(Received for publication, October 3, 1996, and in revised form, December 26, 1996)
From the Institute of Molecular Medicine, Tumor Biology Center,
D-79106 Freiburg, Germany and GSF-Forschungszentrum,
D-81377 München, Germany
The vascular endothelial growth factor (VEGF) receptor FLT-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven Ig-like loops within the extracellular domain. Upon VEGF binding FLT-1 becomes phosphorylated, which has been thought to be preceded by receptor dimerization. To further investigate high affinity binding of VEGF to FLT-1 and ligand-induced receptor dimerization, we expressed in Sf9 cells the entire extracellular domain comprising all seven Ig-like loops: sFLT-1(7) and several truncated mutants consisting of loop one, one and two, one to three, one to four, and one to five. The corresponding proteins, named sFLT-1(1), (2), (3), (4), and (5) were purified. Only mutants sFLT-1(3) to (7) were able to bind 125I-VEGF with high affinity. No binding of VEGF was observed with sFLT-1(1) and sFLT-1(2), indicating that the first three Ig-like loops are involved in high affinity binding of VEGF. The binding of VEGF to sFLT-1(3) could be competed with placenta growth factor (PlGF), a VEGF-related ligand, suggesting that high affinity binding of VEGF and PlGF is mediated by the same or closely related contact sites on sFLT-1. Deglycosylation of the sFLT-1(3), (4), (5), and (7) did not abolish VEGF binding. Furthermore, unglycosylated sFLT-1(3), expressed in Escherichia coli, was able to bind VEGF with similar affinity as sFLT-1(3) or sFLT-1(7), both expressed in Sf9 cells. This indicates that receptor glycosylation is not essential for high affinity binding. Dimerization of the extracellular domains of FLT-1 upon addition of VEGF was detected with all mutants containing the Ig-like loop four. Although sFLT-1(3) was able to bind VEGF, dimerization of this mutant was inefficient, indicating that sites on Ig-like loop four are essential to stabilize receptor dimers.
The vascular endothelial growth factor (VEGF),1 a potent mitogen for endothelial cells, is an important angiogenic factor also involved in the differentiation of endothelial cells and the development of the vascular system (1, 2). It has been shown to be implicated in human diseases such as diabetic retinopathy, rheumatic arthritis, and cancer (3). VEGF in particular appears to be the most important angiogenic factor of many solid tumors, promoting vascularization and formation of metastases (4).
Four different VEGF isoforms have been described so far, all encoded by a single gene: VEGF121, VEGF165, VEGF189, and VEGF206 (5). All different isoforms are secreted dimeric proteins, sharing similarities with platelet-derived growth factor (PDGF) and belong to the family of growth factors containing a cysteine knot motif (6).
Two receptor tyrosine kinases, FLT-1 (7, 8) and KDR/FLK-1 (9, 10), have been identified, which bind VEGF with high affinity. Both receptors belong to the type III tyrosine kinases and are characterized by seven Ig-like loops within their extracellular domain and a split kinase domain within the cytoplasmatic moiety (11). The Ig-like loop motif is a common feature of extracellular domains of membrane-anchored proteins. Members of the immunoglobulin superfamily are often involved in cell surface recognition (12). Both VEGF receptors contain several putative N-glycosylation sites and the apparent molecular weights of the mature proteins suggest that both receptors are extensively glycosylated (8, 10).
The activation of growth factor receptors in general is preceded by the formation of receptor dimers and subsequent receptor phosphorylation. The resulting phosphotyrosine residues are docking sites for signal coupling components such as SH-2 proteins (13). The molecular structures that are responsible for ligand/receptor interaction and ligand-induced dimerization are poorly understood for most receptors. Since the dimeric structure of VEGF is a prerequisite of receptor activation, it can be speculated that one VEGF molecule bridges two receptors via two similar recognition sites, as has been suggested for PDGF (14). Characterization of VEGF binding to its receptors by mutational analysis of the ligand supports the assumption that VEGF has two contact sites for its receptors (15).
Nothing is known about which part of the receptor is involved in ligand binding and which part of the receptor's extracellular domain is involved in VEGF-dependent dimerization. To investigate these questions, we generated several soluble mutants of the extracellular domain of FLT-1, each consisting of a different stretch of Ig-like loops. The data presented here suggest that the recognition site for VEGF is located on the first three Ig-like loops, whereas dimerization is stabilized due to an additional domain located on Ig-like loop four. Glycosylation was found not to be a prerequisite of high affinity binding of VEGF to FLT-1.
Sf9 cells were cultured routinely in 1-liter spinner flasks (Technomara, FRG) in ExCell 400 (JRH Bioscience, UK) without any further supplements. Human umbilical vein endothelial cells (HUVECs) were obtained from PromoCell (FRG) and cultured according to the provided protocol. VEGF165 was expressed in Escherichia coli and purified as described previously (16). VEGF165 was iodinated to a specific activity of 100,000 cpm/ng by using the chloramine T method (17) (Immundiagnostik, FRG). PlGF152 was expressed with the baculovirus/insect cell system as described previously (18). N-Glycosidase F and disuccinimidyl suberate (DSS) were obtained from Boehringer Mannheim (FRG), N-hydroxysuccinimidyl-biotin was from Pierce (FRG).
Expression and Purification of Soluble FLT-1 Receptor MutantsSoluble N-terminal flt-1 fragments were cloned
by polymerase chain reaction from the full-length cDNA clone 3-7
(7) using the upstream primer: 5-GGAATTCCGCGCTCACCATGGTCAGC-3
,
containing an EcoRI site and various downstream primers,
which all contain an artificial stop codon and a BamHI site.
The coding sequences ended at bp 642 for sFLT-1(1), at bp 957 for
sFLT-1(2), at bp 1243 for sFLT-1(3), at bp 1531 for sFLT-1(4), at bp
1915 for sFLT-1(5) and at bp 2497 for sFLT-1(7), respectively. The
polymerase chain reaction products were purified with the Quiax DNA gel
extraction kit (Quiagen, FRG) and subcloned into the baculovirus
transfer vector pVL1392 as EcoRI/BamHI fragments.
Plasmids containing the cDNA were isolated from transfected
bacteria and then used for transfection into Sf9 cells along with
wild-type baculovirus DNA. Recombinant baculoviruses were obtained
using the BaculoGoldTM transfection kit following standard
protocols (Pharmingen, San Diego, CA). For protein expression, Sf9
cells at a density of 2 × 106 cells/ml were infected
with a multiplicity of infection of 10. 72 h after infection,
cell-free conditioned medium was filtered and applied to a
heparin-Sepharose column (Pharmacia, FRG). The column was washed with
either 10 ml of 20 mM Tris-Cl, pH 7.4, for sFLT-1(1) and
(2), or 10 ml of 0.4 M NaCl for sFLT-1(3), or 10 ml of 0.6 M NaCl for sFLT-1(4), (5), and (7). Bound proteins were
eluted by increasing the NaCl concentration by steps of 0.2 M. The monoclonal antibody 7A6 was used to identify all the
various sFLT-1 proteins by Western blotting.
A
0.9-kilobase pair NcoI/BamHI fragment encoding
amino acids 31-338 of human FLT-1 was generated by polymerase chain
reaction and ligated into the bacterial expression vector His-pET (16). Thus the expression plasmid encoded FLT-1 amino acids 31-338 fused to
an N-terminal 6 × His-tag and amino acids Met and Glu, which were
derived from the artifical NcoI site. For bacterial
expression, the plasmid construct was transduced into E. coli strain BL21(DE3) carrying an inducible T7 RNA polymerase gene
(19). Bacterial cultures of 250 ml of LB medium containing 100 µg/ml
ampicillin were grown in shaking flasks at 37 °C to an
A600 of 0.8. Isopropyl--D-thiogalactoside was added to a final
concentration of 0.4 mM, and the culture was grown for
another 3 h. Cells were harvested and washed, and the pellet was
frozen at
80 °C. Upon use, cells were thawed at 37 °C,
resuspended in 25 ml of buffer A (50 mM Tris-HCl, 10 mM 2-mercaptoethanol, 2 mM EDTA, 5% (v/v)
glycerol, 0.2 mg/ml lysozyme, 10 µg/ml DNase I, pH 8.0) and incubated
for 30 min at 22 °C. The suspension was sheared by five high speed
treatments of 20 s in an Ultra-Turrax dispersing apparatus and
incubated for 10 min at 22 °C. The mixture was cooled on ice and
sonicated six times for 15 s with the microtip of a Bronson
Sonifier 250. After the addition of sodium deoxycholate and Nonidet
P-40 to a final concentration of 0.05% and 1% (w/v), respectively,
the mixture was incubated for 10 min at 4 °C and then centrifuged at
10,000 rpm at 4 °C for 30 min. The pellet was resuspended in 25 ml
of buffer A supplemented with 0.05% sodium deoxycholate and 1%
Nonidet P-40 and recentrifuged. The inclusion body pellet was
solubilized in 25 ml of buffer B (6 M guanidine HCl, 0.15 M NaCl, 0.1 M dithiothreitol, 50 mM
NaPO4 buffer at pH 6.5). The solubilized protein was
dialyzed two times against 250 ml of buffer C (6 M urea,
0.1 M dithiothreitol, 50 mM MES at pH 5.5) at
4 °C, and the volume of the dialysate was reduced 20-fold by
ultrafiltration (Millipore Ultrafree-15). The concentrated protein
solution was mixed with 2 volumes of buffer D (6 M urea,
0.5 M cystamine, 0.1 M glycine, 20 mM Hepes at pH 7.4) and incubated for 4 h at 4 °C
with gentle agitation. For refolding, the solution was sequentially
diluted to final 0.3 M urea by the addition of PBS, 50 mM glycine. The refolded sFLT-1(3) protein was concentrated
by ultrafiltration and referred to as sFLT-1(3)E. coli.
For deglycosylation of recombinant sFLT-1 mutants, N-glycosidase F was used. SDS (final concentration, 0.3%) was added to sFLT-1 proteins (250-500 ng in 10 µl, total volume), and the mixture was incubated at 95 °C for 2 min. Then 10 µl of 2 × reaction buffer were added (100 mM sodium phosphate, pH 7.2, 20 mM EDTA, 1% Nonidet P-40) and again incubated at 95 °C for 2 min. After cooling down, N-glycosidase F (0.2 unit/assay) was added, and the mixture was incubated at 37 °C overnight. The reaction was stopped with 4 × SDS sample buffer, and proteins were resolved by SDS-PAGE.
Antibodies and Western Blot AnalysisApproximately 50 µg of sFLT-1(7) dissolved in PBS and emulsified with Freund's complete adjuvant were injected both intraperitoneally and subcutaneously into Lou/C rats and again as a booster 8 weeks after the first immunization. Fusion of the myeloma cell line P3X63-Ag8.653 with rat immune spleen cells was performed essentially as described previously (20). Supernatants were screened for anti-sFLT-1 antibodies using a solid phase enzyme-linked immunosorbent assay. The mAb 7A6 was able to detect the various sFLT-1 mutant proteins and was characterized as a rat IgG2a. For Western analysis, aliquots (20 µl) of the elution fractions of the heparin-Sepharose columns were resuspended in SDS sample buffer and resolved by SDS-PAGE. The gels were electroblotted onto a polyvinylidene difluoride membrane, and blots were blocked for 30 min with 5% milk powder in TBST (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and incubated for 1 h in the same buffer with 1 µg/ml mAb 7A6. The blots were washed twice in water and TBST and incubated for 45 min in TBST containing a 5000-fold diluted peroxidase-conjugated mouse anti-rat IgG/goat anti-rat IgG + IgM (Jackson ImmunoResearch Labs., Inc., West Grove, PA). The blots were washed as described before, and peroxidase-coupled antibodies were visualized using the ECL chemiluminescent Western blotting detection system (Amersham, FRG).
Ligand BlottingFor ligand blotting, 0.5 µg of purified sFLT-1 mutant proteins was mixed with nonreducing SDS sample buffer, and SDS-PAGE was performed. After blotting the gel onto a polyvinylidene difluoride membrane at 4 °C, the filter was blocked in TBS containing 2% bovine serum albumin for 30 min. The filter was incubated with 20 ng/ml 125I-VEGF165 for 2 h. After extensive washing with TBS, the filter was exposed to a Kodak X-Omat x-ray film.
Solid Phase Binding AssayssFLT-1 mutant proteins (0.5-1 µg/well) were absorbed to the surface of 96-well cluster plates (Nunc-Immuno Plate MaxiSorpTM) for 18 h at 4 °C. The plates were washed twice with TBS, nonspecific sites were blocked with 2% bovine serum albumin in TBS for 1 h, and the plates were washed again with TBS. In competition experiments, the binding of 125I-VEGF165 (10 ng/ml) was competed by increasing amounts of recombinant human VEGF165 or PlGF152 as indicated in the figure legends. The wells were washed four times with TBS, bound protein was solubilized in 100 µl of 0.3 M NaOH, 1% SDS, and radioactivity was counted in a gamma-counter (Beckmann, FRG).
For saturation binding curves, biotinylated VEGF was used. Immobilized sFLT-1(3), (7), and sFLT-1(3)E. coli were washed three times with PBS containing 0.1% Tween-20, and nonspecific sites were blocked with 0.5% bovine serum albumin, 0.5% Tween-20 in PBS for 2 h at room temperature. Binding was carried out with increasing amounts of biotinylated VEGF165 for 1 h. Unspecific binding was determined in parallel dishes by the addition of a 100-fold excess of unlabeled VEGF. Biotinylated VEGF was quantified using a streptavidin complexed to alkaline phosphatase (Calbiochem, FRG) and p-nitrophenyl phosphate disodium (Sigma, FRG) as a substrate.
Cell-based Binding AssayBinding assays with endothelial cells were done as described previously (21). Briefly, HUVECs (passage 4) were seeded in growth medium at 5 × 104 cells/well in 24-well cluster plates. After 72 h the cells were washed extensively with binding buffer (Dulbecco's modified Eagle's medium, 25 mM Hepes, 1 mg/ml bovine serum albumin, pH 7.4) and incubated for 2 h at 4 °C with binding buffer containing 1 ng/ml 125I-VEGF165. Increasing amounts of sFLT-1 were used for competition. The cells were washed three times with binding buffer, bound protein was solubilized with 250 µl of 0.3 M NaOH, 1% SDS/well, and radioactivity present in the lysates was quantified.
Chemical Cross-linking of Protein ComplexesFor cross-linking, 5 µg of sFLT-1(3), (4), and (5) were mixed with 100 ng of VEGF165 in PBS, 0.1% Tween 20 in a final volume of 100 µl and incubated on ice for 2 h. DSS was added to a final concentration of 1 mM, and the reaction mixture was incubated for another 15 min. The protein was trichloroacetic acid-precipitated, washed twice with acetone, and resuspended in 30 µl of SDS sample buffer. The proteins were resolved by SDS-PAGE and identified by Western blotting with the mAb 7A6. In a second approach, 125I-VEGF165 (20 ng/assay) was used for the cross-linking reaction with sFLT-1(3), (4), and (5). The gel was vacuum-dried and exposed to a Kodak X-Omat x-ray film.
[3H]Thymidine Incorporation AssayHUVECs (passage 3) were seeded at a density of 1 × 104 cells/well into 48-well cluster plates in growth medium. Cells were allowed to attach overnight at 37 °C. Growth medium was replaced by basal medium (1.7% fetal calf serum), and a constant concentration of VEGF165 (3 ng/ml) together with increasing amounts of the different sFLT-1 proteins were added 24 h later. Incubation was continued for additional 18 h, then 1 µCi of [3H]thymidine (56.7 Ci/mmol, DuPont NEN) was added. Cells were kept at 37 °C for an additional 6 h. Cell monolayers were fixed with methanol, washed with 5% trichloroacetic acid, solubilized in 0.3 M NaOH, and counted by liquid scintillation.
To express the
soluble extracellular domain of the VEGF receptor FLT-1 and its
truncated mutants, constructs were made by introducing stop codons at
various sites of the flt-1 cDNA as shown in Fig.
1. The resulting cDNAs were cloned into the
baculovirus transfer vector pVL1392, and all proteins were expressed in
Sf9 insect cells. Since all constructs contain the N-terminal leader sequence but lack the transmembrane domain, the expressed proteins were
expected to be secreted from the infected Sf9 cells. To enable detection of the expressed proteins, monoclonal antibodies had been
raised against the extracellular domain of FLT-1. The monoclonal antibody 7A6 was found to detect the soluble FLT-1 as well as all
truncated derivatives. Western blot analysis of partially purified
sFLT-1 proteins from conditioned media of infected Sf9 cells confirmed
that all FLT-1 mutants were expressed and secreted (Fig.
2). The apparent molecular masses were estimated from
SDS-PAGE to be 16 kDa for sFLT-1(1), 28 kDa for sFLT-1(2), 45 kDa for
sFLT-1(3), 57 kDa for sFLT-1(4), 72 kDa for sFLT-1(5), and 105 kDa for
sFLT-1(7). sFLT-1(1) and sFLT-1(2) matched more or less the calculated
molecular masses deduced from the amino acid sequence, whereas the
estimated size of sFLT-1(3) to sFLT-1(7) was increased by about 10-20
kDa as compared with the calculated molecular mass. This is most likely due to N-linked sugars since the extracellular domain
contains 12 putative glycosylation sites (see Fig. 1).
Binding of VEGF and PlGF to sFLT-1 Mutants
To investigate
whether the expressed sFLT-1 mutant proteins are able to bind
VEGF165 we used a solid phase binding assay on microtiter
plates. Specific binding of VEGF165 could be observed to
all mutants containing the first three N-terminal Ig-like loops: sFLT-1(3), (4), (5), and (7). No specific VEGF binding could be
obtained with sFLT-1(1) and sFLT-1(2) containing the first or the first
and the second Ig-like loops, respectively (Fig. 3,
inset). In a competition experiment, using increasing amounts of unlabeled VEGF165, comparable high affinity
binding of VEGF to all sFLT-1 mutants containing the first three
Ig-like loop could be demonstrated (Fig. 3). To make sure that lack of ligand binding of the two shorter mutants sFLT-1(1) and sFLT-1(2) was
not due to inefficient immobilization to the microplates, we performed
a cellular binding assay with HUVECs that express both VEGF receptors
KDR and FLT-1 (21). All sFLT-1 mutants containing the first three
Ig-like loops were able to compete efficiently with VEGF165
binding to HUVECs, the mutants sFLT-1(1) and (2), which failed to bind
VEGF in the solid phase assay, also failed to compete with VEGF binding
to endothelial cells (Fig. 4). The slight decrease of
total binding observed with the sFLT-1(1) and sFLT-1(2) preparations is
statistically not significant.
PlGF, similar to VEGF, is a disulfide-bridged homodimer and shares
about 30% identity with VEGF (18, 22). PlGF has been found to bind to
FLT-1 and to displace VEGF from this receptor (23-25). To test whether
this is also true for the soluble extracellular domain of FLT-1, we
performed the solid phase binding assay (Fig. 5).
PlGF152 competed with VEGF165 for binding to
sFLT-1(3) and (7). No difference could be detected between the
full-length extracellular domain and sFLT-1(3), the shortest sFLT-1
mutant still exhibiting VEGF binding. From this we conclude conclude that PlGF and VEGF share similar contact sites on the receptor.
Inhibition of VEGF-mediated DNA Synthesis by sFLT-1 Mutants
Since HUVECs proliferate in response to VEGF, we
investigated the ability of the sFLT-1 mutants to antagonize
VEGF-mediated incorporation of [3H]thymidine into HUVECs
(Fig. 6). A dose-dependent inhibition of
VEGF-stimulated DNA synthesis could be observed with sFLT-1(3), (4),
(5), and (7), confirming the results from the ligand binding studies.
Addition of sFLT-1(1) and sFLT-1(2) had only a minor VEGF-antagonizing
effect. From these experiments we conclude that the presence of Ig-like
loop three is a prerequisite for high affinity binding of VEGF to its
receptor.
Effect of Glycosylation on VEGF Binding
The presence of
putative N-glycosylation sites (Fig. 1) and the obvious
differences between the apparent and calculated molecular weights of
the sFLT-1 mutants (3) to (7) suggest that those sFLT-1 mutants, which
are able to bind VEGF, are released from Sf9 cells as glycosylated
proteins. To investigate whether protein glycosylation has any
influence on VEGF binding, we incubated the recombinant sFLT-1 mutants
with N-glycosidase F. The N-glycosidase F-treated
proteins and appropriate controls were subjected either to Western blot
analysis or ligand blotting (Fig. 7). The Western blot
analysis revealed a significant decrease of the apparent molecular
weight due to the glycosidase treatment, demonstrating that the
polypeptides contain N-linked sugar residues (Fig.
7A). Incubation with radiolabeled 125I-VEGF
identified both the glycosylated and deglycosylated polypeptides as
VEGF binding proteins (Fig. 7B). In several preparations we detected high molecular weight complexes in the absence of VEGF with
the mAb 7A6. The appearance of these complexes varied in individual
preparations of different sFLT-1 mutants. An example is shown for
sFLT-1(5) in Fig. 7, A and B
(arrowheads). The reason for this complex formation is
unclear. Since this effect was most obvious on nonreducing SDS gels, we
speculate that the additional bands might be due to incorrect formation
of disulfide bridges during the recombinant protein expression and
partial proteolytic degradation.
To confirm that VEGF binding does not depend on glycosylation, we used
a different experimental approach, expressing the sFLT-1 sequence
corresponding to amino acid Asp31 to His338 in
E. coli to prevent N-glycosylation. The amino
acid sequence expressed in E. coli thus resembles sFLT-1(3)
lacking the leader peptide. This recombinant protein was found in the
inclusion body fraction of E. coli. The apparent molecular
mass as estimated from SDS-PAGE was approximately 37 kDa, which is very
close to the calculated molecular mass of 35 kDa. For refolding, the
inclusion bodies were first solubilized in the presence of guanidine
HCl. Oxidation and reduction of SH groups was performed to achieve the
formation of proper disulfide bridges. The refolded protein was
referred to as sFLT-1(3)E. coli and subjected to a solid phase
VEGF binding assay (Fig. 8). The
sFLT-1(3)E. coli showed a similar dose dependence of specific
binding as compared with sFLT-1(3) and sFLT-1(7), which had been
expressed in Sf9 cells. The Kd values calculated
from the binding data were estimated to be 47 ng/ml for sFLT-1(7), 71 ng/ml for sFLT-1(3), and 62 ng/ml for sFLT-1(3)E. coli. From
these experiments we conclude that N-glycosylation on the
extracellular domain of sFLT-1 is not a prerequisite of high affinity
VEGF binding.
Dimerization of sFLT-1 Mutants
To test whether the soluble
extracellular domain of the receptor is sufficient for
VEGF-dependent receptor dimerization, we investigated the
formation of dimers in the presence of VEGF. The mutants sFLT-1(3),
(4), and (5) were cross-linked with DSS after incubation without and
with VEGF followed by Western blotting using the mAb 7A6 (Fig.
9A). In the absence of VEGF the sFLT-1
mutants were all found to be in the monomeric form. After incubation
with VEGF, additional high molecular mass complexes were observed. The
sizes of these additional protein complexes were found to be about 66 kDa for sFLT-1(3), 140 kDa for sFLT-1(4), and 180 kDa for sFLT-1(5),
respectively. In the case of sFLT-1(4) and (5) these molecular masses
correspond to a complex of 1 molecule of VEGF and 2 molecules of sFLT-1
mutants. In contrast, the cross-linked sFLT-1(3) complex rather matches
the size of a VEGF-containing monomer and not of a dimer. From these
data we conclude that sFLT-1(3), although binding VEGF with high
affinity, is impaired to form stable dimers.
Similar results can be obtained by cross-linking the sFLT-1 mutants upon incubation with radiolabeled VEGF (Fig. 9B). The autoradiograph of the corresponding SDS-gel shows radiolabeled high molecular mass complexes at about 180 kDa for dimeric, VEGF-containing sFLT-1(5) and at about 140 kDa for dimeric VEGF-containing sFLT-1(4). Under the same experimental conditions the mutant sFLT-1(3) does not show substantial dimerization in the presence of VEGF. The size of the major labeled complex at about 70 kDa corresponds to only 1 molecule of sFLT-1(3) and 1 molecule of VEGF. Only a very weak labeling was found at 120 kDa, the size of the sFLT-1(3) dimer complexed with VEGF. These results suggest that sFLT-1(3) has lost almost completely the potential to form stable VEGF-dependent dimers and that the fourth Ig-like loop harbors a domain that supports and/or stabilizes the ligand-induced formation of receptor dimers.
We expressed different truncated mutants of the extracellular domain of the VEGF receptor FLT-1 to get further insight into the structure/function relationship for VEGF binding and receptor dimerization. The baculovirus/insect cell expression system has already been used to express a soluble extracellular domain of FLT-1, consisting of the Ig-like loops one to six, which retained the full capacity of VEGF binding (26). We used the same expression system to express different sFLT-1 mutants that were constructed by introducing artificial stop codons into appropriate positions, resulting in cDNAs for Ig-like loop one, Ig-like loop one and two, Ig-like loop one to three, Ig-like loop one to four, Ig-like loop one to five, and Ig-like loop one to seven. Monoclonal antibodies were raised against the extracellular domain of FLT-1 to confirm protein expression and to follow protein purification. mAb 7A6 was able to detect all different constructs, indicating that its binding epitope is located within the first N-terminal Ig-like loop (Fig. 2).
To investigate binding of VEGF to the various sFLT-1 mutants, we used
either a solid phase binding assay or an endothelial cell-based
receptor binding assay. High affinity binding of VEGF to sFLT-1(3),
(4), (5), and (7) could be observed with the solid phase binding assay.
The same sFLT-1 mutants were also able to compete with VEGF receptors
on HUVECs for VEGF binding. This is in agreement with the results
obtained with the solid phase binding assay (compare Figs. 3 and 4). No
binding of VEGF to sFLT-1(1) or (2) containing only the first, or the
first and second N-terminal Ig-like loops, respectively, could be
detected in the solid phase assay, and only a very weak competition, if any at all, was observed in cell-based binding assay (Figs. 3 and 4).
Furthermore, VEGF-stimulated DNA synthesis in HUVECs was inhibited in a
dose-dependent fashion by all sFLT-1 mutants except sFLT-1(1) and (2). From this we conclude that the contact site(s) of
VEGF is(are) located within the first three N-terminal Ig-like loops of
sFLT-1. We further hypothesize that especially Ig-like loop three
contributes to VEGF binding, since the deletion of this loop completely
abolishes VEGF binding. Similar results have been reported by others
for c-Kit and the PDGF receptor, two receptors related to the VEGF
receptors and consisting of five Ig-like loops in their extracellular
domain. In each case ligand binding could be mapped to the first three
Ig-like loops (27-29). Further mutational analysis for the PDGF
receptor type revealed that high affinity binding of PDGF is
mediated by Ig-like loops two and three and that loop one defines
specificity between PDGF AA or PDGF BB (27).
PlGF, a growth factor related to VEGF, has been described to bind to FLT-1 (23-25), and heterodimers of VEGF/PlGF have been found to activate VEGF receptors (30). Competition of PlGF with VEGF binding to FLT-1 has been found by others, suggesting that PlGF and VEGF share similar receptor recognition sites (23-25). We confirmed these results by testing the influence of PlGF on VEGF binding to sFLT-1 proteins in the solid phase binding assay (Fig. 5). PlGF was able to displace VEGF from both the entire extracellular domain sFLT-1(7) and sFLT-1(3), the shortest deletion mutant which retained VEGF binding.
The presence of potential N-glycosylation sites within the extracellular domain of FLT-1 and the difference between the apparent molecular weights obtained from SDS-PAGE and the calculated molecular weight, both suggest that sFLT-1(3) and larger sFLT-1 mutants were secreted as glycosylated proteins from infected Sf9 cells (Figs. 1 and 2). We therefore addressed the question, whether glycosylation of FLT-1 participates in the recognition of VEGF. Two lines of evidence demonstrate that glycosylation is not a prerequisite for ligand binding. First, enzymatic deglycosylation of sFLT-1 mutants, using N-glycosidase F, did not abolish VEGF binding (Fig. 7), and second, sFLT-1(3) expressed in E. coli retained the ability to bind VEGF (Fig. 8).
It has been shown, for a variety of receptor tyrosine kinases, that
receptor activation is preceded by receptor dimerization (for review,
see Heldin (13)). The ability of recombinant-purified extracellular
receptor domains to dimerize in the presence of the appropriate ligand
has already been described for the PDGF receptor and
(14). To
investigate whether the soluble extracellular domain of FLT-1 can
dimerize and whether dimerization is VEGF-dependent, we
performed chemical cross-linking experiments of sFLT-1 and VEGF
complexes. Western analysis identifying sFLT-1 mutants with the
monoclonal antibody 7A6 or the use of radiolabeled VEGF revealed that
soluble receptor mutants sFLT-1(4) and (5) are able to form dimers in
the presence of VEGF, whereas sFLT-1(3) associated predominantly in its
monomeric form with VEGF (Fig. 9). This leads us to the conclusion that
Ig-like loop four is of particular importance for the formation of
VEGF-mediated receptor dimerization. Similar observations have been
reported for c-Kit, a receptor consisting of five Ig-like loops on the
extracellular part. Ig-like loop four of c-Kit has been identified as
an intrinsic ligand-dependent dimerization site. Receptor
mutants of c-Kit lacking Ig-like loop four retained their capacity of
ligand binding but no longer formed receptor dimers (31). No
significant differences have been observed with respect to the affinity
for VEGF binding to all sFLT-1 mutants (Figs. 3 and 4). Thus it is
unlikely that Ig-like loop four has a strong impact on ligand
recognition. However, it might be responsible for a direct interaction
of two sFLT-1 monomers. Since efficient dimerization requires the
presence of VEGF, presentation of the active dimerization site seems to
be the consequence of a VEGF-dependent conformational
change. Unlike the other Ig-like loops, loop four lacks the disulfide
bridge, which is thought to stabilize the two
sheets of the Ig-like
loop structure. Thus Ig-like loop four might gain additional
flexibility. However, participation of other domains involved in
formation of receptor dimers cannot be excluded.
The results presented here clearly suggest that VEGF binding can be attributed to the first three N-terminal Ig-like loops and that Ig-like loop four is involved in the formation of receptor dimers.
While this work was under review, the participation of the first three Ig-like loops of FLT-1 in VEGF binding has been confirmed by Davies-Smyth et al. (32).