(Received for publication, October 3, 1995; and in revised form, December 20, 1995)
From the
Vascular endothelial growth factor (VEGF), a potent angiogenic
factor, uses two receptor tyrosine kinases, FLK/KDR and FLT, to mediate
its activities. We have cross-linked I-VEGF
to the cell surface of various tumor cell lines and of human
umbilical vein endothelial cells. High molecular mass (220 and 240 kDa)
and/or lower molecular mass (165 and 175 kDa) labeled complexes were
detected depending on the cell type. The 220- and 240-kDa labeled
complexes were shown to contain FLT and FLK/KDR receptors,
respectively. On the other hand, the 165- and 175-kDa complexes did not
seem to contain FLK/KDR or FLT but instead appeared to contain novel
VEGF receptors with relatively low molecular masses of approximately
120 and 130 kDa. These receptors were further characterized in breast
cancer MDA MB 231 cells (231), which did not form the high molecular
mass complexes and which did not express detectable amounts of flk/kdr or flt mRNA. The 231 cells displayed
one VEGF
binding site, with a K
of 2.8
10
M and
0.95-1.1
10
binding sites per cell. By
comparison, human umbilical vein endothelial cells had two binding
sites, one with a K
of 7.5
10
M, presumably FLK/KDR, and the other
with a K
of 2
10
M, a value similar to the VEGF binding sites on 231
cells. These lower affinity/molecular mass receptors on 231 cells
cross-linked
I-VEGF
but not
I-VEGF
. Accordingly, exon 7 of VEGF, which
encodes the 44 amino acids present in VEGF
that are
absent in VEGF
, was fused to glutathione S-transferase (GST). The GST-VEGF-exon 7 fusion protein bound
to heparin-Sepharose with a similar affinity as VEGF
and
inhibited the binding of
I-VEGF
to 231
cells. Cross-linking of
I-GST-VEGF-exon 7 to 231 cells
resulted in the formation of 150- and 160-kDa labeled complexes that
presumably contained the 120- and 130-kDa lower affinity/molecular mass
VEGF
receptors. It was concluded that certain
tumor-derived cell lines express novel surface-associated receptors
that selectively bind VEGF
via the exon 7-encoded domain,
which is absent in VEGF
.
Vascular endothelial growth factor (VEGF) ()was
initially purified from the conditioned media of folliculostellate
cells (1) and a variety of tumor cell lines as a potent
angiogenic factor and mitogen for endothelial cells (EC) in
vitro(2, 3) . An inducer of blood vessel
permeability was concurrently purified from the conditioned medium of
U937 cells that was found to be the same as VEGF and was named vascular
permeability factor(4, 5) .
Several studies point to VEGF as being an important regulator of angiogenesis. For example, VEGF expression is up-regulated in tissues undergoing vascularization during embryogenesis and during the female reproductive cycle(6, 7) . High levels of VEGF are found in various types of tumors in response to tumor-induced hypoxia but not in normal tissue(8, 9, 10, 11, 12) . A recent study has directly linked VEGF to vascularization-dependent tumor growth by showing that treatment of tumors with monoclonal antibodies directed against VEGF resulted in a dramatic reduction in tumor angiogenesis and tumor mass(13) .
Four different VEGF
isoforms, consisting of 121, 165, 189, and 206 amino acids, are
produced as a result of alternative splicing from a single gene
containing eight exons(14, 15, 16) . The
active form of VEGF is a homodimer, and all four isoforms display a
similar ability to induce EC proliferation(15) . Although all
VEGF isoforms are synthesized with a signal peptide that allows
secretion, they differ in their localization probably as a result of
differential affinities for heparan sulfate proteoglycans that are
found on the cell surface and in the extracellular
matrix(15, 17, 18) . VEGF and
VEGF
have a high affinity for heparan sulfate and are
mostly cell and extracellular matrix-associated. VEGF
,
which has a lower affinity for heparan sulfate, is partially released
into the culture medium and partially found on the cell surface and in
the extracellular matrix. VEGF
is the only VEGF isoform
that does not bind to heparin and is exclusively secreted into the
culture medium(15, 17) .
VEGF binds to specific
receptor tyrosine kinases, KDR and FLT, that are expressed by EC and by
several types of non-EC such as human melanoma cells, NIH3T3 cells,
HeLa cells, and Balb/c 3T3
cells(19, 20, 21, 22, 23) .
The flt and kdr cDNAs were cloned from human
libraries prepared from placenta and EC,
respectively(24, 25) . KDR has a molecular mass of 190
kDa(26) , and FLT has a lower molecular mass, which is 160
kDa(27) . KDR, whose mouse homologue is known as FLK, is the
only known VEGF receptor that is found on the cell surface of bovine
aortic endothelial cells(28) . On the other hand, some melanoma
cell lines have been shown to synthesize FLT(29) . Efficient
binding of I-VEGF
to its receptors on EC
requires the presence of heparin-like molecules on the cell
surface(20) . Heparin has differential effects on the binding
of
I-VEGF
to FLK/KDR and FLT. Soluble
heparin seems to enhance the binding of
I-VEGF
to EC synthesizing FLK/KDR (20, 30) but has the
opposite effect on the binding of
I-VEGF
to
melanoma cells synthesizing FLT(28, 29) .
I-VEGF
binds to
2 macroglobulin and as
a consequence is not able to bind its receptors on HUVEC. Heparin
interferes with this binding and is able to restore the receptor
binding capability of
I-VEGF
(30, 31) .
The binding of VEGF to VEGF receptors on non-EC does not seem to induce cell proliferation. Rather, VEGF induces motility of monocytes(32) , differentiation of osteoblasts(33) , production of insulin by beta cells(34) , and disorganization of actin stress fibers in Balb/c 3T3 cells(23) . Recent studies with cells expressing endogenous or transfected flt and flk/kdr have suggested that VEGF activities, e.g. mitogenicity, chemotaxis, and morphological changes are mediated by FLK/KDR but not through FLT, even though both receptors undergo phosphorylation upon binding of VEGF(35, 36, 37, 38) . Placenta growth factor, a recently purified VEGF homologue (39) that binds to FLT, has no effect on the proliferation rate of EC(36, 40) . These results suggest that although VEGF can bind to multiple receptors on the cell surface, the subsequent VEGF-mediated responses may be different.
Cross-linking of I-VEGF
to EC and non-EC results in the
formation of multiple
I-VEGF
-VEGF receptor
labeled complexes, with higher molecular masses of about 220-240
kDa and lower molecular masses of about 165-175
kDa(19, 20, 22) . However, to date it has not
been clear which of the known VEGF receptors are contained in the
various labeled complexes. In this report, we demonstrate (i) that the
high molecular mass complexes found in EC and melanoma cells contain
FLK or FLT; (ii) that the lower molecular mass complexes do not appear
to contain FLK or FLT but rather VEGF receptors of relatively lower
affinity; (iii) that the lower affinity/molecular mass receptors found
on tumor cells are isoform-specific in that they bind
I-VEGF
but not
I-VEGF
, in agreement with previous results
obtained for HUVEC(41) ; and (iv) that the binding of
I-VEGF
to these receptors is mediated by
the VEGF exon 7-encoded domain, which is present in VEGF
but not VEGF
.
Figure 1:
Cross-linking of -VEGF
to the surface of HUVEC and
tumor-derived cell lines.
I-VEGF
(5 ng/ml)
was bound and cross-linked to subconfluent cultures of HUVEC (lanes
1 and 2), 231 cells (lanes 3 and 4),
LNCaP cells (lanes 5 and 6), and MDA-MB-453 cells (lanes 7 and 8) in 6-cm dishes. The binding was
carried out in the presence (lanes 2, 4, 6,
and 8) or the absence (lanes 1, 3, 5, and 7) of 1 µg/ml heparin. The cells were
lysed, and proteins were resolved by 6% SDS-PAGE as described under
``Experimental Procedures.''
Figure 2:
Immunoprecipitation of I-VEGF
cross-linked complexes containing
VEGF receptors.
I-VEGF
(5 ng/ml) was bound
and cross-linked to subconfluent cultures of HUVEC (lanes
1-3), 231 cells (lanes 4-6), EP-mel cells (lanes 7-9), and RU-mel cells (lanes
10-12) in 10-cm dishes. The binding was carried out in the
presence of 1 µg/ml heparin, except for RU-mel. The cells were
lysed, and immunoprecipitation was performed with anti-FLK (lanes
2, 5, 8, and 11) and anti-FLT (lanes 3, 6, 9, and 12) antibodies
as described under ``Experimental Procedures.'' Samples of
cell lysate were kept aside before the antibodies were added to serve
as controls (lanes 1, 4, 7, and 10). The immunocomplexes were resolved by 6% SDS-PAGE as
described in the legend to Fig. 1.
Figure 3:
Analysis of VEGF binding
sites on 231 cells and HUVEC. A, increasing amounts of
I-VEGF
(1.5-80 fmol) were added to
subconfluent cultures of 231 cells (circles) and HUVEC (triangles) in 48-well dishes. The binding to 231 cells was
carried out in the presence (closed circles) or the absence (open circles) of 1 µg/ml heparin. Nonspecific binding was
determined by competition with a 200-fold excess of unlabeled
VEGF
. After binding, the cells were washed and lysed, and
the cell-associated radioactivity was determined using a
counter.
The results shown in A were analyzed by the method of
Scatchard, and best fit plots for HUVEC (B), 231 cells (C), and 231 cells in the presence of heparin (D)
were obtained using the LIGAND program.
Figure 4:
The binding of I-VEGF
to 231 cells and HUVEC in the
presence of excess growth factors.
I-VEGF
(4 ng/ml) was bound to subconfluent cultures of 231 cells (hatched bars) and HUVEC (solid bars) in 48-well
dishes in the presence of 400 ng/ml of unlabeled VEGF
,
VEGF
, PIGF, or platelet-derived growth factor. After
binding, cells were washed and lysed, and the cell-associated
radioactivity was determined using a
counter. The counts obtained
are expressed as the percentage of the counts obtained compared with a
phosphate-buffered saline (PBS)
control.
VEGF is an isoform that is
similar to VEGF
in its mitogenicity for EC, but unlike
VEGF
it is not heparin-binding(15) . A 100-fold
excess of VEGF
inhibited the binding of
I-VEGF
to HUVEC by 75% but did not inhibit
binding of
I-VEGF
to 231 cells (Fig. 4). In addition, unlike
I-VEGF
(Fig. 5, lanes 1 and 2),
I-VEGF
(Fig. 5, lanes 3 and 4) did not form any cross-linked complexes with 231 cells. As
a control to ensure that
I-VEGF
was
bioactive,
I-VEGF
bound to HUVEC but to
form only the 240-kDa complex, presumably containing FLK/KDR, (Fig. 5, lanes 5 and 6), confirming previous
results(41) . Thus, it appears that VEGF
binds
only to high affinity receptors such as FLK/KDR to form the 240-kDa
complex, whereas VEGF
can bind to FLK/KDR and FLT and in
addition to lower affinity/molecular mass receptors to form the 165-
and 175-kDa complexes.
Figure 5:
Cross-linking of I-VEGF
and
I-VEGF
to the surface of 231 cells and HUVEC.
I-VEGF
(5 ng/ml) (lanes 1, 2, 7, and 8) or
I-VEGF
(10 ng/ml) (lanes 3-6) were bound and cross-linked
to subconfluent cultures of 231 cells (lanes 1-4) and
HUVEC (lanes 5-8) in 6-cm dishes. The binding was
carried out in the presence (lanes 2, 4, 6,
and 8) or the absence (lanes 1, 3, 5, and 7) of 1 µg/ml heparin. Cells were lysed,
and proteins were resolved by a 6% SDS-PAGE as described in the legend
to Fig. 1.
Figure 6: Heparin affinity chromatography of the GST-ex 7 fusion protein. GST-ex 7 fusion protein was prepared and purified by glutathione-agarose affinity chromatography as described under ``Experimental Procedures,'' and 300 µg were applied to a TSK-heparin column. Heparin-bound proteins were eluted with a linear gradient of 0.2-1.2 M NaCl (A). An aliquot from the peak fraction (number 58) was resolved by 15% SDS-PAGE, which was silver-stained and photographed (B).
Figure 7:
Competition of I-VEGF
binding to 231 cells by the GST-ex 7
fusion protein.
I-VEGF
(4 ng/ml) was bound
to subconfluent cultures of 231 cells in 48-well dishes in the presence
of 2 and 5 µg/ml GST-ex 7 fusion protein (fraction 58) (solid
bars) or 5 µg/ml GST protein (hatched bar). The cells
were washed and lysed, and the cell-associated radioactivity was
determined in a
counter. The counts obtained are expressed as the
percentage of the counts obtained in a control experiment that used
phosphate-buffered saline (PBS, open
bar).
Figure 8:
Cross-linking of I-GST-ex 7
fusion protein to the surface of 231 cells.
I-GST-ex 7
(10 ng/ml) was bound and cross-linked to subconfluent cultures of 231
cells in 6-cm dishes. The binding was carried out in the presence (lane 2) or the absence (lane 1) of 1 µg/ml
heparin. The cells were lysed, and proteins were resolved by 6%
SDS-PAGE as described in the legend to Fig. 1.
In recent years, there has been growing evidence that VEGF
plays a major role in regulating angiogenesis during normal development
and in
tumors(6, 11, 13, 38, 46) .
VEGF binds to specific high affinity receptors, FLK/KDR and FLT, which
mediate VEGF responses(26, 27) , and which were
initially shown to be associated with various types of EC (25, 47, 48) . In addition to EC,
VEGF also binds to receptors on the surface of cell types
such as HeLa, human melanoma, and
NIH3T3(20, 22, 23) . Analysis of
I-VEGF
-VEGF receptor cross-linking patterns
have demonstrated that there are multiple VEGF
binding
sites, for example, on EC and on melanoma
cells(19, 20, 22) . However, the actual
identities of the multiple VEGF receptors in these cross-linked
complexes have not been determined, and thus our goal in this study was
to characterize the VEGF receptors in the various complexes.
Accordingly,
I-VEGF
was cross-linked to the
cell surface of several cell types. Analysis of the cross-linking
products revealed the presence of several
I-VEGF
-cross-linked complexes of higher
(220 and 240 kDa) and lower (165 and 175 kDa) molecular mass. HUVEC
formed both the higher and lower molecular mass complexes as shown
previously(20, 41) , as did several melanoma cell
types(22, 29) . On the other hand, the breast
cancer-derived cell line, MDA MB 231 (231 cells), and the prostate
carcinoma-derived cell-line, LNCaP, formed only the lower molecular
mass complexes. There was no case in which the high but not the low
molecular mass complexes were formed.
Immunoprecipitation studies
with anti-FLK/KDR and anti-FLT antibodies were used to identify the
VEGF receptors on the various cell types. The 240-kDa
complex formed by
I-VEGF
binding to HUVEC
was shown to contain FLK/KDR, confirming previous results(41) ,
whereas the 220-kDa complex formed with RU-mel cells was shown to
contain FLT. Although EC have been reported to express flt mRNA(47, 48) , we have not been able to identify
FLT as part of the
I-VEGF
-cross-linked
complexes formed with HUVEC or bovine aortic endothelial cells. On the
other hand, immunoprecipitation with anti-FLK/KDR and anti-FLT
antibodies showed no cross-reactivity with proteins in the 165- and
175-kDa complexes in 231 cells, consistent with similar results for
HUVEC(41) , suggesting that these complexes contain
VEGF
receptors that are different than FLK/KDR and FLT.
Based on the molecular mass of a VEGF
dimer, we estimated
the size of these lower molecular mass VEGF
receptors to
be 120 and 130 kDa.
We chose 231 cells to characterize the lower
molecular mass VEGF receptors because they do not produce
FLK/KDR- or FLT-containing complexes and do not express flk/kdr or flt mRNA. Scatchard analysis of
the binding of
I-VEGF
to 231 cells revealed
the presence of one class of binding sites that bind VEGF
with a K
of 2.8
10
M and 0.95-1.1
10
binding
sites/cell. Heparin induced a 2-fold increase in the binding of
I-VEGF
to 231 cells by increasing the
number of binding sites/cell without significantly changing the
affinity for VEGF
. On the other hand, HUVEC displayed two
binding sites for VEGF
, with K
values of 7.5
10
and 2.0
10
M, respectively. These results indicate
that the VEGF
binding sites on 231 cells have an affinity
for VEGF
similar to the lower affinity binding sites on
HUVEC. Because both cell types produce labeled complexes of 165 and 175
kDa upon cross-linking with
I-VEGF
, it
seems that the lower molecular mass VEGF
receptors
detected on the cell surface of 231 cells may be the same as the lower
affinity VEGF
receptors found on HUVEC. Accordingly, we
have designated these binding sites as lower affinity/molecular mass
VEGF receptors.
Although excess nonlabeled VEGF inhibited
I-VEGF
binding to 231
cells, a 100-fold excess of VEGF
did not. In addition, no
radiolabeled complexes, neither of higher nor lower molecular mass,
could be detected upon cross-linking of
I-VEGF
to 231 cells. As a control,
I-VEGF
was shown to be active in that it formed a 240-kDa complex,
presumably containing FLK/KDR, upon cross-linking to HUVEC. On the
other hand,
I-VEGF
did not form 165- and
175-kDa complexes with HUVEC, consistent with the 231 cell results. The
ability of
I-VEGF
, but not
I-VEGF
, to form 165- and 175-kDa complexes
with HUVEC has been reported recently(41) . Taken together,
these results demonstrate a VEGF isoform specificity of binding in
which
I-VEGF
is capable of binding to
FLK/KDR but not to the lower affinity/molecular mass receptors, whereas
I-VEGF
binds to both. Thus, VEGF
activities may be specific for FLK/KDR and/or FLT, whereas
VEGF
has a wider range of activities by using more
receptor types.
Because the only structural difference between
VEGF and VEGF
resides in the 44-amino acid
insert encoded by exon 7(14) , we assumed that this domain
might mediate the binding of VEGF
to 231 cells. To test
this hypothesis, a chimeric protein made of GST and the exon 7-encoded
domain of VEGF
was prepared. This GST-ex 7 fusion protein
bound to heparin-Sepharose with a similar affinity as
VEGF
(17) , indicating that the heparin-binding
domain of VEGF
is localized to the exon 7-encoded domain.
In addition, GST-ex 7 competed with the binding of
I-VEGF
to 231 cells and could bind and be
cross-linked directly to the lower affinity/molecular mass receptors.
Taken together, these results indicate that the exon 7-encoded domain
is responsible for both VEGF
heparin-binding and the
binding to the lower affinity/molecular mass receptors.
An important
question to consider is whether the 120- and 130-kDa lower affinity
receptors expressed by 231 cells are novel or whether they may be
truncated forms of FLK/KDR or FLT lacking about 40-50 kDa in
molecular mass. The latter possibility is unlikely because (i)
VEGF does not bind to these receptors on 231 cells or EC
but is fully capable of binding to FLK/KDR on EC (Fig. 5) (41) and to FLT on melanoma cells(29) , suggesting that
the lower affinity/molecular mass receptors do not contain the
extracellular domain of FLK or FLT. However, it is possible that FLT or
FLK could be truncated in such a way that would allow VEGF
but not VEGF
binding, but this possibility would
suggest different binding sites for the two VEGF isoforms, which has
yet to be demonstrated; (ii) the 120- and 130-kDa lower affinity
receptors are not recognized by anti-FLK or anti-FLT antibodies. These
are polyclonal antibodies directed against the C-terminal 20 and 17
amino acids of FLK and FLT, respectively. Thus, as a minimum the lower
affinity receptors do not appear to contain the C-terminal of FLK or
FLT, although it is possible that they do contain some other
cytoplasmic sequences; and (iii) the 231 cells do not express
detectable levels of flk/kdr or flt mRNA.
Taken together, the circumstantial evidence suggests that the lower
affinity/molecular mass VEGF
receptors are probably not
related to FLK or FLT. However, this question will not be resolved
definitively until these receptors are purified or cloned and sequence
information is available.
We have not yet determined the biological
role of the lower affinity/molecular mass VEGF receptors.
To date, we have not been able to demonstrate
VEGF
-induced proliferation or protein phosphorylation of
231 cells. It may be that these receptors mediate other effects of
VEGF
such as migration (49) or morphological
changes(23) . Alternatively, they may potentiate the responses
of FLK/KDR or FLT to VEGF
. In addition, it has been
demonstrated that although VEGF
and VEGF
are both released from cells, some VEGF
is also
associated with the cell surface, possibly due to its interaction with
heparan sulfate proteoglycans(17, 18) . Thus, the
binding of VEGF
to the lower affinity/molecular mass
VEGF
receptors via its exon 7-encoded domain suggests
that these proteins might contain heparan sulfate because exon 7
contains within it the heparin-binding domain of VEGF
However, the lower affinity/molecular mass VEGF
receptors on 231 cells are probably not heparan
sulfate-containing proteoglycans because heparin augments rather than
inhibits their
I-VEGF
binding and the
relative sharpness of the 165- and 175-kDa complexes is not
characteristic of proteoglycans.
In summary, we have characterized a
new class of lower affinity/molecular mass VEGF isoform-specific
receptors found on EC and tumor cell surfaces, that bind VEGF but not VEGF
. Their structure and function is,
however, unclear at present. Purification of these receptors is now
underway in order to better determine their role in modulating
VEGF
activity.