From the Departments of Cardiovascular Research and
Immunology, Genentech, Inc.,
South San Francisco, California 94080
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ABSTRACT |
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Flt-1 is one of two receptor tyrosine kinases through which the angiogenic factor vascular endothelial growth factor (VEGF) functions. Placenta growth factor (PlGF) is an additional ligand for Flt-1. The second immunoglobulin-like domain in the extracellular domain of Flt-1 has previously been identified as the region containing the critical ligand-binding determinants. We analyzed the contribution of charged residues within the first three domains of Flt-1 to ligand binding by alanine-scanning mutagenesis. Domain 2 residues Arg159, Glu208 and His223-Arg224 (together) affect both VEGF and PlGF binding, while Glu137, Lys171, His223, and Arg224 affect PlGF but not VEGF. Several charged residues, especially Asp187, are important in maintaining the structural integrity of domain 2. In addition, some residues in domain 3 contribute to binding (Asp231) or provide for additional discrimination between ligands (Arg280-Asp283).
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INTRODUCTION |
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Vascular endothelial growth factor (VEGF)1 is a major effector of vasculogenesis and angiogenesis (1-3). The pivotal role it serves is emphasized by the fact that loss of a single allele results in embryonic lethality (4, 5). VEGF activities are mediated by the endothelial cell-specific receptor tyrosine kinases, Flk-1/KDR and Flt-1 (2, 6, 7). Although structurally similar, the two receptors appear to invoke different signal transduction pathways, as indicated by the distinctive phenotypes associated with inactivation of each receptor gene (8, 9). Mouse embryos null for Flk-1 died in utero at day 8.5 and displayed a lack of endothelial cell precursors (8). Mice lacking Flt-1 also died in utero at day 8.5. However, although endothelial cells were present, they failed to organize into normal vascular channels (9).
Most of the work in the field has focused on the Flk-1/KDR receptor due to its strong VEGF-induced tyrosine autophosphorylation and its role in mitogenesis (10-13). Less is understood about the role of Flt-1. Although Flt-1 does exhibit VEGF-induced tyrosine phosphorylation, the signal is very weak compared with that seen with Flk-1/KDR (12, 14), and Flt-1 has not been shown to be directly involved in endothelial cell mitogenesis. However, Flt-1 also binds a VEGF-like molecule placenta growth factor (PlGF), which does not bind to Flk-1/KDR (15, 16). PlGF has minimal activity in endothelial cell growth assays and vascular permeability assays (16, 17), consistent with the concept that activation of Flk-1/KDR is required for both functions. However, in monocytes that express Flt-1, but not Flk-1/KDR, stimulation by either VEGF or PlGF leads to the production of tissue factor and chemotaxis (18, 19). Recently, naturally occurring PlGF/VEGF heterodimers have been identified (17, 20). Consistent with the ability of VEGF but not PlGF to bind to Flk-1/KDR, it has been shown that the PlGF/VEGF heterodimers can bind Flk-1/KDR (17). Cao et al. (21) also demonstrated that PlGF/VEGF heterodimers were approximately 50% less potent than VEGF homodimers in mitogenic assays but were equally potent in stimulating cell migration. In vivo mouse corneal neovascularization assays revealed that the PlGF/VEGF heterodimers could induce new vessel growth with similar vessel lengths as VEGF homodimers, although vessel density was reduced (21).
Keyt et al. (22) have shown that a plasmin-cleaved product of VEGF165, consisting of the first 110 NH2-terminal amino acids, is able to bind both Flk-1/KDR and Flt-1, suggesting that the binding determinants for both receptors reside within that region. Further analysis identified several charged residues that appeared to be associated with receptor binding (13). Residues Arg82, Lys84, and His86 were found to be critical for binding to Flk-1/KDR, while negatively charged amino acids Asp63, Glu64 and Glu67, were associated with Flt-1 binding (13).
Recently, we and others demonstrated that of the seven Ig-like domains within the extracellular domain (ECD) of Flt-1, the second Ig-like domain is critical for ligand binding (23-25). In the context of a recombinant chimeric receptor, the second domain of Flt-1 was sufficient for either VEGF or PlGF to bind and initiate a signal transduction cascade (23). We also demonstrated that a soluble receptor immunoadhesin (26) possessing only the first three ECD domains was sufficient to bind VEGF with the same affinity as the full-length ECD (23). In addition, PlGF can compete with VEGF for binding to constructs possessing the first three domains of Flt-1, further supporting the concept that PlGF and VEGF share similar contact sites on the receptor (23-25).
To better understand VEGF and PlGF action, analysis of the Flt-1 ligand-binding site would be useful. In the present study, using alanine-scanning mutagenesis, we examine the role of charged residues within the first three domains of Flt-1 in ligand binding. We demonstrate that at least one charged residue within the second domain serves a critical role in maintaining proper conformation of the domain and that only two of the charged residues within the second domain appear to be directly involved in ligand interaction. Furthermore, we examine the contribution of both the second and third domains of Flt-1 in distinguishing VEGF from PlGF binding.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis and Expression of Flt(1-3)IgG
Variants--
Mutants were created via oligonucleotide-directed
mutagenesis performed according to the method of Kunkel (27). A plasmid vector, pHEBO23 (28), containing cDNA encoding the signal peptide and first three Ig-like domains of Flt-1 fused to the human heavy chain
IgG1 (23) was used for mutagenesis and expression. Variants were
screened and verified by sequence analysis using the Dye Terminator
Cycle Sequencing Ready Reaction kit (Perkin Elmer) followed by
application of the Sequencher 3.01 program (Gene Codes Corp.).
Double-stranded DNA encoding each selected variant was prepared using
the Qiagen DNA purification kit (catalog number 12163, QIAGEN Inc.) and
used for transfection into CEN4 cells via calcium phosphate
precipitation. Forty-eight hours post-transfection, conditioned medium
was collected from transiently expressing cells, and the concentration
of soluble receptor was determined by an enzyme-linked immunosorbent
assay detecting human Fc-IgG.
Preparation of 125I-Labeled VEGF165, PlGF152, and Rabbit Anti-mouse IgG-- Radiolabeling of Escherichia coli-derived recombinant human VEGF165, recombinant human PlGF152 purified from conditioned medium of stably expressing CEN4 cells, and purified rabbit anti-mouse IgG (Cappel) was performed using a modification of the chloramine T-catalyzed iodination reaction (29). A typical reaction consisted of 10 µg of VEGF, 3 µg of PlGF, or 10 µg of rabbit anti-mouse IgG in phosphate-buffered saline containing 0.01% Tween 20 added to a half-volume of 1 M Tris-HCl, pH 7.5, with 0.01% Tween 20, followed by the addition of 0.5 mCi of sodium iodide-125 (NEN Life Science Products). The reaction was catalyzed with 10 µg of chloramine T (Aldrich), resuspended in 100 mM sodium phosphate buffer, pH 7.5, and incubated at room temperature for 1 min. Following the termination of the reaction by the addition of 20 µg of sodium metabisulfate (Aldrich) dissolved in sodium phosphate buffer, the radiolabeled protein was isolated on a PD-10 Sephadex G-25 column (Pharmacia Biotech Inc.) that was preequilibrated with phosphate-buffered saline containing 0.5% bovine serum albumin and 0.01% Tween 20. The average specific activity of the iodinated recombinant human VEGF165 and recombinant human PlGF152 was 29.01 × 106 cpm/µg and 54.65 × 106 cpm/µg, respectively. Rabbit anti-mouse IgG labeled with a specific activity of 29.5 × 106 cpm/µg.
Soluble Receptor Binding Assays--
Assays were performed as
described previously (23) with a few modifications. In brief, 96-well
breakaway immunoabsorbent assay plates (Nunc) were coated overnight at
4 °C with 1 µg/well rabbit IgG anti-human Fc-IgG (Cappel) in 50 mM sodium carbonate buffer, pH 9.6. Prior to the addition
of 100 µl of binding reaction per well, the wells were blocked with
phosphate-buffered saline containing 10% fetal bovine serum (buffer B)
at room temperature for 1 h. Binding reactions consisted of 10 ng/ml soluble receptor-IgG, either 125I-labeled recombinant
human VEGF165 or 125I-labeled recombinant human
PlGF152 (~9000 cpm/100-µl reaction), and cold
competitor protein where indicated. The reactions were assembled and
allowed to equilibrate overnight at 4 °C prior to loading into the
coated wells. Incubation in the wells proceeded either for 4 h at
25 °C or overnight at 4 °C. Following removal of the supernatant
from the wells and several washes with buffer B, the amount of bound
protein was determined by counting individual wells in a -counter.
In addition, the unbound radiolabeled proteins in the supernatant was
also determined. The data were analyzed and reported as percentage
relative to the amount bound to wild-type Flt(1-3)IgG. For competition
studies, a four-parameter nonlinear curve-fitting program
(Kaleidagraph, Synergy Software) was used to determine the point of
inflection.
Antibody Binding-- Monoclonal antibodies were generated against the Flt-1 ECD region (Hybridoma Group, Genentech, Inc.). For binding, 1 ng of a receptor-IgG variant was incubated with 2 ng of monoclonal antibody (mAb) and 125I-labeled rabbit anti-mouse IgG (~6000 cpm/100-µl reaction) in buffer B at 4 °C overnight. The reactions were then placed in immunoabsorbent assay plates that had been treated as described above, and incubation continued to proceed either for 4 h at 25 °C or overnight at 4 °C. After the removal of unbound supernatant and several washes with buffer B, the amount of monoclonal antibody bound to receptor-IgG variant was determined by counting the amount of radiolabeled material bound to the well. The results were then normalized to that bound to the wild-type Flt(1-3)IgG.
Molecular Modeling of the Flt-1 Binding Domain--
The model of
human Flt-1 domain 2 (Flt-1d2) was based on the crystal structure of
telokin (30). The sequence alignment of human Flt-1 domain 2, as well
as human KDR domain 2, with telokin is shown in Fig.
1. Telokin was transformed into Flt-1d2
in three steps. First, all residues in the -strands were changed to
the Flt-1d2 sequence using the INSIGHT-II program (Biosym Technologies, San Diego, CA). If possible, conformations of Flt-1d2 side chains were
kept similar to those of telokin; otherwise, they were based on
INSIGHT-II rotamer libraries, packing, and hydrogen bond
considerations. Second, loop structures connecting the
-strands were
gleaned from a search of loops of similar size from the Protein Data
Bank (31) using the loop search algorithm in the INSIGHT-II program. Finally, the relatively large segment connecting
-strands C and E
(20 residues long; Phe172-Gly191) was modeled
in several conformations by a combination of loop searching and manual
adjustment.
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RESULTS |
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Alignment of VEGF and PlGF Sequences-- The sequences of mature, secreted human VEGF165 and PlGF152 were aligned based upon the 10 conserved cysteine residues (Fig. 2). PlGF shares 47% sequence identity with VEGF within the first 110 residues and 63% similarity overall. Two of the negatively charged residues previously identified as being important in the interaction of VEGF with Flt-1, Asp63 and Glu64, are conserved in PlGF, whereas a histidine replaces Glu67, which has also been implicated in VEGF binding to Flt-1 (13). It is also noteworthy that positively charged VEGF residues identified as being involved in Flk-1/KDR binding (Arg82, Lys84, and His86) are not conserved in PlGF (13, 33).
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Charge-to-Alanine Mutagenesis within the First Three Domains of Flt-1-- Based on the involvement of charged residues in the binding site on VEGF, we opted to examine the influence of charged residues within the first three domains of Flt-1 on ligand binding. Thirty-five mutants of the Flt(1-3)IgG soluble receptor were generated where 1-5 neighboring charged amino acids were replaced with alanine(s) (Table I). This analysis focused on both acidic and basic amino acids (Asp, Glu, Lys, Arg, and His). Each of the resulting mutant receptors was then tested for its ability to bind either 125I-VEGF or 125I-PlGF relative to wild-type Flt(1-3)IgG. The overall ligand binding profiles of the variant receptors were very similar (Table I). The receptors that bound the least amount of ligand (AS170, AS180, and AS187) all had mutations occurring within the second domain.
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Analysis of the Effects of Individual Charge-to-Alanine Mutations-- Since several of the multiple alanine mutations resulted in an impaired conformation, we further analyzed domain 2 by separating some of the clusters of mutations and generating variants of Flt(1-3)IgG, which had single residue charge-to-alanine changes. The variants were tested for their ability to bind both VEGF and PlGF. The single residue mutations can be separated into three classes: those that had minimal or no effect on binding (D175A, K182A, R183A, R189A, K190A, and K200A), those that affected binding of both VEGF and PlGF, and those that affect binding of PlGF but not VEGF (Table I).
Although several individual charge-to-alanine mutants did exhibit decreases in the amount of both VEGF and PlGF bound (R159A, K170A, D187A, and E208A), only D187A showed a significant reduction in bound ligand (Table I). D187A also bound poorly to the panel of antibodies and hence is probably conformationally impaired. In the model of Flt-1 domain 2, the Asp187 side chain accepts hydrogen bonds from the Arg189 and Lys190 backbone nitrogens, thereby stabilizing a single turn of
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Role of Domain 3 in Binding--
Although domain 2 may be the
primary site of binding for VEGF and PlGF (23-25), amino acids located
in domain 3 that are near domain 2 might also influence ligand binding.
Examination of the ligand binding profiles of the alanine scan mutants
in domain 3 highlights two mutants: AS231 and AS279 (Table I).
Asp231 should be at the N-terminal end of -strand A in
domain 3 (Fig. 1) near the domain 2 interface. This mutation decreased
the amount bound of both VEGF and PlGF by about 50% but did not
perturb binding to the antibodies directed against domain 2 (Table I).
AS279 includes residues 279-283 of Flt-1 domain 3 and resulted in a 30% reduction in the amount of VEGF binding; PlGF appeared to be more
sensitive to the charge-to-alanine changes, since the amount of ligand
bound decreased by 60% (Table I). Interestingly, the antibody
screening showed that domain 1 was not affected by the alterations, but
there was a slight increase in the amount of binding by the antibodies
that recognized domain 2 (Table I). When residues 279-283 were instead
changed to the sequence found in the KDR receptor,
R280D/R281L/I282K/D283T (F280-283K), the difference between VEGF and
PlGF binding by this mutant receptor was further enhanced compared with
AS279 (where all charges were changed to alanine) (Table I). Although
the amount of VEGF bound was comparable with wild type, there was a
slight reduction in the binding affinity for VEGF as evidenced by the
mutant:wild type IC50 ratio of 0.68 ± 0.16.
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DISCUSSION |
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Examination of the charge-to-alanine scans emphasized that the
domain 2 of Flt-1 is important for ligand binding, in agreement with
previous work (23-25). Residues Arg159,
Glu208, and His223/Arg224 affect
both VEGF and PlGF binding, while Glu137,
Lys171, His223, and Arg224 affect
PlGF but not VEGF. Most of these residues (except Arg159)
are located on the side of domain 2 comprising -strands C, F, and G
(Fig. 4). The
-sheet consisting of strands C, F, and G also contains
many exposed hydrophobic residues (Ile145,
Phe172, Leu204, and Leu221) (Fig.
4) that were not evaluated in this study. Another patch of hydrophobic
residues (Val155, Ile185, and
Ile193) are located adjacent to Asp187 (Fig.
4).
Several charged residues were ascertained to play a role in maintaining the structural integrity of domain 2, as determined by monoclonal antibody binding. Interactions between an antibody and antigen (in this case a receptor) are dependent on shape complementarity of the interacting surfaces as well as direct contact with the epitope site (36, 37). Disruption of either the epitope site or a receptor's structural conformation can lead to loss of antibody binding (38-40). Utilizing several antibodies that recognize different epitopes may help in distinguishing between these possibilities (36-40).
Asp187 may be required to maintain the conformation of the
loop between -strands D and E; the binding of monoclonal antibodies, which recognize either the first or second domains of Flt-1, was reduced when either the negative charge or side chain size was altered
(Table I). The role of other residues, e.g.
Lys170 and Glu201, was less pronounced than
that of Asp187, since the binding of the anti-Flt-1
antibodies was reduced much less than for the D187A mutant (Table
I).
Two important residues in domain 2 are His223 and Arg224. When these residues were changed to alanine, either individually or together, the resulting variants did not show any significant structural changes as determined by antibody binding (Table I). There was, however, a dramatic difference in receptor binding between VEGF and PlGF. PlGF binding was much more sensitive than VEGF to the removal of the positive charges in Flt-1 (Table I). Two other residues in domain 2, Glu137, distant from His223 and Arg224 (Fig. 4), and Lys171 also discriminate between VEGF and PlGF (Table I).
Residues in domain 3 also contribute to the interaction. D231A affected binding of both VEGF and PlGF, while residues 280-283 discriminated between the two ligands. The latter, when changed to the sequence of human KDR receptor, affected PlGF binding more than VEGF binding, suggesting that this segment of amino acids may contribute in preventing PlGF from binding to KDR.
The crystal structure of VEGF has recently been reported (33, 41). VEGF forms an antiparallel homodimer covalently linked by two disulfide bridges. Hence, each homodimer contains two binding sites located at each end of the dimer, and each binding site has residues contributed by both monomers: Ile43, Ile46, Gln79, Ile83, Lys84, and Pro85 from one monomer and Phe17 and Glu64 from the second monomer (33). The presence of five hydrophobic residues in the binding site on VEGF implies that part of the binding site on the receptors also contains hydrophobic residues. Hydrophobic residues were not evaluated in the present study, but several hydrophobic residues lie among His223/Arg224, Lys171, and Glu137, residues determined to be important for ligand binding and discrimination.
Although crystal structure information on PlGF is not available, based on the similarities between VEGF and PlGF (41), one might predict that a PlGF/VEGF heterodimer would also form in an antiparallel fashion with receptor binding sites at each end of the dimer. Speculating that a PlGF/VEGF heterodimer could bind Flt-1, one might hypothesize that mutations affecting binding to both homodimers (R159A, E208A, and H223A/R224A) would also affect binding to the heterodimer. It is more difficult to predict how mutations that affect PlGF homodimer binding but not VEGF binding (E137A, K171A, H223A, and R224A) would bind a heterodimer. Screening of the receptor mutants generated in this study with a PlGF/VEGF heterodimer will provide more information as to how a heterodimer may interact with Flt-1. In addition, alanine-scanning mutagenesis of PlGF will help define critical residues within that molecule involved in receptor binding.
Since VEGF binds to both Flt-1 and KDR, the Flt-1 residues involved in binding VEGF might be expected to have an identical or similar character in the KDR receptor. Comparison of the amino acid sequences of the two receptors, however, shows that this is not the case. While Glu208 and Asp231 are conserved, Arg159 is a leucine in KDR and His223 and Arg224 are both valine in KDR (Fig. 1). Of the Flt-1 residues that discriminate between VEGF and PlGF, Glu137 is an alanine in KDR and Lys171 is an arginine. Even more interesting is that several of the amino acids that help maintain structure are conserved in both Flt-1 and KDR: Asp187, Asp180, and Glu141 (aspartic acid in KDR). This suggests that domain 2 of Flt-1 and KDR are structurally similar but that the orientation of VEGF when it binds to domain 2 differs between Flt-1 and KDR. To determine if such a difference does occur, the crystal structure of KDR/VEGF must be compared with the crystal structure of the complex of Flt-1 domain 2 with VEGF; only the latter structure has been solved (42).
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ACKNOWLEDGEMENTS |
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We thank the following groups at Genentech for their invaluable support: the DNA Synthesis Group for oligonucleotides used in mutagenesis reactions, the Hybridoma Group for generating and purifying the monoclonal antibodies, the Sequencing Group for running the sequencing gels, and Assay Services for information on the Fc-detecting enzyme-linked immunosorbent assay. We also acknowledge Drs. A. M. de Vos and C. Wiesmann (Department of Protein Engineering, Genentech) for helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Cardiovascular Research, Genentech, Inc., 1 DNA Way, S. San Francisco, CA 94080. Tel.: 650-225-2968; Fax: 650-225-6327.
1 The abbreviations used are: VEGF, vascular endothelial growth factor; PlGF, placenta growth factor; ECD, extracellular domain; mAb, monoclonal antibody.
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REFERENCES |
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