From the Cardiology Division, Department of Medicine,
and the Department of Pathology, Albert Einstein College of Medicine,
Bronx, New York 10461 and § Sibtech,
Newington, Connecticut 06111
Received for publication, January 26, 2001, and in revised form, March 9, 2001
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ABSTRACT |
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The vascular endothelial growth
factor (VEGF) receptor tyrosine kinase subtype kinase insert domain
receptor (KDR) contains seven extracellular Ig-like domains, of
which the three most amino-terminal contain the necessary structural
features required for VEGF binding. To clarify the functional role of
KDR Ig-like domains 4-7, we compared VEGF-induced signaling in human
embryonic kidney and porcine aortic endothelial cells expressing native
versus mutant receptor proteins in which Ig-like domains
4-7, 4-6, or 7 had been deleted. Western blotting using an
anti-receptor antibody indicated equivalent expression levels for each
of the recombinant proteins. As expected, VEGF treatment robustly
augmented native receptor autophosphorylation. In contrast, receptor
autophosphorylation, as well as downstream signaling events, were
VEGF-independent for cells expressing mutant receptors.
125I-VEGF165 bound with equal or better
affinity to mutant versus native receptor, although the
number of radioligand binding sites was significantly reduced because a
significant percentage of mutant, but not native, receptors were
localized to the cell interior. As was the case for native KDR,
125I-VEGF165 binding to the mutant receptors
was dependent upon cell surface heparan sulfate proteoglycans, and
125I-VEGF121 bound with an affinity equal to
that of 125I-VEGF165 to the native and mutant
receptors. It is concluded that KDR Ig-like domains 4-7 contain
structural features that inhibit receptor signaling by a mechanism that
is independent of neuropilin-1 and heparan sulfate proteoglycans. We
speculate that this provides a cellular mechanism for blocking unwanted signaling events in the absence of VEGF.
Vascular endothelial growth factor
(VEGF)1 has for a number of
reasons received attention as a key angiogenesis activator (for review,
see Ref. 1). Its expression correlates both temporally and spatially
with the onset of angiogenesis in several normal and pathophysiological
situations (2-5), VEGF elicits a strong angiogenic response in a
variety of in vivo experimental models (6, 7), and its
actions are largely restricted to vascular endothelial cells (8, 9). An
essential role for VEGF in tumor angiogenesis and ischemia-related
retinal disorders has been demonstrated by the findings that
neutralizing VEGF antibodies or dominant-negative VEGF receptors
inhibit both angiogenesis and the progression of these diseases
(10-12).
VEGF exhibits high affinity binding to two distinct endothelial cell
receptor tyrosine kinases, the fms-like tyrosine kinase Flt1 (13, 14)
and the kinase insert domain containing receptor KDR (15, 16). Both
receptors possess insert sequences within their catalytic domains and
seven immunoglobulin-like domains in the extracellular regions, and
they are related to the PDGF family of receptor tyrosine kinases.
Although expression of both VEGF receptor types occurs in adult
endothelial cells including human umbilical vein endothelial cells,
recent findings suggest that KDR and not Flt-1 is able to mediate the
mitogenic and chemotactic effects of VEGF (17, 18). VEGF binding to KDR
stimulates other cellular responses including enhancement of expression
of matrix-degrading enzymes (19), inhibition of apoptosis (20), and
regulation of nitric-oxide synthase expression (21). A number of cell
signaling proteins that participate in the diverse biological functions of VEGF have been identified, including NCK, PLC The very earliest steps in KDR-mediated signal transduction, and the
KDR structural features that allow those steps, are just starting to be
clarified. Based upon the wealth of experimental results obtained using
other receptor tyrosine kinases, it is generally accepted that the
molecular mechanism by which VEGF initiates signaling is through
receptor dimerization followed by receptor autophosphorylation (for
review, see Ref. 24). VEGF is a covalent dimer, and part of the
mechanism by which it causes KDR dimerization is by binding two
receptor monomers simultaneously (25-27). KIT and PDGF receptors are
growth factor receptors structurally similar to KDR, and in addition to
the interactions between ligand and receptor, receptor-receptor
interactions are necessary for receptor dimerization (28, 29). The
fourth Ig-like domains of these receptors mediate the receptor-receptor
interactions. Whether receptor-receptor interactions are necessary for
KDR dimerization is not known.
There are other reasons for suspecting that the extracellular domain of
KDR contains structural features that participate in receptor
activities that are independent of ligand binding. VEGF is expressed as
five alternatively spliced isoforms (30, 31), and VEGF165
but not VEGF121 binding to KDR requires two other cell
surface molecules; these are heparan sulfate proteoglycans (HSPGs) (32,
33) and neuropilin-1 (34). Although HSPG and neuropilin-1 interactions
with VEGF165 participate in this response, an interaction
with KDR has not been ruled out. If this were the case, then it may be
expected that deletion of the relevant KDR sequences would alter
VEGF165 binding properties.
In this study we investigated the consequence of deleting amino acid
sequences contained within KDR Ig-like domains 4-7 on receptor
function. The experimental results led to the conclusion that this
region contains structural features that inhibit receptor dimerization
and signaling, and the binding of VEGF to KDR relieves that inhibition,
allowing for receptor activation. We propose that this mechanism acts
to prevent unwanted receptor dimerization in the absence of growth factor.
Cell Culture--
Transfected porcine aortic endothelial (PAE)
and human embryonic kidney epithelial (HEK293) cells were cultured in
DMEM containing 10% newborn calf serum, 0.4 µg/ml puromycin, 10 units/ml penicillin, and 10 µg/ml streptomycin.
Antibodies--
Rabbit anti-KDR antibody was isolated in our
laboratory and targets a polypeptide sequence within the KDR cytosolic
domain (35). Anti-NCK, anti-PLC Site-directed Mutagenesis of the KDR cDNA--
The
experimental strategy for constructing mutant KDR cDNAs involved
the introduction of NotI restriction sites in both the region of KDR between Ig-like domain 7 and the membrane-spanning domain
(nucleotides 2195-2202) and the regions between either Ig-like domain
3 and 4 (nucleotides 968-975) or Ig-like domains 6 and 7 (nucleotides
1961-1968). Mutagenesis was done using the native KDR cDNA cloned
into an expression vector (36) and a U.S.E. mutagenesis kit (Amersham
Pharmacia Biotech.). The resulting plasmids were digested with
NotI and religated. All mutations were confirmed by DNA
sequencing. The resulting vectors were transfected into HEK293 and PAE
cells, and stable transfectants were selected and expanded as described
previously (35).
Western Blotting--
For examination of the effect of VEGF on
receptor autophosphorylation, and NCK and PLC Iodination of VEGF121--
1 µg of
VEGF121 (R & D Systems) was added to a reaction buffer
containing 20 mM sodium phosphate buffer, pH 7.2, and 0.5 mCi of Na125I. The final volume was 120 µl. To this
mixture were added 8 µg of freshly dissolved chloramine T, and the
reaction was allowed to proceed for 45 s at room temperature. The
reaction was quenched by the addition of 50 µl of sodium
metabisulfite (2 mg/ml) and 50 µl of 10 mM KI. The
solution was then centrifuged through a Sepharose G-25 spin column
twice, and 30 µl of 1% gelatin was added. A small aliquot of the
sample was precipitated with trichloroacetic acid to ensure the
complete removal of unincorporated radiolabel.
125I-VEGF Binding Assay--
Transfected cells were
grown until confluent on 24-well (4 × 105 cells/well)
cell culture plates. The cells were washed twice with PBS, and 0.2 ml
of DMEM containing 0.15% gelatin and 25 mM HEPES, pH 7.4, was added. 125I-VEGF165 (Amersham Pharmacia
Biotech) or 125I-VEGF121 was added at
concentrations ranging from 1 to 2,000 pM, and 10 mM VEGF and other additions were made to appropriate dishes, thus adjusting the final volume to 0.25 ml. Cells were incubated for either 1 h at room temperature or 4 h at
4 °C. A 50-µl sample of the medium for each well was taken to
determine the concentration of free radioligand, and the cells were
washed three times with ice-cold PBS containing 0.1% BSA. The cells
were extracted from the dishes by incubating them for 30 min with 1% Triton X-100 in 100 mM sodium phosphate, pH 8.0, and
radioactivity of the extracts was determined in a gamma counter.
Specific 125I-VEGF binding is defined as the difference
between the presence and absence of 10 nM nonradioactive
VEGF. Duplicate samples were analyzed. Binding affinities were
calculated by Scatchard (37) analysis.
Immunostaining Assay--
Transfected PAE cells were plated on
eight-well Permanox plastic chamber slides coated with 0.2% gelatin
and grown until 70% confluent. 50 ng/ml VEGF165 was added
to certain wells for 20 min. The cells were washed twice with PBS and
then fixed in 3.5% formaldehyde in PBS for 10 min at room temperature.
The cells were incubated with Hanks' solution containing 1% BSA and
0.1% glycine for 5 min at room temperature, and the solution was
aspirated; this step was repeated. Cells were incubated with Hanks',
0.1% Triton X-100, and 1% BSA for 10 min and then washed in Hanks' and 1% BSA three times. Blocking buffer (Hanks', 1% BSA, and 10% goat serum) was added, and the cells were incubated for 1 h.
Anti-KDR or anti-Tyr(P) antibodies were added to appropriate
coverslips, which were incubated for 1 h at room temperature. The
slides were washed three times with Hanks and 1% BSA followed by a
90-min incubation with secondary Cy-3-conjugated goat anti-mouse or
fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies (1:40
dilution) (Jackson Laboratories). After washing three times with
Hanks' and 1% BSA, cells were viewed under a Nikon fluorescent microscope.
VEGF-independent Signaling in KDR(Ig1-3)-, KDR(Ig1-6)-, and
KDR(Ig1-3, 7)-expressing HEK293 and PAE Cells--
The KIT and PDGF
receptor tyrosine kinases contain structural features adjacent to the
ligand binding domains which are necessary for receptor dimerization
(28, 29), and their deletion prevents both ligand binding and
ligand-induced signaling. In an attempt to determine if complementary
domains within KDR function in a similar manner, three mutant receptor
(Fig. 1) cDNA constructs were made in
which either Ig-like domains 4-7 (KDR(Ig1-3)), Ig-like domain 7 (KDR(Ig1-6)), or Ig-like domains 4-6 (KDR(Ig1-3, 7)) were deleted.
KDR and KDR(Ig1-3) were expressed in HEK293 cells, and KDR,
KDR(Ig1-3), KDR(Ig1-6), and KDR(Ig1-3, 7) were expressed in PAE
cells. Analysis of Western blots of cell extracts using an anti-KDR
antibody reveals the expression of native and mutant receptor protein
in these cells at the expected sizes (Fig.
2).
The ability of VEGF to stimulate receptor autophosphorylation in cells
expressing native and mutant receptors was then examined (Fig.
3). The protocol for this experiment
involved treating cells with or without 50 ng/ml VEGF,
immunoprecipitation with anti-Tyr(P) antibodies, and immunoblotting
with the anti-KDR antibody. For HEK293 and PAE cells expressing native
KDR, there is no tyrosine phosphorylation of receptor in the absence of
VEGF, and growth factor treatment induces a robust response (Fig. 3,
A and B). Unexpectedly, KDR(Ig1-3), KDR(Ig1-6),
and KDR(Ig1-3, 7) are heavily phosphorylated in control cells, and
VEGF does not enhance that phosphorylation (Fig. 3, A,
C, and D). For KDR(Ig1-3)-expressing cells, VEGF
treatment results in some experiments in a decrease in the level of
receptor phosphorylation (Fig. 3A); the reason for this is
not known. For the experiment shown in Fig. 3C, two molecular weight proteins are visualized in samples prepared from KDR(Ig1-3)-expressing PAE cells; this was not observed for all experiments. The structural differences between these proteins are not
known, although they could represent receptor degradation or receptor
proteins containing different degrees of tyrosine phosphorylation.
Receptor autophosphorylation allows for the recruitment of cellular
signaling proteins to the receptor and subsequent signal transduction.
We therefore tested whether cell signaling pathways that are stimulated
in endothelial cells by VEGF are activated in the mutant
KDR-transfected cells in the absence of growth factor. Transfected
HEK293 cells were incubated with and without VEGF, cell extracts were
immunoprecipitated with anti-Tyr(P) antibodies, and immunoblotting was
done using antibodies to two signaling proteins (NCK and PLC
As shown in Fig. 5, the activation of
more downstream signaling events is also independent of VEGF in cells
transfected with mutant receptors. VEGF-induced assembly of focal
adhesions plays a necessary role in the signaling pathway leading to
endothelial cell migration (38). Fig. 5, A and B,
demonstrates that this effect is observed in KDR-transfected PAE cells
because VEGF stimulates the number and increases the size of focal
adhesions as visualized by immunofluorescent staining using the
anti-Tyr(P) antibody. In the absence of VEGF treatment, both the number
and size of focal adhesion complexes in KDR(Ig1-3)-expressing cells
are identical to that seen for VEGF-treated KDR-expressing cells (Fig.
5C). VEGF had no effect on focal adhesion complex assembly
in cells expressing KDR(Ig1-3) (data not shown).
125I-VEGF165 Binding to Native and Mutant
Receptors--
The region of KDR which is involved in direct VEGF
binding has been mapped to Ig-like domains 1-3 (39). To determine
whether amino acid sequences in Ig-like domains 4-7 can regulate
growth factor binding, we performed
125I-VEGF165 radioligand binding assays to
cells expressing native and mutant receptors. KDR-expressing HEK293 and
PAE cells exhibit a high number of specific
125I-VEGF165 binding sites (Fig.
6). The affinity of binding to
transfected HEK293 cells was 85 pM (Fig. 6A),
which is similar to that which has been reported for endothelial cells
expressing endogenous KDR (40, 41). The affinity of binding to
transfected PAE cells is significantly weaker (1,200 pM)
(Fig. 6B). This result was observed for several individual
expanded cell lines derived from two separate transfection protocols
(data not shown). The molecular reasons why KDR expressed in these
cells exhibits this weak affinity is not clear, although other
investigators have made similar observations (42).
125I-VEGF165 binds to KDR(Ig1-3)-expressing
HEK293 cells with an affinity similar to that observed for native
receptor (Fig. 6A). In contrast,
125I-VEGF165 binds to KDR(Ig1-3)- and
KDR(Ig1-6)-expressing PAE cells with affinities that are significantly
greater than that observed for native receptor (Fig. 6B).
Radioligand bound to KDR(Ig1-3) with a greater affinity than to
KDR(Ig1-6), and at an affinity that is similar to that observed for
the KDR(Ig1-3)- and KDR-transfected HEK293 cells. In addition, the
number of 125I-VEGF165 binding sites is reduced
significantly in PAE cells expressing mutant, versus native,
receptor, a result that was found when analyzing several separate
stably transfected cell lines.
A comparison of the results shown in Figs. 2 and 6 indicates that
although the number of mutant KDR proteins expressed in PAE cells is
similar to that for native KDR proteins, the number of
125I-VEGF165 binding sites is significantly
less for cells expressing mutant receptor. It is known that
VEGF165 stimulates the internalization of KDR in
endothelial cells by a mechanism that requires receptor autophosphorylation (35). In view of the finding that KDR(Ig1-3) autophosphorylation and signaling are VEGF-independent (Figs. 3, 4, and
5), we reasoned that KDR(Ig1-3) internalization may also be
VEGF-independent. This would account for the decreased number of
125I-VEGF165 binding sites in cells expressing
mutant receptor because the radioligand does not penetrate the cell
membrane and only binds to surface receptors.
This hypothesis was tested using immunofluorescence protocols to
examine receptor distribution in KDR- and KDR(Ig1-3)-expressing PAE
cells. In cells expressing native receptor, there is weak KDR staining
in the cell cytoplasm, and the cell periphery is clearly outlined,
suggesting cell surface receptor (Fig.
7A). In contrast, staining of
KDR(Ig1-3)-expressing cells indicates an intense spotty receptor
distribution within the cytoplasm, and the line of fluorescence marking
the cell periphery is not visible (Fig. 7B). These results
are consistent with the hypothesis that the low number of
125I-VEGF165 binding sites in cells expressing
mutant receptor is because these receptors are contained in the
distinct domains within the cell interior and a relatively less number
at the cell surface.
KDR(Ig1-3) Dimerization Is VEGF-independent--
Receptor
tyrosine kinase dimerization is the earliest step in the signaling
pathway by which growth factors activate cells. In view of the finding
that KDR(Ig1-3) autophosphorylation and signaling are observed in the
absence of VEGF treatment, we asked whether KDR(Ig1-3) might exist
constitutively in the dimeric form. VEGF-treated and control KDR- and
KDR(Ig1-3)-expressing HEK293 cells were solubilized and then subjected
to cross-linking analysis using disuccinimidyl suberate. The
solubilization step was included because of the results indicating that
the majority of KDR(Ig1-3) is internal.
The results from affinity cross-linking KDR-expressing cells did not
reveal the presence of covalent receptor dimers, even after VEGF
treatment (Fig. 8, lanes
1-4). There may be two reasons for receptor dimers not being
observed in the presence of growth factor. First, the
SDS-polyacrylamide gels were run in the presence of 2-mercaptoethanol,
and so noncovalent receptor dimers will have dissociated during sample
preparation. Second, the inefficiency of the cross-linking step may
prevent detection of covalently dimerized receptors.
The results were very different for KDR(Ig1-3)-expressing cells, where
it was found that greater than 50% of receptors were cross-linked,
even in the absence of growth factor (Fig. 8, lanes 7 and
8). These results suggest that deletion of Ig-like domains 4-7 induces activation of receptor tyrosine kinase activity by allowing receptor dimerization in the absence of VEGF binding.
Requirement of Cell Surface HSPGs for KDR Dimerization--
It is
concluded from the data described above that Ig domains 4-7 contain
structural features that inhibit KDR autophosphorylation and signaling,
and deletion of this domain relieves that inhibition. We hypothesize
further that the molecular mechanism by which VEGF activates KDR is by
relieving the inhibitory action of Ig domains 4-7.
The binding of VEGF165 to KDR is dependent upon HSPGs (32,
33). Although it is generally thought that this effect is mediated by
an interaction of HSPG with growth factor, a necessary interaction of
HSPG with KDR for receptor dimerization is possible. A heparin binding
domain has been identified between KDR Ig domains 6 and 7, and it was
suggested that interactions of HSPG with this domain might facilitate
VEGF binding and receptor dimerization (33). This hypothesis is made
more reasonable by reports demonstrating that HSPG interactions with
both bFGF and its receptor participate in growth factor binding (43,
44). It was proposed that the functional role for HSPG binding domains
on the bFGF receptor is to prevent receptor dimerization in the absence
of growth factor and that HSPG binding to these domains facilitates
bFGF-induced signaling (44).
Experiments were designed to test the hypothesis that HSPG interactions
with KDR participate in relieving the inhibition of signaling caused by
Ig-like domains 4-7, thereby facilitating the ability of VEGF to
activate KDR autophosphorylation and cell signaling. Two strategies
were taken. First, we tested whether heparin is required for
125I-VEGF121 binding to KDR-expressing cells.
The rationale for this was that if HSPG functions through an
interaction with KDR, then HSPGs will be required for both
VEGF121 and VEGF165 binding to receptor.
Second, we asked whether HSPGs are required for
125I-VEGF165 binding to KDR(Ig1-3)- and
KDR(Ig1-6)-expressing cells. The rationale for this was that if HSPG
binding to Ig-domains 4-7 participates in relieving an inhibitory
effect, then deletion of these domains would remove the need for HSPG
in 125I-VEGF165 binding.
A preliminary experiment was done to compare the interaction of
VEGF121 versus VEGF165 with KDR and
mutant KDR-expressing cells. VEGF121 treatment of
KDR-expressing HEK293 (Fig.
9A) and PAE (data not shown)
cells results in receptor autophosphorylation at a degree equivalent to
that observed using VEGF165.
125I-VEGF121 bound to KDR- and
KDR(Ig1-3)-expressing HEK293 (data not shown) and PAE (Fig.
9B) with an affinity similar to that seen for
125I-VEGF165 (compare Fig. 9B with
Fig. 6B).
Commercial heparin can substitute for cellular HSPGs in allowing for
VEGF165 binding to KDR, although an effect of heparin on
125I-VEGF165 binding is maximal only when HSPGs
are depleted (32). We took the strategy of blocking HSPG biosynthesis
by treating cells with chlorate, an inhibitor of ATP sulfurylase and
hence of the production of phosphoadenosine phosphosulfate, the active sulfate donor for sulfotransferases (45). Chlorate abolishes sulfation
on proteins and carbohydrate residues in cells without inhibiting
protein synthesis or cell growth (46).
KDR-expressing HEK293 cells were cultured to 40% confluence on 10-cm
dishes, and 70 mM chlorate was added for 24 h. The
cells were then plated on 24-well dishes and cultured for 24 h in
medium containing 70 mM chlorate.
125I-VEGF165-radioligand binding indicated that
the cells lose 75% of cell surface binding sites compared with control
cells not incubated with inhibitor (Fig.
10A). The addition of
heparin restored radioligand binding to the chlorate-treated cells. In
contrast, chlorate treatment had no effect on
125I-VEGF121 binding to these cells (Fig.
10B), indicating that HSPGs are not required for binding of
this isoform.
These results argue against the hypothesis that HSPGs function to
interact with sites within Ig-like domains 4-7 and argue that HSPGs do
not participate in relieving the inhibitory actions of these domains.
This conclusion is supported by the results of testing for an HSPG
requirement in 125I-VEGF165 binding to cells
expressing KDR(Ig1-3) (Fig. 10C). As observed for cells
expressing native receptor, chlorate treatment inhibited radioligand
binding to mutant receptor, and heparin restored the binding. Heparin
had no effect on 125I-VEGF121 binding to
KDR(Ig1-3) (Fig. 10D).
The accepted model for activation of receptor tyrosine kinases is
that ligand-induced receptor dimerization juxtaposes the cytoplasmic
tyrosine kinase domains, resulting in trans-phosphorylation of target
amino acids and initiating signaling cascades. Our results indicate
that there are further molecular interactions that regulate growth
factor-induced receptor activation. More specifically, for KDR there
are structural features in the extracellular domain which prevent
unwanted receptor activation in the absence of VEGF binding.
Fig. 11 shows two molecular mechanisms
that may mediate this effect. Model A predicts that in the absence of
growth factor, there are structural features within the KDR
extracellular domain which repel receptor-receptor interactions, thus
preventing receptor dimerization. The binding of VEGF to KDR
neutralizes these structural features, either by causing a
conformational change in the receptor or by VEGF-KDR interactions, thus
allowing for receptor dimerization. Model B predicts that in the
absence of VEGF, the three-dimensional structure of the KDR monomer is
such that interactions between monomers will not allow the catalytic
domains to come close enough together to allow for
trans-phosphorylation. VEGF causes a conformational change in the
receptor monomers so that necessary structural features within the
catalytic domain become sufficiently juxtaposed to allow
trans-phosphorylation. Both of these models would be consistent with
our results showing that deletion of KDR Ig-like domains 4-7 leads to
VEGF-independent receptor activation. For model A, the mutant receptors
no longer contain the structural features that repel receptor-receptor
interactions, thus allowing for receptor signaling. For model B, the
mutant receptors no longer contain the conformational constraints that
prevent the catalytic domains to juxtapose, and so the receptor would
be active.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
mitogen-activated protein kinase, phosphoinositide 3-kinase,
focal adhesion kinase, and paxillin (22, 23).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and anti-phosphotyrosine (PY20)
monoclonal antibodies were from Transduction Laboratories.
Peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse
immunoglobulins were from Amersham Pharmacia Biotech.
tyrosine
phosphorylation, transfected HEK293 and PAE cells were grown on 10-cm
dishes until subconfluent. The cells were incubated in serum-free DMEM
with 1 mM Na3VO4 for 1 h at
4 °C; 50 ng/ml VEGF was then added for 4 h at 4 °C. The cells were scraped from the dishes, pelleted, and suspended in 2 ml of
lysis buffer (10 mM Tris-HCl, pH 8.0, 50 mM
NaCl, 0.2 mM Na3VO4, 30 mM Na4P2O7, 30 mM NaF, 1 mM EDTA, 1 µg/ml leupeptin, 0.7 µg/ml pepstatin A, and 1% Triton X-100). After a 1-h incubation at
4 °C, the lysates were pelleted, and anti-Tyr(P) was added to the
supernatant. The samples were incubated for 4 h at 4 °C, and
the antibodies were then immobilized on protein A-Sepharose beads
(Sigma). The beads were washed five times with lysis buffer and boiled
in 40 µl of SDS-polyacrylamide gel electrophoresis sample loading
buffer. Proteins were separated on SDS-polyacrylamide gels and
transferred to nitrocellulose (Bio-Rad). The filters were then blocked
in Tris-buffered saline containing 0.2% Tween 20 plus 5% dry milk at
4 °C overnight and probed with specific antibodies for 4 h at
room temperature. After washing, the filters were incubated with
horseradish peroxidase-linked anti-rabbit or anti-mouse IgG. The
reactions were visualized through enhanced chemiluminescence (ECL;
Amersham Pharmacia Biotech).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic illustration of wild-type and
altered KDR. Ig-like domains 4-7, 7, or 4-6 were deleted to
generate the deletion mutants KDR(Ig1-3), KDR(Ig1-6), and KDR(Ig1-3,
7), respectively. The mutagenesis strategy involved the introduction of
NotI restriction sites into wild-type KDR cDNA, and
NotI indicates the mutagenesis sites in the figure.
TM indicates the transmembrane domain.
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Fig. 2.
Expression of native and mutant receptors in
HEK293 and PAE cells. Transfected HEK293 (panel A) or
PAE (panel B) cells were grown to confluence on 10-cm cell
culture dishes. The cells were lysed, anti-KDR immunoprecipitates were
prepared, and immunoblotting was done using anti-KDR.
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Fig. 3.
Effect of VEGF on native and mutant KDR
autophosphorylation. Transfected HEK293 (panel A) or
PAE (panels B-D) were grown to confluence on 10-cm dishes.
All subsequent steps were done at 4 °C. The medium was replaced with
3 ml of serum-free DMEM containing 1 mM
Na3VO4 plus 20 mM HEPES, pH 7.4. After 1 h, 50 ng/ml VEGF165 was added to certain
dishes for 4 h. The cells were lysed, anti-Tyr(P)
immunoprecipitates prepared, and immunoblotting was done using
anti-KDR.
) that
are activated by growth factor in endothelial cells (22) (Fig.
4). Neither PLC
(Fig. 4A)
nor NCK (Fig. 4B) is tyrosine phosphorylated in
KDR-expressing cells, and VEGF stimulates a robust response. For
KDR(Ig1-3)-expressing cells, both of the signaling proteins are
phosphorylated in the absence of growth factor. As is found for
KDR(Ig1-3) autophosphorylation (Fig. 3A), VEGF treatment
decreases the level of tyrosine phosphorylation of both NCK and PLC
;
the mechanisms that account for this are not known.
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Fig. 4.
NCK and PLC tyrosine
phosphorylation is VEGF-independent for KDR(Ig1-3)-expressing HEK293
cells. This experiment was done exactly as described for Fig. 3
except that immunoblotting was done using anti-PLC
(panel
A) or anti-NCK (panel B).
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Fig. 5.
Focal adhesion complex assembly is
VEGF-independent for KDR(Ig1-3)-expressing PAE cells. KDR
(panels A and B)- and KDR(Ig1-3) (panel
C)-expressing PAE cells were plated on eight-well Permanox plastic
chamber slides coated with 0.2% gelatin and grown until they were
confluent. Cells were either untreated (panels A and
C) or treated with 50 ng/ml VEGF (panel B) for 20 min. Cells were fixed, permeabilized, and incubated with anti-Tyr(P)
antibody. Immmunofluorescent staining was performed as described under
"Experimental Procedures." The results shown are representative of
at least 10 different fields observed in each experiment and of three
similar independent experiments.
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Fig. 6.
125I-VEGF165
binding to native and mutant KDR-expressing
cells. Transfected HEK293 (panel A) or PAE (panel
B) cells were grown to confluence on 24-well dishes. Radioligand
binding was done using serum-free medium containing 0.15% gelatin, 20 mM HEPES, pH 7.4, and 125I-VEGF165
(1-2,000 pM). 10 nM nonradioactive
VEGF165 was added to certain wells to define nonspecific
binding. After a 1-h incubation at room temperature, an aliquot of the
medium was removed to calculate total radioactivity, and the remaining
medium was aspirated. The wells were washed three times with ice-cold
PBS, and the cells were lysed in PBS containing 1% Triton X-100. The
samples were transferred to gamma counting tubes and radioactivity
determined. The data were analyzed as described (37).
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Fig. 7.
KDR and KDR(Ig1-3) distribution in KDR- and
KDR(Ig1-3)-expressing PAE cells. KDR (panel A)- and
KDR(Ig1-3) (panel B)-expressing PAE cells were plated on
eight-well Permanox plastic chamber slides coated with 0.2% gelatin
and grown until they were confluent. Cells were fixed, permeabilized,
and incubated with anti-KDR antibody. Immmunofluorescent staining was
performed as described under "Experimental Procedures." The results
shown are representative of at least 10 different fields observed in
each experiment and of three similar independent experiments.
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Fig. 8.
KDR(Ig1-3) dimerization is
VEGF-independent. KDR (lanes 1-4)- and KDR(Ig1-3)
(lanes 5-8)-expressing HEK293 cells were grown on six-well
dishes until confluent. The medium was replaced with serum-free DMEM
containing 1 mM Na3VO4, and the
cells were incubated 1 h at 4 °C. 50 ng/ml VEGF was added to
certain wells (lanes 2, 4, 6, and
8), and all wells were incubated 4 h at 4 °C. The
medium was aspirated, and the cells were washed two times with ice-cold
PBS. The cells were scraped from the wells, pelleted, and suspended in
200 µl of PBS containing 0.5% Triton X-100. 15 min later, 1 mM disuccinimidyl suberate (DSS) was added to
some samples (lanes 3, 4, 7, and
8), and all samples were incubated at room temperature for
20 min. Gel sample buffer was added, and the samples were boiled for 5 min. Immunoblotting was done using anti-KDR antibody.
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Fig. 9.
125I-VEGF121
binding to transfected cells and its effect on
receptor autophosphorylation. Panel A shows the effects
of VEGF165 and VEGF121 on receptor
autophosphorylation in KDR-expressing HEK293 cells. The experiment was
done exactly as described for Fig. 3. Panel B shows the
results of 125I-VEGF121 binding to KDR- and
KDR(Ig1-3)-expressing PAE cells. The experiment was done exactly as
described for Fig. 6.
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Fig. 10.
Effect of chlorate treatment on
125I-VEGF165 and
125I-VEGF121 binding to
KDR- and KDR(Ig1-3)-transfected HEK293 cells. KDR (panels
A and B) and KDR(Ig1-3) (panels C and
D) were cultured until 40% confluent on 10-cm dishes. 70 mM chlorate was then added to certain dishes, and the cells
were cultured for 24 h. The cells were replated on 24-well dishes
in medium containing 70 mM chlorate and cultured for
24 h. Control cells were prepared in an identical manner, but
chlorate was not added. 125I-VEGF165
(panels A and C) and
125I-VEGF121 (panels B and
D) binding was done as described in Fig. 6. 10 µg/ml
heparin was added to certain samples as indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Hypothetical models for how KDR Ig-like
domains 4-7 might block receptor signaling in the absence of
VEGF. Details of the models are described under
"Discussion."
Model A predicts that there are specific peptide sequences within the KDR extracellular domain which prevent VEGF-independent signaling. Model B, on the other hand, predicts that the three-dimensional structure of Ig-like domains 4-7 is important, and so mutations throughout this region would most likely disrupt the inhibitory effect. Therefore, our findings are more consistent with model B because all three mutant receptors showed VEGF-independent receptor activation.
The proposed mechanism by which VEGF stimulates KDR is similar to how certain nonreceptor kinases (e.g. protein kinase A and protein kinase C) are activated by their substrates. Both of these kinases contain regulatory and catalytic domains, and binding of either cAMP or diacylglycerol to the regulatory domains causes a conformational change in the protein allowing for catalytic activity (47, 48). Our experimental results indicate that VEGF activates KDR in an analogous manner. This conclusion is strengthened by the fact that separation of the regulatory domain from the catalytic domain of protein kinases A and C, either by expression of recombinant catalytic domains (49, 50), or in the case of protein kinase C, proteolytic digestion (51), leads to substrate-independent kinase activity. In an analogous manner, a recombinant KDR cytosolic domain is catalytically active (52). There are most likely differences in the molecular interactions by which KDR versus the other protein kinases are activated by substrate, as the conformational changes in the regulatory domains caused by cAMP and diacylglycerol allow for the exposure of ATP-binding peptides within the catalytic domains (53, 54). It is more difficult to envision a similar mechanism for VEGF activation of KDR because VEGF and ATP binding to KDR occur on opposite sides of the cell membrane.
Two other receptor tyrosine kinases also contain structural features within their extracellular domains which function to block ligand-independent receptor dimerization. A mutant PDGFA receptor lacking Ig-like domain 3 results in growth factor-independent receptor dimerization and activation (55), and an Ig-like domain contained within the extracellular domain of the TrkA receptor serves a similar function (56). Interestingly, the structural features of the PDGF receptor A and TrkA receptors that participate in these effects are contained within the receptor's ligand binding domains. This is different from that seen for KDR because the constitutively active receptor mutants maintain 125I-VEGF binding activity.
PDGF receptor and cKIT are receptor-like tyrosine kinases that contain five extracellular Ig-like domains. Each of these receptors contains structural features within their fourth Ig-like domain which participate in receptor-receptor interactions and are required for receptor dimerization. The deletion of these domains blocks both high affinity growth factor binding as well as receptor signaling. The results shown in Figs. 4, 5, and 6 demonstrating that deletions of KDR Ig-like domains 4-7 have no effect on 125I-VEGF binding and allow for KDR activation, would indicate that this structural feature is not contained within the KDR Ig-like domains 4-7. We cannot rule out that KDR Ig-like domains 1-3 contain structural features that facilitate receptor-receptor interactions, and the results of Fig. 8 demonstrating VEGF-independent dimerization of KDR(Ig1-3) are consistent with this.
The results of Fig. 6 indicate that the KDR structural domains that mediate growth factor binding are contained within Ig-like domains 1-3. A similar conclusion was made in certain previous studies measuring 125I-VEGF165 binding to a soluble chimeric protein consisting of various deletion mutants of the KDR extracellular domain fused to the Fc-portion of human IgG (39, 40). One previous report (57) used this soluble receptor chimera and concluded that Ig-like domain 4 is also required for VEGF binding. The difference between these results and the others is most likely accounted for by the fact that 22 fewer amino acids were used in defining the Ig-like domain 3-COOH boundary, and so the recombinant KDR Ig-like 1-3 protein used lacked important structural features required for VEGF binding. This hypothesis is supported by the recently reported (58) finding that KDR amino acids 313 and 315, which are contained within the relevant 22-amino acid peptide, interact directly with VEGF and are required for binding.
The experimental results shown in Figs. 6 and 7 indicate that the vast majority of mutant receptors expressed in PAE cells are internal. It was suggested under "Results" that this is because once the receptors autophosphorylate at the cell surface, they internalize. We cannot rule out an alternative conclusion that a majority of newly synthesized mutant receptors never reach the cell surface either because they lack appropriate trafficking domains or because of receptor activation prior to reaching the cell surface. Our results do clearly demonstrate that a certain population of newly synthesized mutant receptors is expressed at the cell surface because we can measure 125I-VEGF binding. Evidence indicating that these cell surface receptors are functional is that focal adhesion complex assembly, which is dependent upon the KDR-mediated recruitment of NCK to the cell surface (59), is VEGF-independent in the KDR(Ig1-3)-expressing PAE cells.
The molecular interactions that account for the lower affinity of 125I-VEGF binding to KDR-transfected PAE versus HEK293 cells is not clear. The fact that high affinity binding is seen for cells expressing mutant receptors suggests that the interactions are mediated, in part, by KDR Ig-like domains 3-7. PAE cells do not express neuropilin-1 (34), but this most likely does not account for the observed effects because neuropilin-1 does not interact with 125I-VEGF121 (34), which also binds with low affinity to these KDR-expressing PAE cells.
In summary, the results of this study indicate that the role of VEGF in
stimulating KDR activation is more complex than simply allowing for
receptor monomers to come into close contact with each other. In
addition to ligand binding regions within the extracellular domain of
KDR, there are also regions that act to prevent VEGF-independent dimerization and thus unwanted signaling events. VEGF functions, in
part, to relieve this inhibitory effect. The molecular interactions that allow for VEGF activation of KDR are different from those involved
in PDGF and colony-stimulating factor-1 activating their receptors
because deletion of KDR Ig-like domain 4 does not prevent high affinity
VEGF binding.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01 CA86289-01.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: Cardiology Division, Dept. of Medicine, Forchheimer 715, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2774; Fax: 718-430-8989; E-mail: terman@aecom.yu.edu.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M100763200
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; KDR, kinase insert domain receptor; PDGF, platelet-derived growth factor; PLC, phospholipase C; HSPG, heparan sulfate proteoglycan; HEK, human embryonic kidney; PAE, porcine aortic endothelial; DMEM, Dulbecco's modified Eagle's medium; Tyr(P), phosphotyrosine; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
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