S-transferase-Exon 7-8 fusion protein competes for
[125I]VEGF165 binding to both VEGFR-2 and
Npn-1, (18) and reduces signaling in response to both
VEGF165 and VEGF121 (16). Because VEGF121 does not bind to Npn-1 (18, 23), it is possible
that the antagonistic effect of this protein is due to direct
antagonism of signaling through VEGFR-2. Indeed, the results of the
deletion analysis on the activity of this fusion protein (16) are
inconsistent with the known structure of the heparin-binding region of
VEGF165 (48, 49), suggesting that this reagent may not
represent the Npn-1-binding domain of VEGF165 and, by
inference, that the protein is not an Npn-1 antagonist. Similar to what
was observed for the glutathione S-transferase-Exon 7-8
protein, we also find that a peptide derived from the Exon 6 region of
PlGF152 (21) can compete for the binding of
[125I]VEGF165 to Npn-1, with a proportional
competition for [125I]VEGF165 binding to
VEGFR-2 in HUVEC cells (Fig. 7A). The proportional decrease
in binding to VEFGR-2 in HUVEC probably represents a decrease in
binding to VEGFR-2 in the VEGFR-2·Npn-1 complex because this
concentration of the PlGF152 Exon 6 peptide does not
substantially compete for binding at VEGFR-2 when it is expressed in
the absence of Npn-1 (Fig. 7B). Notably, this peptide only
antagonizes the signaling of VEGF165 and does not affect
VEGF121-mediated phosphorylation of VEGFR-2 (Fig. 8).
Together, these data suggest that a blockade of binding to the
VEGFR-2·Npn-1 complex can antagonize VEGF signaling in an
isoform-specific manner and that isoform-specific binding to the
VEGFR-2·Npn-1 complex can explain the increased potency of
VEGF165 versus VEGF121 in HUVEC.
These data stand in contrast to that observed for PlGF139
and PlGF152, where access to the complex of VEGFR-1 with
Npn-1 (41) does not appear to confer a signaling advantage for the
Npn-1-binding PlGF isoform (PlGF152) (21).
If formation of the VEGFR-2·Npn-1 complex does not result in a higher
affinity state of the receptor, how is the increased potency of
VEGF165 relative to VEGF121 explained? In Fig.
9 we propose a mechanism wherein
formation of the VEGFR-2·Npn-1 complex can serve to potentiate
signaling through VEGFR-2. VEGFR-2 is thought to bind to
VEGF121 across the dimer interface, at the opposite poles
of the ligand dimer (50-52) (Fig. 9A). VEGF165
binds to VEGFR-2 and Npn-1 through distinct epitopes, with the VEGFR-2 binding epitope similar to that observed with VEGF121, and
the Npn-1 binding epitope occurring symmetrically in the Exon-7-8 regions of the VEGF165 dimer (Fig. 9B). Unlike
VEGF121, VEGF165 can cluster VEGFR-2 through
its ability to bind VEGFR-2 in a manner similar to VEGF121
as well as through the preformed Npn-1·VEGFR-2 complex that binds
via Exon 7.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter (38). For
stable expression in Balb/c 3T3 A31 cells, hVEGFR-2, GFP, and
hNeuropilin-1 cDNAs were generated and subcloned into the pBMN-Z
family of retroviral vectors. The pLTRMCSIRES-GFP (referred to as the
Mock vector) and pLTRhFLK1(full)IRES-GFP constructs are a bi-cistronic
modification of the pBMN-Z vector, containing a GFP marker or
containing GFP + hVEGFR-2. The hNpn-1 cDNA was subcloned into the
pBMN-Z-I-Neo vector.
counter.
Generation of competition curves and IC50 values
(concentration of unlabeled ligand that reduces radioligand binding to
halfway between the upper and lower plateaus of a competition curve)
were performed using the Prism software package (GraphPad Software
Inc., San Diego, CA).
Saturation analysis results from multiple cell lines
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
11 M (log EC50 =
10.11 ± 0.113, n = 14) and for VEGF121 of
2.45 × 10
9 M (log EC50 =
8.61 ± 0.091, n = 14) in stimulating
phosphorylation of VEGFR-2, with a representative experiment
illustrated in Fig. 1. The reported
similarity in affinity between VEGF165 and
VEGF121 at the isolated VEGFR-2 receptor is not an artifact
of producing the protein as a receptor body (15), because when the
full-length receptor is expressed in cells similar results are observed
(Fig. 2A). In Fig.
2A, VEGFR-2 is overexpressed in COS-1 cells, and the ability
of these two ligands to compete for
[125I]VEGF165 binding is measured. In the
COS-1 cells overexpressing the full-length VEGFR-2, the
IC50 values for VEGF165 (IC50 = 6.34 × 10
10 M) and VEGF121
(IC50 = 3.12 × 10
10 M)
binding are nearly identical, indicating similar affinity to VEGFR-2
when it is expressed in isolation. Surprisingly, VEGF121 has only limited ability to compete for
[125I]VEGF165 binding in HUVEC (Fig.
2B), despite the presence of functional VEGFR-2 in these
cells (Fig. 1). Because the HUVEC are reported to contain Npn-1 (18),
we examined whether the binding observed in HUVEC could be reproduced
in the COS-1 cell system. As expected, VEGF121 does not
compete in COS-1 cells where Npn-1 is expressed alone (Fig.
2C) but limited or no competition for binding is also
observed in cells expressing Npn-1 in concert with VEGFR-2 (Fig. 2,
B and D). The data from the COS-1 cells expressing defined receptor populations suggest that the relative inability of VEGF121 to compete for binding in HUVEC may be
attributed to excess Npn-1 expression relative to VEGFR-2. We therefore
explored the possibility that VEGFR-2 and Npn-1 form a co-receptor
complex and that the preferential ability of VEGF165 to
signal through this complex is responsible for the increased potency of
VEGF165 relative to VEGF121 in the VEGFR-2
autophosphorylation assay.
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Fig. 1.
VEGF165 and VEGF121
exhibit distinctly different EC50 values in the VEGFR-2
phosphorylation assay. HUVEC cells were stimulated with ligand for
5 min at 37 °C, lysed, and immunoprecipitated with an antibody
specific to VEGFR-2 (R2.2). Following SDS-PAGE separation and transfer,
the blots were probed using an anti-phosphotyrosine (pY)
antibody (4G10; Upstate Biotechnology Inc.) and developed using
standard ECL techniques (upper panel). These same blots were
then stripped and reprobed using an antibody specific for VEGFR-2
(R2.2, lower panel). The films were scanned and quantitated
using Image Quant (Molecular Dynamics). The nonlinear regression
analysis (Prism) of the phosphotyrosine (pY)/R2 signal ratio
yields EC50 values of 34.9 pM and 2.09 nM for VEGF165 and VEGF121,
respectively. This experiment has been repeated 13 times with similar
results.
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Fig. 2.
Examination of the binding affinity of
VEGF165 (squares) and VEGF121
(circles) in cells expressing various receptor
complements. Cells with various VEGF receptor backgrounds were
incubated in the presence of 382 pM (COS-1, where the
average Kd = 339 pM or 2 nM
for VEGFR-2 and Npn-1, respectively; see Table I) or 287 pM
(HUVEC, where the average Kd = 169 pM;
see Table I) [125I]VEGF165 and increasing
concentrations of cold VEGF165 or VEGF121 to
equilibrium (4 h) at 4 °C as described under "Experimental
Procedures." A, COS-1 cells expressing VEGFR-2
(IC50 VEGF165 = 634 pM,
VEGF121 = 312 pM). B, endogenous
HUVEC cells known to express both VEGFR-2 and Npn-1 (IC50
VEGF165 = 79.5 pM, VEGF121 = no
displacement). C, COS-1 cells expressing Npn-1
(IC50 VEGF165 = 1090 pM,
VEGF121 = no displacement). D, COS-1 cells
expressing both VEGFR-2 and Npn-1 (IC50 VEGF165 = 2890 pM, VEGF121 = no displacement). The
IC50 values were calculated by fitting the data to a
four-parameter logistic equation (Prism software). The data points
represent the averages ± S.E. of triplicate determinations. This
experiment has been repeated twice with similar results.
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Fig. 3.
Affinity labeling and immunoprecipitation of
the VEGFR-2·Npn-1 receptor complex. A, COS-1 cells
transiently overexpressing the indicated receptors were
affinity-labeled at 4 °C for 4 h with a tracer concentration
greater than the Kd (704 pM
[125I]VEGF165) to allow for better labeling
of the receptors. The cells were then lysed, immunoprecipitated
(IP) using the receptor-specific antibody listed (for
VEGFR-2, R2.2; for Npn-1, C-19), separated by SDS-PAGE, and developed
using the Storm System (Molecular Dynamics). The left panel
demonstrates that the Npn-1 doublet is co-precipitated with
affinity-labeled VEGFR-2 when the two receptors are co-expressed. The
right panel demonstrates that VEGFR-2 is co-precipitated in
the complex when an Npn-1 specific antibody is used. In both
panels, a high molecular mass band is observed that may
represent the VEGFR-2·Npn-1 complex (see text). This experiment has
been repeated three times with similar results. B, Western
blot of matched lysates depicts the relative receptor expression level
achieved (20 µg of total protein was loaded per lane).
Blotting antibodies are R2.2 for VEGFR-2 and the Npn-1 number 30 antibody for Npn-1.
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Fig. 4.
Affinity labeling and immunoprecipitation of
the VEGFR-2·Npn-1 receptor complex in endogenous HUVEC cells.
HUVEC cells were affinity-labeled at 4 °C for 4 h with a tracer
concentration greater than the Kd (700 pM [125I]VEGF165). The cells were
lysed, immunoprecipitated (IP) using the receptor-specific
antibody listed (for VEGFR-2, R2.2; for Npn-1, C-19), separated by
SDS-PAGE, and developed using the Storm system (Molecular Dynamics).
The left lane demonstrates that a triplet of
affinity-labeled bands were co-precipitated with affinity-labeled
VEGFR-2. The right lane confirms the identity of the
top two bands of the triplet as being Npn-1 because an
identical doublet is immunoprecipitated using an Npn-1 specific
antibody (right panel). The asterisk represents
the Npn-1 band not immunoprecipitated by the Npn-1 antibody (see text).
Similar to what is observed in Fig. 3, a high molecular mass band is
also present in the HUVEC immunoprecipitates, and this band may
represent the VEGFR-2·Npn-1 complex (see text). This experiment has
been repeated three times with similar results.
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Fig. 5.
Ligand-independent complex formation.
A, COS-1 expressing the indicated receptors or HUVEC cells
were incubated to equilibrium in the presence or the absence of 500 pM VEGF165 at 4 °C for 4 h. The cells
were then cross-linked, lysed, and immunoprecipitated (IP)
with the receptor-specific antibody (for VEGFR-2, R2.2; for Npn-1,
C-19). The immunoprecipitations were then separated by SDS-PAGE,
transferred, and probed with a receptor-specific antibody. The blotting
antibodies are R2.2 for VEGFR-2 and the Npn-1 number 30 antibody for
Npn-1. This experiment has been repeated twice with similar results.
B, Western blot analysis of the same COS-1 cells expressing
the receptors indicated or HUVEC cells (20 µg of total protein was
loaded per lane). The blotting antibodies are R2.2 and Npn-1
number 30 for VEGFR-2 and Npn-1, respectively.
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Fig. 6.
The affinity of VEGF165
to VEGFR-2 is not increased in the presence of
Npn-1. A, Balb/c 3T3 A31 cells stably expressing the
indicated receptors are incubated to equilibrium with increasing
concentrations of [125I]VEGF165 in the
presence or the absence of a saturating dose of unlabeled
VEGF165 (30 nM). The curves depicted
represent specific binding, where the error bars are the
standard error of the mean for triplicate determinations.
Squares, D7R2·Npn-1 cells, Bmax = 98,750 ± 3,436 sites/cell, Kd = 119 ± 15 pM; circles, PGBMGH (Mock) cells,
Bmax = 38,990 ± 3,135 sites/cell,
Kd = 123 ± 36 pM;
triangles, Npn-1(2) cells, Bmax = 78,390 ± 3,381 sites/cell, Kd = 184 ± 25 pM; diamonds, D7R2 cells,
Bmax = 60,800 ± 2,080 sites/cell,
Kd = 145 ± 17 pM. The errors
associated with the curve fitting parameters represent approximate
errors in the estimates for the curves. B,
Western blot analysis of matched lysates demonstrating relative
receptor levels (20 µg of total protein was loaded per
lane). The blotting antibodies are R2.2 and Npn-1 number 30 for VEGFR-2 and Npn-1, respectively. The binding experiment has been
repeated at least three times with similar results (Table I). The
Western blot analysis has been repeated at least six times with similar
results.
10
M) is similar to that observed in the D7R2 cells (2.91 × 10
10 M), despite the much higher Npn-1
background in the D7R2 cells (Table I and Figs. 3B,
5B, and 6B). Furthermore, the affinity at Npn-1
in the Balb/c cells (4.17 × 10
10 M) is
similar to that at VEGFR-2 expressed in the COS-1 cells (Table I). This
indicates that the affinity of VEGF165 is similar at either
receptor, making it even less likely that we missed detection of a
subpopulation of high affinity sites that is created upon co-expression
of the two receptors.
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Fig. 7.
The PlGF152 Exon 6 peptide
competes for binding at the VEGFR-2·Npn-1 complex as well as at Npn-1
alone. A, experimental design is the same as for Fig.
4, with the following exceptions. The tracer (420 pM
[125I]VEGF165 (still
greater than the Kd; see Table I)) is added
simultaneously in the presence or the absence of the indicated
concentration of unlabeled VEGF165 or the
PlGF152 Exon 6 peptide, and the NP1ECD4 antibody was used
to immunoprecipitate (IP) Npn-1. Both VEGF165
and the PlGF152 peptide compete for binding at the
VEGFR-2·Npn-1 complex. The PlGF152 Exon 6 peptide also
competes for binding at the Npn-1 bands (lane 6). This
experiment has been repeated once with similar results. B,
experimental design is the same as for Fig. 2 except that 201 pM [125I]VEGF165 was used as
tracer. Left panel, the PlGF152 Exon 6 peptide
(circles) does not compete for
[125I]VEGF165 binding in VEGFR-2 transfected
cells, whereas it completely competes for tracer binding in Npn-1
transfected cells (right panel, circles). This
experiment has been repeated three additional times with similar
results. The IC50 values for VEGF165 are
7.25 × 10 10 M and 4.33 × 10
10 M for VEGFR-2 and Npn-1, respectively.
The PlGF152 Exon 6 peptide competes for binding at Npn-1
with an IC50 value of 1.34 × 10
5
M.
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Fig. 8.
The PlGF152 peptide antagonizes
VEGF165 signaling without affecting VEGF121
signaling in HUVEC cells. The experimental design is the same as
for Fig. 1, with the exception that the PlGF152 peptide was
added to the indicated samples at a final concentration of 100 µM. If the PlGF152 peptide were acting solely
as an antagonist of VEGFR-2, it would shift both the
VEGF165 and VEGF121 dose-response curves to the
right. In contrast, only the VEGF165 response
curve is affected. This implies that the PlGF152
Exon 6 peptide is specifically affecting signaling through the
VEGFR2·Npn-1 complex. The nonlinear regression analysis (Prism) of
the phosphotyrosine (pY)/R2 signal ratio reveals the
following: VEGF165 EC50 = 283 pM;
VEGF165 + PlGF152 Exon 6 peptide
EC50 = 1.06 nM; VEGF121
EC50 = 1.84 nM; VEGF121 + PlGF152 Exon 6 peptide EC50 = 2.38 nM. This experiment has been repeated once with similar
results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit and the Ret receptor tyrosine
kinase (RTK) co-receptor complex, where ligand binding is also observed to the non-receptor tyrosine kinase
subunit in the absence of the
RTK, and co-expression of the
subunit with the RTK does not result
in an increase in ligand binding affinity (46). Hence, Npn-1 does not
appear to function as an affinity converter for VEGF165 in
concert with VEGFR-2, as has been observed for other multi-component
receptor systems (47). This is in contrast to the role played by Npn-1
in the regulation of ligand binding affinity of Sema3A to the
Npn-1·Plexin1 co-receptor complex (26).
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Fig. 9.
Model of complex formation between VEGFR-2
and Npn-1 in the presence of different VEGF isoforms.
A, VEGFR-2 contacts VEGF121 across the dimer
interface, at the opposite poles of the ligand dimer (50-52).
B, VEGF165 can cluster VEGFR-2 through its
ability to bind VEGFR-2 in a manner similar to VEGF121, as
well as through the VEGFR-2·Npn-1 complex that binds via
Exon 7.
The model incorporates the experimental data we have described but also incorporates data from a variety of other receptor systems. It has been demonstrated that the concentration of receptors at the cell surface can affect the position of the dose-response curve for G-protein-coupled receptors (53-55), as well as for RTK (56, 57) ligands, with detection of the ligand-mediated response occurring at progressively lower ligand concentrations as receptor expression increases. By analogy, the ability of VEGF165 to bind to both VEGFR-2 and Npn-1 may serve to increase the local concentration of VEGFR-2 upon binding VEGF165 relative to that achieved with VEGF121 (Fig. 9) because Npn-1 has the potential to form a ligand-independent complex with VEGFR-2 (Fig. 5). It is also possible that Npn-1 could function to increase the local ligand concentration in the vicinity of VEGFR-2 (58) and that the multivalency of VEGF165 relative to VEGF121 could serve to increase the avidity of the ligand to VEGFR-2, resulting in increased activity at lower ligand concentrations for VEGF165 relative to VEGF121. The latter provides an explanation for the limited ability of VEGF121 to compete for the binding of [125I]VEGF165 in cells co-expressing VEGFR-2 with Npn-1 (Fig. 2).
There is precedence for the preclustering of different members of
multi-component receptors (59-61), wherein ligand binding to different
receptor clusters can have different signaling consequences (61, 62).
Such precedence is also available for the receptor tyrosine kinases. In
the case of the Ephrin receptors, the oligomeric state of the ligand
has been demonstrated to confer signaling specificity through the EphB1
and EphB2 receptors in endogenous endothelial cell systems, with
cellular attachment and recruitment of the low molecular mass
protein-tyrosine phosphatase to the receptor cytoplasmic domain only
occurring in higher order ligand-receptor oligomers (63). Both
platelet-derived growth factor receptor and VEGFR-2 have been
demonstrated to exist in a ligand-independent complex with the
v
3 integrin through an interaction of the
RTK with the
3 extracellular domain, although
association of VEGFR-2 with
v
3 requires
the presence of the
v integrin receptor subunit (64).
This finding indicates that oligomerization of receptor subunits need
not occur through enzyme-substrate interactions of the cytoplasmic
domains. The attachment of endothelial cells to vitronectin potentiates
signaling through VEGFR-2, and an antibody to the integrin
3 subunit that does not antagonize cellular adhesion is
capable of inhibiting the ligand-stimulated phosphorylation of VEGFR-2
as well as signaling downstream of VEGFR-2 (65). Because VEGFR-2 has
the potential to be preassociated with
v
3 on the cell surface (64) and because the anti-integrin
3
antibody does not interfere with the ability of
[125I]VEGF165 to bind to VEGFR-2 (65), the
mechanism for the inhibitory effect of this antibody on
VEGFR-2-mediated signaling may also be due to an interference with
formation of higher order VEGFR-2 oligomers, similar to what we have
hypothesized for the consequences of inhibition of VEGF165
binding to Npn-1 within the VEGFR-2·Npn-1 receptor complex.
In summary, we have demonstrated that Npn-1 forms a co-receptor complex
with VEGFR-2 in the presence of VEGF165 ligand, both in
heterologous systems as well as in an endogenous endothelial cell
system. Surprisingly, formation of this complex does not result in
formation of a higher affinity state of the receptor for
VEGF165; therefore the difference in the potency of
VEGF165 versus VEGF121 to stimulate
VEGFR-2 cannot be explained by alterations in receptor affinity that
are specific to VEGF165. Using a peptide derived from the
Npn-1-binding region of PlGF152 (21), we demonstrate that
antagonism of VEGF165 binding to Npn-1 interferes with
signaling specifically through the VEGFR-2·Npn-1 complex, without an
effect on signaling of VEGF121 through VEGFR-2. Because a
concurrent up-regulation of VEGF165 with VEGFR-2 and Npn-1
correlates with increased vascular density in certain pathologies (66,
67), it is tempting to speculate that antagonism of signaling through the VEGFR-2·Npn-1 receptor complex may attenuate pathological angiogenesis without affecting the function of VEGFR-2 in the quiescent
vasculature (68, 69).
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ACKNOWLEDGEMENTS |
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We thank Drs. Thomas J. Daly and Nicholas Papadopoulos (Regeneron Pharmaceuticals) for the production and purification of the recombinant VEGFR-2:Fc used to produce the monoclonal antibodies used in our studies, Dr. Rocco J. Rotello and Ms. Jennifer M. Kennedy (Procter & Gamble Pharmaceuticals) for the preparation of the J5F4A2 monoclonal antibody, and Dr. David M. Valenzuela (Regeneron Pharmaceuticals) for the VEGFR-2 cDNA and retroviral constructs used for the creation of the stable cell lines reported herein. We also thank Drs. Kevin G. Peters (Procter & Gamble Pharmaceuticals), David D. Ginty (Johns Hopkins University), and George D. Yancopoulos (Regeneron Pharmaceuticals) for their helpful comments and discussions during the preparation of this manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Cardiovascular Research, Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd., Mason, OH 45040. Tel.: 513-622-2552; Fax: 513-622-1433; E-mail:
rosenbaum.js@pg.com.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M102315200
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; Npn, neuropilin; RTK, receptor tyrosine kinase; GFP, green fluorescent protein; PlGF, placenta growth factor; HUVEC, human umbilical vein endothelial cell; BSA, bovine serum albumin; PAGE, polyacrylimide gel electrophoresis.
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