From the Department of Biology, Technion, Israel
Institute of Technology, Haifa, 32000, Israel and the
Department
of Genetics, Institute of Medical Science, University of Tokyo,
Minato-Ku, Tokyo 108, Japan
Received for publication, August 1, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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The products of the neuropilin-1 (Np-1)
and neuropilin-2 (Np-2) genes are receptors for factors belonging to
the class 3 semaphorin family and participate in the guidance of
growing axons to their targets. In the presence of heparin-like
molecules, both receptors also function as receptors for the
heparin-binding 165-amino acid isoform of vascular endothelial growth
factor (VEGF165). Both receptors are unable to bind
to the 121-amino acid isoform of vascular endothelial growth factor
(VEGF121), which lacks a heparin-binding domain.
Interestingly, complexes corresponding in size to
125I-VEGF121·neuropilin complexes are
formed when 125I-VEGF121 is bound and
cross-linked to porcine aortic endothelial cells co-expressing
VEGFR-1 and either Np-1 or Np-2. These complexes do not seem to
represent complexes of 125I-VEGF121 with a
truncated form of VEGFR-1, presumably formed as a result of the
presence of Np-1 or Np-2 in the cells, because such truncated forms
could not be detected with anti-VEGFR-1 antibodies. Antibodies directed
against VEGFR-1 co-immunoprecipitated the 125I-VEGF121·Np-2 sized cross-linked complex
along with 125I-VEGF121·VEGFR-1 complexes
from cells expressing both VEGFR-1 and Np-2 but not from control cells,
indicating that VEGFR-1 and Np-2 associate with each other. To perform
the reciprocal experiment we have expressed in porcine aortic
endothelial cells a Np-2 receptor containing an in-frame
myc epitope at the C terminus. Surprisingly, the
myc-tagged Np-2 receptor lost most of its
VEGF165 binding capacity but not its semaphorin-3F binding
ability. Nevertheless, when Np-2myc was co-expressed in
cells with VEGFR-1, it partially regained its VEGF165
binding ability. Antibodies directed against the myc
epitope co-immunoprecipitated
125I-VEGF165·Np-2myc and
125I- VEGF165·VEGFR-1 complexes from cells
co-expressing VEGFR-1 and Np-2myc, indicating again that
VEGFR-1 associates with Np-2. Our experiments therefore indicate
that Np-2, and possibly also Np-1, associate with VEGFR-1 and that such
complexes may be part of a cell membrane-associated signaling complex.
The various forms of the growth factors belonging to the
VEGF1 family (VEGF, PlGF,
VEGF-B, VEGF-C, and VEGF-D) act as inducers and modulators of
angiogenesis in vivo (1-3). The active forms of the VEGF
family members are synthesized as homodimers (4, 5) or as heterodimers
with other VEGF family members such as PlGF (6). Targeted disruption of
the VEGF gene has shown that angiogenesis is severely disrupted even in
heterozygous animals containing a single functional allele of the VEGF
gene. It is therefore believed that the maintenance of correct levels
of VEGF in vivo is critical for the development of the
cardiovascular system (7, 8). Five splice forms of human VEGF ranging
from 121 to 206 amino acids (VEGF121-VEGF206)
have been characterized (4, 5, 9, 10). These differ primarily in the
presence or the absence of the heparin-binding domains encoded by exons 6 and 7, giving rise to forms that differ in their heparin and heparan-sulfate binding ability (11). Likewise, other VEGF family members such as PlGF and VEGF-B are also expressed in several forms
that differ in their heparin binding ability. For example, the peptide
encoded by exon 6 of PlGF is found only in PlGF-2 and confers a heparin
binding ability to PLGF-2, whereas PlGF-1 does not bind to heparin
(12).
All VEGF isoforms bind to the tyrosine-kinase receptors VEGFR-1
(flt-1) (13) and VEGFR-2 (KDR/flk-1) (14). The
binding of VEGF to VEGFR-2 initiates intracellular signal transduction (1, 15-18) and is correlated with the induction of endothelial cell
proliferation, migration, and in vivo angiogenesis (19, 20).
By contrast, the activation of VEGFR-1 does not seem to result in the
induction of cell proliferation or angiogenesis, although exceptions
have been observed (21, 22). However, the activation of VEGFR-1 seems
to enhance cell migration (20, 23-25). Both receptors play pivotal
roles in embryonic vasculogenesis and angiogenesis. Embryos lacking the
VEGFR-2 gene die before birth because differentiation of endothelial
cells does not take place and blood vessels do not form (26). In
contrast, disruption of the gene encoding the VEGFR-1 receptor did not
prevent the differentiation of endothelial cells in homozygous animals,
but the development of functional blood vessels from these endothelial cells was severely impaired (27).
Endothelial cells also contain another type of VEGF receptor possessing
a lower mass than either VEGFR-2 or VEGFR-1 (28, 29). It was
subsequently found that these smaller VEGF receptors of the endothelial
cells are isoform-specific receptors that bind VEGF165 but
not VEGF121 (30). Additional experiments revealed several
types of prostate and breast cancer-derived cell lines that express
unusually large amounts of these isoform-specific receptors (31). The
receptors were purified from such cells using affinity chromatography
on VEGF165 affinity matrices followed by partial protein
sequencing and were found to be the products of the neuropilin-1 (Np-1)
gene (32). It was subsequently observed that the heparin-binding form
of placenta growth factor (PlGF-2) and VEGF-B, two additional members
of the VEGF family of growth factors, are also able to bind to Np-1
(33, 34). When the role of Np-1 as a VEGF receptor was discovered, it
was already known that Np-1 functions in the nervous system as receptor
for sema-3A. sema-3A is a Np-1 agonist that causes repulsion of growing tips of axons (35, 36). It was recently observed that sema-3A is also
able to inhibit migration of endothelial cells (37). These results
indicate that signaling via Np-1 affects angiogenesis and possibly the
development of the cardiovascular system. Targeted disruption of the
Np-1 gene resulted in severe cardiovascular defects, confirming these
suspicions (38-40). Np-1 is part of a gene family that includes the
closely related receptor Np-2. In the nervous system Np-2 is activated
by another class 3 semaphorin, sema-3F, which also induces the
repulsion of axons that express Np-2 (41). We have recently observed
that Np-2 is also able to bind VEGF165 and PlGF-2 but not
VEGF121. However, unlike Np-1, Np-2 was also able to
interact with the VEGF145 form of VEGF. VEGF145
lacks the peptide encoded by exon 7 of VEGF, which is included in
VEGF165, but contains instead the heparin-binding domain
encoded by exon 6 of the VEGF gene (9, 42).
The neuropilins have a short intracellular domain and are unlikely to
function as independent receptors. Indeed, no responses to
VEGF165 were observed when cells expressing either Np-1 or Np-2 but no other VEGF receptors were stimulated with
VEGF165 (32, 42). It was recently found that plexins form
complexes with neuropilins and that these complexes mediate signal
transduction by sema-3A (43, 44). It is possible that neuropilins
associate with additional cell surface molecules to form complexes that transduce VEGF signaling. We present evidence indicating that Np-2 and
possibly also Np-1 form complexes with the VEGFR-1 receptor and that
the formation of these complexes changes the binding characteristics of
neuropilins so that they are now able to bind VEGF121, a
splice form that is not recognized by neuropilins in cells that do not
express VEGFR-1.
Materials--
Antibodies directed against the intracellular
tyrosine-kinase domain of VEGFR-1 were obtained from Santa Cruz
Biotechnology. Immunoprecipitating antibodies that bind to the
extracellular part of VEGFR-1 were generated as previously described
(45). Antibodies directed against the myc epitope were
purchased from Santa Cruz Biotechnology. Np-2 and Np-1 expressing PAE
cells were generated as previously described (42). A sema-3F expression plasmid was kindly given to us by Dr. David Ginty by permission from
Dr. Marc Tessier-Lavigne. This construct contains the semaphorin-3F cDNA fused in-frame to alkaline phosphatase at the N terminus of
sema-3F (41). LipofectAMINE was purchased from Life Technologies, Inc.
Generation of Anti-Np-2 Antibodies--
A 780-base pair Np-2
Sph-1/Pst-1 cDNA fragment was ligated into the bacterial expression
vector pQE-30 (Qiagen). This plasmid was used for the production of the
recombinant, His6-tagged 30-kDa peptide according to the
manufacturer's instructions. The peptide was cleaned from bacterial
cell extracts using nickel affinity chromatography according to the
instructions of the vendor and purified again using SDS/PAGE.
The gel was electroblotted onto nitrocellulose, and the band containing
the peptide was cut out, solubilized in Me2SO, and
used to immunize rabbits. The antiserum was purified on a protein A
affinity column followed by affinity purification on a column to which
the recombinant peptide was coupled using a previously described method
(46). The antibody was eluted from the column using 0.1 M
glycine at pH 3. The antibody generated in this manner recognized Np-2
specifically in Western blots and did not recognize other proteins even
when these proteins contained a His6 tag but did not
immunoprecipitate Np-2 (data not shown).
Cell Lines and cDNAs--
PAE cells (20) were kindly
provided by Dr. Carl Heldin. The PAE·VEGFR-1 cell lines were
generated by transfecting PAE cells with the VEGFR-1 expression vector
BCMGSneo-hu-flt-1 (23), and selection of VEGFR-1 expressing cell lines
was done using 0.5 mg/ml G418. The cells were continuously maintained
in medium containing 0.25 mg/ml G418. Cell lines expressing VEGFR-1 and
Np-2 were generated by co-transfection of VEGFR-1 expressing cells with
the PECE/Np-2 expression vector and with the pBabe-puro vector (47).
Stable cell lines were isolated by double selection with 0.5 µg/ml
puromycin and 0.25 mg/ml G418. For the generation of PAE cells
expressing VEGFR-1 and Np-1, the VEGFR-1 expression vector
BCMGSneo-hu-flt-1 was transfected into the previously described
PAE·Np-1 cells (32), and VEGFR-1 expressing cell lines were selected
using 0.5 mg/ml G418. Transfections were carried out using
LipofectAMINE according to the instructions of the vendor. Human
umbilical vein-derived endothelial cells were cultured as previously
described (30).
Construction and Expression of Np-2myc in PAE
Cells--
The primer
5'-GCTCTAGAGGGCCCTCACAGATCCTCCTCTGAGATGAGTTTTTGTTCAGCCTCGGAGCAGCACTTTTG-3'
containing the myc epitope (underlined) and the primer
5'-CAACCTCAGGGTCTGGCGCC-3' were used to introduce a myc
epitope after the last amino acid of Np-2a22 (41). The primers were
used to amplify the modified C terminus using the Np-2 cDNA (42) as
a template. The myc epitope-containing fragment was ligated
back into the Np-2 cDNA using a unique NarI site and an
Xba1 site donated by the plasmid (pcDNA3.1/hygro).
Following sequencing, the complete expression vector
(pcDNA3.1-hygro/Np-2myc) was transfected into PAE or
PAE·VEGFR-1 cells using LipofectAMINE. Stable cell lines were
selected using hygromycin (0.3 mg/ml). In the case of the
PAE·VEGFR-1·Np-2myc cells the selective medium also
contained G418 (0.5 mg/ml).
Binding and Cross-linking--
VEGF121 and
VEGF165 were produced using the baculovirus expression
system and iodinated as described (30, 48). Binding of
125I-VEGF165 or
125I-VEGF121 to cells was carried out
essentially as previously described (42). The water-soluble
cross-linker BS3 was used to cross-link bound VEGFs to
receptors. BS3 was dissolved in PBS to a final concentration of 0.2 mM and applied to cells. The
cross-linking procedure was done essentially as previously described
(30). All experiments were performed at least twice with similar results.
Immunoprecipitation of Complexes Cross-linked to
125I-VEGF121 or
125I-VEGF165 Using Anti-VEGFR-1 or Anti-myc
Epitope Antibodies--
Anti-VEGFR-1 antibodies directed against the
extracellular domain of VEGFR-1 (45) or commercial anti-myc
epitope antibodies were used in immunoprecipitation experiments.
125I-VEGF121 or
125I-VEGF165 was bound and cross-linked to
cells expressing various VEGF receptors. Following cross-linking, the
cells were lysed using lysis buffer (20 mM Tris/HCl, pH
7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 0.5 mM Na3VO4, 1 mM
dithiothreitol, and protease inhibitors (2 µg/ml of leupeptin
and aprotinin and 1 mM phenylmethylsulfonyl fluoride)) for
10 min. at 4 °C. The lysate was cleared by centrifugation, and
protein content was measured using the Bio-Rad protein assay according
to the instructions of the vendor. Equal amounts of protein from the
different lysates were taken for immunoprecipitation. The lysates
were incubated overnight at 4 °C with anti-myc or anti-VEGFR-1 antibodies. Protein A-Sepharose was added the next day and
incubated with the antibody for 2 h at 4 °C, and the beads were
subsequently washed three times with cold PBS. SDS/PAGE sample buffer
was then added to the beads, and the beads were boiled for 3 min. The
supernatant was then separated using SDS/PAGE followed by
autoradiography and phosphorimaging analysis. All experiments were
performed at least twice with similar results.
Western Blot Analysis--
Detection of VEGFR-1 by Western blot
analysis was performed using a commercial anti-VEGFR-1 antibody (Santa
Cruz Biotechnology) directed against the intracellular tyrosine-kinase
domain of the receptor using the ECL system (Amersham Pharmacia
Biotech) as previously described (9). Detection of Np-2 in Western
blots was performed similarly using our affinity purified anti-Np-2 rabbit derived polyclonal antibodies. All experiments were performed at
least twice with similar results.
Production and Binding of sema-3F--
The production of sema-3F
was done essentially as described (41). The Np-2 expressing PAE cells
or parental PAE cells were grown in gelatin-coated 48-well dishes to
confluence. The cells were washed once with PBS and incubated with 0.1 ml of conditioned medium from transiently transfected COS-7 cells
expressing alkaline phosphatase (AP)-tagged sema-3F. The conditioned
medium was supplemented with 1 mg/ml gelatin, 10 mM HEPES
buffer, pH 7.3, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride prior to the binding
experiment. The cells were incubated with the conditioned medium at
room temperature for 2 h, washed three times with PBS, and then
fixed with 4% paraformaldehyde for 20 min at room temperature. After
fixation, the dishes were washed three times with PBS and incubated at
65 °C for 1 h to inactivate endogenous phosphatases. The cells
were rinsed with AP substrate buffer containing 0.05 M
Na2CO3, 1 mM MgCl2, pH
9.8, and detection of alkaline phosphatase activity was done using the
same buffer containing 1 mg/ml p-nitrophenyl phosphate for 3 or 10 h at room temperature. Absorption of generated nitrophenol
was measured in a spectrophotometer at 405 nm. All experiments were
performed at least twice with similar results.
Np-2 Appears to Gain a VEGF121 Binding Ability When
Co-expressed with VEGFR-1--
We have previously found that
recombinant Np-2 is unable to bind 125I-VEGF121
when it is expressed on its own in PAE cells (Fig.
1A, lane 1) (42).
When 125I-VEGF121 was bound and cross-linked to
PAE cells expressing either VEGFR-1 (PAE·VEGFR-1 cells) or VEGFR-1
and Np-2 (PAE·VEGFR-1·Np-2 cells), two diffuse complexes of ~190
and ~210 kDa were formed, corresponding to the expected masses of
125I-VEGF121·VEGFR-1 complexes (Fig.
1A, lanes 2 and 3). Surprisingly, we
have found that two additional labeled complexes corresponding in mass
to the expected masses of 125I-VEGF121·Np-2
complexes (~140 and ~160 kDa) were formed in PAE·VEGFR-1·Np-2 cells (Fig. 1A, lane 3, arrow). This
observation suggested that the presence of VEGFR-1 affects the ligand
binding specificity of Np-2, enabling
125I-VEGF121 binding, and that VEGFR-1 and Np-2
may perhaps form complexes. This effect on
125I-VEGF121 binding seems to be specific to
VEGFR-1 because we could not detect binding of
125I-VEGF121 to Np-2 when
125I-VEGF121 was bound and cross-linked to PAE
cells co-expressing VEGFR-2 and Np-2 (not shown). On closer examination
we have also noticed that a very weak cross-linked complex
corresponding in size to a 125I-VEGF121·Np
complex is usually formed when 125I-VEGF121 is
bound and cross-linked to PAE·VEGFR-1 cells (see faint bands at the
level of the arrows in Fig. 1A (lane
2) and in Fig. 1B (lane 1)). This complex
may perhaps represent binding of 125I-VEGF121
to residual endogenous neuropilins and was never observed in parental
PAE cells or in PAE cells transfected with empty expression vectors.
The related Np-1 receptor is also unable to bind
125I-VEGF121 when it is expressed in PAE cells
on its own (32). However, cross-linked complexes corresponding in size
to 125I-VEGF121·Np-1 complexes were observed
following the binding of 125I-VEGF121 to cells
co-expressing VEGFR-1 and Np-1, indicating that Np-1 and VEGFR-1 may
also form complexes (Fig. 1B, lane 2, arrow). Indeed, such complexes have been recently observed
using a completely different experimental approach (49).
Western Blot Analysis Does Not Reveal Smaller Forms of VEGFR-1 in
Cells Expressing Both VEGFR-1 and Np-2--
Although the ~140- and
~160-kDa complexes observed in the previous experiment correspond in
size to 125I-VEGF121·Np-2 complexes, it was
still possible that these cross-linked complexes do not represent
complexes of 125I-VEGF121 and Np-2 but rather
complexes formed as the result of the binding of
125I-VEGF121 to truncated forms of VEGFR-1.
Such truncated VEGFR-1 forms could perhaps be generated as a result of
the presence of Np-2 in the cells. To exclude this possibility,
extracts from parental PAE cells, PAE·VEGFR-1 cells, and
PAE·VEGFR-1·Np-2 cells were analyzed for the presence of shorter
forms of VEGFR-1 using commercial antibodies directed against the
tyrosine-kinase domain of VEGFR-1. The cells were solubilized and
subjected to SDS/PAGE chromatography followed by Western blot analysis.
Two VEGFR-1 high molecular weight forms of ~170 and ~190 kDa were
easily detected in PAE·VEGFR-1 and in PAE·VEGFR-1·Np-2 cells but
not in Np-2-expressing PAE cells (Fig.
2). A band of ~140 kDa was observed in
all of the PAE-derived cells including the parental nontransfected
cells (not shown). This band probably represents nonspecific binding of
the antibody to an unknown antigen and has a mass that is higher than
that of Np-2 (Fig. 2). These experiments therefore indicate that it is
unlikely that PAE·VEGFR-1·Np-2 cells express smaller forms of
VEGFR-1 as a result of the presence of Np-2 in these cells.
Antibodies Directed against VEGFR-1 Co-immunoprecipitate a Labeled
Complex Corresponding in Mass to a
125I-VEGF121·Np-2 Complex--
The
experiments described above suggested that VEGFR-1 may be able to form
complexes with Np-1 and Np-2. To test this hypothesis directly, we used
antibodies directed against VEGFR-1 to co-immunoprecipitate Np-2 from
PAE cells co-expressing VEGFR-1 and Np-2 receptors. 125I-VEGF121 was bound and cross-linked to
PAE·Np-2, PAE·VEGFR-1, or PAE·VEGFR-1·Np-2 cells. The cells
were then lysed, and radiolabeled complexes were immunoprecipitated
using anti-VEGFR-1 antibodies. This method takes advantage of the high
sensitivity afforded by the use of 125I-labeled VEGF but
cannot be used to determine whether complex formation is
VEGF-dependent or not (50). This experiment revealed that
the ~140- and ~160-kDa
125I-VEGF121-containing complexes seen in the
experiment shown in Fig. 1 could be immunoprecipitated along with the
125I-VEGF121·VEGFR-1 complex from
PAE·VEGFR-1·Np-2 cells but not from PAE·VEGFR-1 cells (Fig.
3B, lane 5 versus lane
6). The anti-VEGFR-1 antibodies did not immunoprecipitate a
similar 125I-VEGF165·Np-2 complex from cells
expressing Np-2 but no VEGFR-1, indicating that the anti-VEGFR-1
antibodies do not cross-react with Np-2 (Fig. 3B, lane
4). It should be noted that the PAE·Np-2 cells used in this
control experiment contain large amounts of Np-2 as revealed by
125I-VEGF165 binding/cross-linking (Fig.
3A, lane 1). These results therefore indicate
that VEGFR-1 forms complexes with Np-2.
Np-2 Tagged with a myc Epitope at the C Terminus Loses its
VEGF165 Binding Ability--
To obtain further independent
experimental evidence for the formation of complexes between VEGFR-1
and Np-2, we have attempted to immunoprecipitate VEGFR-1 from cells
expressing both VEGFR-1 and Np-2 using our anti-Np-2 affinity purified
antibodies. However, our antibodies turned out to be poor precipitating
antibodies. We have therefore tagged Np-2 by expressing a
myc epitope in-frame at the end of the intracellular
C-terminal domain of Np-2. When we expressed the Np-2myc
construct in PAE cells the cDNA directed the expression of tagged
Np-2 as revealed in Western blots employing anti-Np-2 antibodies (Fig.
4A, lane 2). The amount of
Np-2myc in the transfected cells was about 5-fold lower than
the amount of Np-2 in our Np-2 expressing PAE cells (Fig.
4A, lane 1). However, in contrast to our
expectations, we could not detect specific 125I-VEGF165 binding to the Np- 2myc
expressing PAE cells (Fig. 4B). Only at very high
concentrations was there some residual specific binding. The maximal
specific binding observed was at least 40-fold less than the specific
binding of 125I-VEGF165 to PAE·Np-2 cells
(Fig. 4B). Surprisingly, the PAE·Np-2myc cells
were still able to bind sema-3F. The amount of sema-3F bound per cell
was about 5-fold lower than the binding of sema-3F to PAE·Np-2 cells
(Fig. 4C) and therefore in good agreement with the
experiment shown in Fig. 4A. These experiments therefore
indicate that the attachment of the myc epitope selectively
inhibits the binding of 125I-VEGF165 to Np-2
but does not affect significantly the binding of sema-3F to
Np-2myc. This experiment indicates indirectly that sema-3F
and VEGF165 may bind to different domains on the
extracellular part of Np-2. This conclusion was further supported by an
experiment showing that the binding of sema-3F to PAE·Np-2 cells
could not be inhibited by the inclusion of 2 µg/ml of
VEGF165 in the binding reaction (Fig.
5).
Np-2myc Partially Regains Its 125I-VEGF165
Binding Properties in the Presence of VEGFR-1, and Antibodies Directed
against the myc Epitope Tag of Np-2 Co-precipitate VEGFR-1--
The
previous experiments have indicated that the addition of the
myc tag inhibits the VEGF binding ability of Np-2.
Nevertheless, when 125I-VEGF165 was bound and
cross-linked to PAE cells co-expressing Np-2myc and VEGFR-1,
we noted that 125I-VEGF165 formed complexes
corresponding in size to VEGF165·Np-2myc complexes (Fig. 6A, lane
2). It therefore seems that the presence of VEGFR-1 enables
Np-2myc to regain at least part of the
125I-VEGF165 binding ability of untagged Np-2.
We have therefore attempted to co-immunoprecipitate VEGFR-1 from cells
co-expressing Np-2myc and VEGFR-1 using an antibody directed
against the myc epitope.
125I-VEGF165 was bound and cross-linked to the
cells, and the anti-myc antibody was then used in the
immunoprecipitation. As expected from the previous experiments, the
anti-myc antibody was able to co-precipitate
125I-VEGF165·Np-2myc and
125I-VEGF165·VEGFR-1 complexes from the cells
(Fig. 6B, lane 3). Because
125I-VEGF165 did not bind to Np-2myc
in cells lacking VEGFR-1, no complex could be precipitated from such
cells, nor did the anti-myc antibody precipitate any
cross-linked complexes from cells co-expressing VEGFR-1 and native Np-2
following the cross-linking of 125I-VEGF165 to
such cells (Fig. 6B, lanes 2 and
4). These observations therefore support the results
obtained using the anti-VEGFR-1 antibodies (Fig. 3B,
lane 6) indicating that VEGFR-1 is able to form complexes
with Np-2.
Np-1 and Np-2 were originally found to function as receptors for
several class 3 semaphorins that repel growing tips of axons during the
development of the nervous system (35, 36, 41). These discoveries were
followed by experiments that have demonstrated that Np-1 and Np-2 can
function in addition as receptors for VEGF165, one of the
heparin-binding forms of the angiogenic factor VEGF (32, 42). These
experiments indicated that the neuropilins may play a role in
cardiovascular biology, in addition to their role in the nervous
system. In the case of Np-1 these expectations were verified when it
was shown that targeted disruption of the Np-1 gene results in severe
cardiovascular defects (38). In agreement with this observation it was
found that the Np-1 agonist sema-3A inhibits migration of endothelial
cells (37), but the consequences of the binding of VEGF to Np-1 are not
completely clear as yet. In contrast, mice lacking functional Np-2
receptors are viable, and no vascular defects were reported so far (51, 52). Nevertheless, the absence of vascular defects in these gene
targeted mice does not necessarily preclude a physiological role for
these receptors in vascular biology because the absence of a phenotype
may be explained by redundancy with other signaling pathways.
We have not been able to demonstrate any biological responses to
VEGF165 in PAE cells expressing either recombinant Np-1 or recombinant Np-2 receptors and no other types of VEGF receptors (32,
42). These observations suggested that for the transduction of VEGF
signals the neuropilins may perhaps have to associate with other
membrane-bound proteins. Neuropilins possess short intracellular
domains, and it was demonstrated that binding of sema-3A to Np-1 is not
sufficient for induction of sema-3A mediated growth cone collapse (53).
It was indeed found that neuropilins form complexes with plexin
receptors to be able to transduce semaphorin signals (43, 44). Our
binding/cross-linking experiments and co-immunoprecipitation
experiments indicate that in addition, Np-2 can form complexes with
VEGFR-1. Our experiments also suggest that Np-1 too can associate with
VEGFR-1. This was recently verified in a manuscript that was published
during the preparation of this manuscript in which complexes between
Np-1 and VEGFR-1 were observed using completely different methods
(49).
The mechanism by which VEGFR-1 enables the binding of
VEGF121 to Np-1 and Np-2 is unclear. The binding of VEGFR-1
to the neuropilins may induce a neuropilin conformation that binds
VEGF121 with increased affinity. It is possible that
VEGF121 binds initially to VEGFR-1, placing the bound
VEGF121 in close proximity to neuropilins in cells
co-expressing both receptor types and effectively increasing the
affinity of the neuropilins toward VEGF121. The effect of VEGFR-1 on VEGF121 binding may therefore be similar to the
potentiating effect that heparin-like molecules have on the binding of
VEGF165 to neuropilins (32, 33, 42).
VEGFR-1 and Np-2 may be able to form complexes prior to the addition of
VEGF. Alternatively, it is possible that VEGFR-1 binds to Np-2 only
subsequent to the binding of VEGF to VEGFR-1. We have not been able to
differentiate between these two possibilities. We have attempted to
detect co-immunoprecipitated Np-2 or VEGFR-1 using Western blot
analysis, but we have failed regardless of whether the cells were
exposed or not to VEGF prior to the immunoprecipitation. It is possible
that these experiments failed because the sensitivity of the assays was
insufficient or because the VEGFR-1·Np-2 complexes are sensitive to
the detergents used during the solubilization of the cells, making the
detection of VEGFR-1·Np-2 complexes by less sensitive techniques than
the technique we have used difficult. To circumvent these problems we
have therefore used antibodies to immunoprecipitate recombinant
receptors that have been covalently cross-linked to
125I-VEGF prior to the immunoprecipitation using a
previously described method (50). The method we used utilized the high
sensitivity afforded by the use of 125I but did not allow
us to determine whether complex formation between Np-2 and VEGFR-1 was
VEGF-dependent or not.
We have no data regarding the biological significance of VEGFR-1·Np-2
complexes at this stage. The formation of complexes between Np-2 and
VEGFR-1 may contribute to VEGF-induced signal transduction by VEGFR-1.
If that assumption is correct, then it may provide a clue to a puzzling
observation. Mice deficient in VEGFR-1 expression die before birth
because of severe cardiovascular defects (27). In contrast, mice that
retain the extracellular and trans-membrane domains of VEGFR-1 but lack
the signaling tyrosine-kinase domain develop normally (54). It is
unclear how the extracellular domain of VEGFR-1 is able to restore the
normal embryonic development of mice. It is possible that the
extracellular domain is required for VEGF sequestration, so as to limit
the activity of VEGF. Alternatively, the extracellular domain may
associate with another membrane protein to form a signaling
holo-receptor. It is possible that Np-2 and Np-1 may perhaps
participate in the formation of such a putative VEGFR-1 containing
holo-receptor.
In the course of our experiments we have found that when a
myc epitope is inserted in-frame after the conserved
SEA terminal tripeptide of Np-2a, the modified
Np-2myc receptor loses most of its VEGF165
binding ability. It was shown that Np-1 and Np-2 can form homodimers
and heterodimers (55). Formation of such dimers may perhaps be required
for high affinity binding of VEGF to neuropilins. The insertion of the
myc epitope may perhaps interfere with dimer formation and
consequently with VEGF binding. Interestingly, the VEGF165
binding ability of Np-2myc was restored to some extent in
cells co-expressing VEGFR-1, perhaps because following complex formation a high affinity VEGF-binding conformation of
Np-2myc is favored. Interestingly, the sema-3F binding
properties of Np-2 were not affected by the introduction of the
myc epitope, perhaps because the sema-3F-binding domain of
Np-2 seems to be distinct from the VEGF-binding domain as suggested by
the results of the competition experiments.
To conclude, our experiments indicate that VEGFR-1 forms complexes with
Np-2 and possibly also with Np-1. The presence of VEGFR-1 changes the
specificity of VEGF binding, allowing VEGF121 to bind to
Np-2. However, the biological function of these VEGFR-1·Np-2 complexes is still unclear. In addition our experiments indicate that
changes in the intracellular domain of Np-2 can affect VEGF binding to
Np-2 and provide evidence indicating that VEGF and semaphorins bind to
nonoverlapping sites in the extracellular part of Np-2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
VEGF121 binding to Np-1 and Np-2
is enabled in PAE cells co-expressing VEGFR-1. A, PAE
cells expressing recombinant Np-2 (lane 1), recombinant
VEGFR-1 (lane 2), or both receptors (lane 3) were
incubated with 30 ng/ml 125I-VEGF121 for 2 h at 4 °C in the presence of 1 µg/ml heparin. At the end of the
experiment, bound 125I-VEGF121 was cross-linked
to the cells using BS3 as described. Lysates of cells
containing equal amounts of protein were separated using SDS/PAGE, and
the gel was then autoradiographed as previously described (30). The
arrow points to the putative
125I-VEGF121·Np-2 complex. B, PAE
cells expressing recombinant VEGFR-1 (lane 1) or PAE cells
co-expressing recombinant Np-1 and VEGFR-1 (lane 2) were
incubated with 30 ng/ml 125I-VEGF121 for 2 h at 4 °C in the presence of 1 µg/ml heparin. At the end of the
binding, bound 125I-VEGF121 was cross-linked to
the cells. Lysates of cells containing equal amounts of protein were
separated using SDS/PAGE, and the gel was then autoradiographed as
described. The arrow points to the putative
125I-VEGF121·Np-1 complex.
View larger version (42K):
[in a new window]
Fig. 2.
Co-expression of Np-2 and VEGFR-1 in PAE
cells does not result in the production of smaller VEGFR-1
species. PAE cells expressing recombinant VEGFR-1 (lane
1), recombinant neuropilin-2 (lane 3), or both
receptors (lane 2) were lysed using lysis buffer. Extracts
were clarified by centrifugation and separated using SDS/PAGE. The
proteins were electroblotted to nitrocellulose and probed with a
commercial anti-VEGFR-1 antibody (Santa Cruz Biotechnology.). A
secondary anti-rabbit antibody coupled to peroxidase was used to detect
bound primary antibody. Bound secondary antibody was detected using the
ECL method.
View larger version (50K):
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Fig. 3.
Antibodies directed against VEGFR-1
co-immunoprecipitate a labeled complex corresponding in mass to a
125I-VEGF121·Np-2 complex. A,
125I-VEGF165 (10 ng/ml) (lane 1) or
125I-VEGF121 (30 ng/ml) (lanes 2 and
3) were bound to PAE cells expressing Np-2 (lane
1), VEGFR-1 (lane 2), or Np-2 and VEGFR-1 (lane
3) in the presence of 1 µg/ml heparin. Binding was carried out
2 h at 4 °C. Bound 125I-VEGF was cross-linked
(CL) to the cells, which were subsequently lysed as
described. Equal amounts of protein representing about 10% of the
lysates were chromatographed on a 6% SDS/PAGE gel, which was
subsequently dried and autoradiographed. B, the remaining
90% of the lysate from each of the binding/cross-linking reactions
described under A was subjected to immunoprecipitation
(IP) using an anti-VEGFR-1 antibody as described under
"Experimental Procedures." Immunoprecipitates were solubilized in
SDS/PAGE sample buffer, chromatographed on a 6% SDS/PAGE gel, and
autoradiographed. Lane 4, immunoprecipitate from a
PAE·Np-2 cell lysate to which 125I-VEGF165
was bound and cross-linked. Lane 5, immunoprecipitate from a
PAE·VEGFR-1 cell lysate to which 125I-VEGF121
was bound and cross-linked. Lane 6, immunoprecipitate from a
PAE·VEGFR-1·Np-2 cell lysate to which
125I-VEGF121 was bound and cross-linked.
View larger version (22K):
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Fig. 4.
myc epitope-tagged Np-2 looses its
VEGF165 binding ability but not its sema-3F binding
properties. A, PAE cells expressing recombinant Np-2
(lane 1), Np-2myc (lane 2), VEGFR-1
and Np-2 (lane 3), VEGFR-1 and Np-2myc
(lane 4), or VEGFR-1 alone (lane 5) were grown to
confluence in 5-cm dishes (~3 × 106 cells/dish) and
lysed. Lysates were chromatographed on a 6% SDS/PAGE gel. The amount
of lysate loaded in each lane was equivalent to 2 × 106 cells. The proteins were transferred to nitrocellulose
by electroblotting and probed with an affinity purified anti-Np-2
antibody. A secondary antibody coupled to peroxidase was used to detect
bound primary antibody. Bound secondary antibody was detected using the
ECL detection method. B, PAE cells expressing Np-1, Np-2, or
Np-2myc were grown in gelatin-coated 24-well dishes to
confluence. Increasing concentrations of
125I-VEGF165 were bound in duplicates to the
cells in the presence of 1 µg/ml heparin at 4 °C for 2 h as
described to determine total binding. Nonspecific binding was measured
in the presence of 1 µg/ml VEGF165. At the end of the
binding, the cells were washed three times with PBS containing 1 mg/ml
bovine serum albumin and lysed by the addition of 0.5 ml of 0.5 N NaOH. Aliquots of 0.4 ml were then counted in a
counter. Nonspecific binding was subtracted from total binding to
calculate specific binding values. The nonspecific binding did not
exceed 15% of the total binding values. The experiment was repeated
twice, and the error bars represent the standard deviations
from the mean. C, control PAE cells transfected with the
pBabePuro plasmid (42) as well as PAE cells expressing recombinant
Np-2, Np-2myc, VEGFR-1 and Np-2, or VEGFR-1 and
Np-2myc were grown to confluence in gelatin-coated 48-well
dishes. The cells were incubated with conditioned medium containing
AP-tagged sema-3F as described. Following washing, heat-resistant
alkaline phosphatase activity of cell bound sema-3F was measured as
described. Binding to pBabe-puro vector transfected PAE cells was 0.08 optical density units (O.D.) and was subtracted from the total
binding values to calculate the specific binding values shown. The
experiment was repeated twice in triplicate with similar results, and
the error bars represent the standard deviations from the
mean.
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Fig. 5.
VEGF165 does not inhibit the
binding of sema-3F to Np-2. PAE cells expressing Np-2, VEGFR-1, or
VEGFR-1 and Np-2 as well as control PAE cells transfected with the
pBabePuro plasmid (42) were grown in gelatin-coated 48-well dishes to
confluence. The cells were incubated with conditioned medium containing
AP-tagged sema-3F at room temperature for 3 h. The binding was
performed in the absence (black columns) or the presence of
2 µg/ml VEGF165 (open columns). The cells were
then washed and assayed for bound heat stable AP activity as described.
The experiment was repeated twice in triplicate with similar results.
The error bars represent the standard deviations from the
mean. O.D., optical density units.
View larger version (39K):
[in a new window]
Fig. 6.
An antibody directed against the
myc epitope co-immunoprecipitates VEGFR-1 from cells
co-expressing Np-2myc and VEGFR-1. A,
PAE cells expressing Np-2myc (lane 1), VEGFR-1
(lane 3), or Np-2myc and VEGFR-1 together
(lane 2) were grown to confluence in 6-cm dishes.
125I-VEGF165 (10 ng/ml) was bound to the cells
in the presence of 1 µg/ml heparin for 2 h at 4 °C. Bound
125I-VEGF was then cross-linked to the cells. The cells
were subsequently lysed. Equal amounts of protein were chromatographed
on a 6% SDS/PAGE gel, which was subsequently dried and
autoradiographed. B, PAE cells expressing VEGFR-1
(lane 1), Np-2 (lane 5), Np-2myc
(lane 4), Np-2 and VEGFR-1 (lane 2), or
Np-2myc and VEGFR-1 (lane 3) were grown to
confluence in 10-cm dishes. 125I-VEGF165 (10 ng/ml) was bound to the cells for 2 h at 4 °C in the presence
of 1 µg/ml heparin. At the end of the experiment, bound
125I-VEGF was cross-linked to the cells using
BS3 as described. Following cross-linking, the cells were
lysed, and immunoprecipitation was carried out using
anti-myc antibodies as described under
"Experimental Procedures." Precipitated 125I-labeled
complexes were solubilized using SDS/PAGE sample buffer and
chromatographed on a 6% SDS/PAGE gel, and the gel was then dried and
autoradiographed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by a grant from the Israel Academy of Sciences (to G. N.).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
972-4-8294216; Fax: 972-4-8225153, E-mail:
gera@techunix.technion.ac.il.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M006909200
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; BS3, bis(sulfosuccinimidyl) suberate; VEGFR-1, vascular endothelial growth factor receptor-1; VEGFR-2, vascular endothelial growth factor receptor-2; PAGE, polyacrylamide gel electrophoresis; VEGF165, 165-amino acid form of vascular endothelial growth factor; VEGF121, 121-amino acid form of vascular endothelial growth factor; Np-1, neuropilin-1; Np-2, neuropilin-2; PAE, porcine aortic endothelial cells; sema, semaphorin; PlGF, placenta growth factor; PBS, phosphate-buffered saline; AP, alkaline phosphatase..
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