(Received for publication, September 14, 1995; and in revised form, December 11, 1995)
From the
Vascular endothelial growth factor (VEGF) expression in various
cell types is induced by hypoxia and other stimuli. VEGF mediates
endothelial cell proliferation, angiogenesis, vascular growth, and
vascular permeability via the endothelial cell receptors, kinase insert
domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and
FLT-1. Alanine-scanning mutagenesis was used to identify a positively
charged surface in VEGF that mediates binding to KDR/Flk-1.
Arg, Lys
and His
, located in a
hairpin loop, were found to be critical for binding KDR/Flk-1, while
negatively charged residues, Asp
, Glu
, and
Glu
, were associated with FLT-1 binding. A VEGF model
based on PDGFb indicated these positively and negatively charged
regions are distal in the monomer but are spatially close in the dimer.
Mutations within the KDR site had minimal effect on FLT-1 binding, and
mutants deficient in FLT-1 binding did not affect KDR binding.
Endothelial cell mitogenesis was abolished in mutants lacking KDR
affinity; however, FLT-1 deficient mutants induced normal
proliferation. These results suggest dual sets of determinants in the
VEGF dimer that cross-link cell surface receptors, triggering
endothelial cell growth and angiogenesis. Furthermore, this mutational
analysis implicates KDR, but not FLT-1, in VEGF induction of
endothelial cell proliferation.
VEGF ()is a specific mitogen for vascular endothelial
cells in vitro and a potent angiogenic and vascular
permeability enhancing factor in
vivo(1, 2, 3, 4, 5) .
VEGF, also known as vascular permeability factor, is expressed in
response to hypoxia and other stimuli by a variety of differentiated
cells(1, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14) .
VEGF expression has also been found in numerous human and rodent
transformed
cells(3, 4, 15, 16, 17, 18, 19) .
VEGF is encoded by a single gene, but it exists in four isoforms of
121, 165, 189, and 206 amino acids due to alternative mRNA
splicing(20, 21) . The two low molecular weight forms,
VEGF
and VEGF
, are secreted as soluble
factors, while the other higher molecular weight forms, VEGF
and VEGF
, are secreted but remain bound to
extracellular matrix(22) . Of the four isoforms, VEGF
is the most abundantly expressed splice variant.
VEGF is a heparin binding glycoprotein with a single glycosylation
site (at Asn
) and is secreted as a homodimer of
approximately 45 kDa(3, 17) . VEGF
can
be cleaved by plasmin to yield VEGF
, which is equipotent
to VEGF
with respect to mitogenic activity on endothelial
cells(22, 23) . The amino acid sequence of
VEGF
exhibits limited but significant homology (17%) with
PDGF. All eight cysteine residues, which are involved in intra- or
interchain disulfides, are conserved among these growth factors. A cDNA
sequence encoding a similar protein was identified by screening a human
placental cDNA library(24) . Placental growth factor (PLGF),
with 47 and 18% sequence homology with VEGF and PDGF, respectively, is
recognized as a member of the PDGF/VEGF family of growth factors.
Two tyrosine kinases, fms-like tyrosine kinase (FLT-1) and the kinase insert domain-containing receptor (KDR/Flk-1), have been identified as high affinity VEGF receptors(25, 26, 27) . The murine homolog of KDR, fetal liver kinase 1 (Flk-1) has 85% homology with the human VEGF receptor(28) . These receptors are localized on the cell surface of various endothelial cell types(29, 30) . The FLT-1 and KDR/Flk-1 genes encode tyrosine kinase receptors, characterized by an extracellular domain containing seven immunoglobulin-like domains and a split tyrosine kinase intracellular domain(31) . In many aspects, the VEGF receptors are structurally and functionally similar to the PDGF receptors, which have five immunoglobulin-like repeats in the extracellular domain(32, 33) . In addition to VEGF, PLGF has also been demonstrated to bind FLT-1 with high affinity, but not to KDR/Flk-1 (34, 35) . VEGF is a potent endothelial cell mitogen in vitro, while PLGF is 3 orders of magnitude less potent at inducing endothelial cell proliferation(4, 15, 16, 17, 18) . However, PLGF can significantly potentiate the in vitro and in vivo activity of low concentrations of VEGF(34) .
To gain a better understanding of the biological activity of VEGF,
we began an analysis of structure/activity relationships using
site-directed mutagenesis of VEGF. In the current study, we report on
the mutational analysis of VEGF and identify the receptor binding
determinants for KDR/Flk-1 and FLT-1. Furthermore, the epitope mapping
of a neutralizing monoclonal antibody is also described. We used the
strategy of charged-to-alanine scanning mutagenesis to evaluate the
role of various sites on VEGF in receptor binding. To confirm the
results of alanine scanning, we have also introduced novel
glycosylation sites in VEGF to inhibit receptor binding with additional
carbohydrate. We report that KDR and FLT-1 receptors bind to different
sites on VEGF, which may serve to dimerize tyrosine
kinase receptors resulting in endothelial mitogenesis, proliferation,
and initiation of angiogenesis and vasculogenesis.
Figure 1:
Alignment of VEGF,
PLGF
, PDGFa, and PDGFb. The receptor binding domain of
VEGF(1-110) is shown aligned with the corresponding sequence of
mature, secreted PLGF(9-118). The numbering on top is that of
secreted VEGF. The sequence of PDGFa that is shown(71-185)
contains part of the amino-terminal propeptide that is removed by a
processing cleavage at Arg
-Ser
, which
yields the mature PDGFa. Similarly, the sequence of PDGFb shown
above(72-186) contains 10 amino acids of the amino-terminal
propeptide that are removed by cleavage at
Arg
-Ser
, which yields the mature form
of PDGFb. Note that amino acid numbering of PDGF is based on the cDNA
sequence with Met at position 1, whereas the sequences of VEGF and PLGF
are based on the numbering of the mature, secreted
polypeptide.
Figure 2: Quantitation of VEGF mutants by monoclonal- and polyclonal-based ELISA. Aliquots of conditioned cell media with VEGF or VEGF mutants were analyzed by immunoassay using two types of ELISA. A polyclonal anti-VEGF antibody combined with a monoclonal antibody (mAb 5F8, specific to the carboxyl-terminal domain of VEGF) yielded a sandwich-type immunoassay that was relatively unaffected by mutations in the receptor-binding domain of VEGF (1-110 region). Alternatively, a dual monoclonal-based ELISA with mAbs 5F8 and A4.6.1 was used to quantify the VEGF mutants. The immunoassay results of multiple transfections (2-10 replicates) were averaged for each mutant and compared in the figure. Mutations that were associated with loss of A4.6.1 monoclonal immunoreactivity, R82A, R82N/I83L/K84S, R82A/K84A/H86A, and H90A/E93A are indicated by the respective mean residue numbers 82, 83, 84, and 91.5.
Figure 3:
SDS-PAGE immunoblot of VEGF mutants.
Transient transfection supernatants from 293 cells containing
approximately 10-20 ng of VEGF or VEGF mutant were analyzed by
nonreduced SDS-PAGE. The gels were transferred and blotted as described
under ``Experimental Procedures,'' using a panel of five
murine monoclonal anti-human VEGF antibodies identified
as 2E3, 4D7, A4.6.1, 5C3, and 5F8.
Figure 4:
Receptor binding of alanine scan mutants
of VEGF. The VEGF mutants were expressed in 293 cell
culture, and the conditioned cell medium was used to displace wild-type
VEGF from binding receptor-IgG chimeras. The mean residue number is the
average of the amino acid positions that were altered by mutation. The
values are expressed as the concentration required to half-maximally
inhibit (IC
) the binding of radiolabeled CHO-derived VEGF
to KDR-IgG (panel A) or FLT-1 IgG (panel B). The
binding assays were done in the presence (filled circles) or
the absence (open boxes) of heparin at 10 µg/ml. These
experiments were performed in triplicate; errors bars indicate
standard deviation.
Many of the
mutant proteins exhibited binding comparable with wild-type VEGF. In
fact, the binding to KDR for 19 out of 25 alanine scan mutants was
similar to that of wild-type VEGF; the average IC for
mutants with wild-type phenotype was 29 ± 18 pM (n = 19). Three mutants (E42A, E44A, and D63A) exhibited an
apparent 4-6-fold increased binding to KDR receptor compared with
wild-type VEGF. Since this effect (for two of these mutants) was
observed for FLT-1 binding as well, the potential exists for under
quantitation of selected mutants by the polyclonal/monoclonal ELISA.
However, the most significant effect on binding was observed with the
R82A, K84A, H86A mutant of VEGF, which exhibited 1000-fold decreased
affinity for the KDR receptor in the absence of heparin, relative to
that of wild-type VEGF (Fig. 4A). Interestingly, in the
presence of heparin, the binding of this triple mutant was only 10-fold
decreased compared with that of wild-type VEGF. These results are
consistent with VEGF binding to KDR as a function of two sites of
interaction, a heparin-independent site in the 1-110 dimer and a
heparin-dependent binding site in the 111-165 domain. When the
binding of VEGF
to KDR is mediated entirely by the
1-110 region (in the absence of heparin) the mutations at 82, 84,
and 86 severely compromise the binding of VEGF to KDR.
To evaluate the relative contribution of individual residues, single amino acid substitution mutants of VEGF were constructed. The single mutations, R82A, K84A, and H86A were found to display more modest decreases with respect to KDR binding (Table 1). R82A VEGF exhibited wild-type KDR binding, while K84A VEGF and H86A VEGF were approximately 5.5- and 1.8-fold decreased in binding compared with that of wild-type VEGF, respectively. While the 84 position of VEGF was most dominant in the triple alanine mutant, the combination of mutations at 82, 84 and 86 clearly exhibited a synergistic effect on the interaction with the KDR receptor. In addition to the major KDR binding determinant, a minor site was observed in the 63-67 region. The triple mutant, D63A/E64A/E67A VEGF, was 2.4- and 3.0-fold reduced in binding to KDR in both the presence and absence of heparin. The single amino acid mutant E64A VEGF exhibited 7-fold decreased affinity for KDR, which implicated this site more than neighboring mutations in this region, i.e. D63A and E67A. Although modest in comparison to the major effects observed with R82A/K84A/H86 VEGF, the most potent effects with single alanine replacement of charged amino acids were observed for E64A VEGF and K84A VEGF.
We inserted potential neoglycosylation sites to
observe the effects of carbohydrate addition at or near putative
receptor binding sites. Surface accessible sites were considered
optimal in exterior loops or turns as predicted on the basis of the
crystal structure of PDGFb dimer (50) . One such site
(42-44 region) was selected as a control since no receptor
binding determinants were identified in this region by
charged-to-alanine scanning mutagenesis. The neocarbohydrate site in
E42N/E44S VEGF was apparently glycosylated, as indicated by the
increased molecular weight observed on SDS-PAGE immunoblots (Fig. 3). The N-linked carbohydrate at position 42 did
not interfere with binding to KDR or FLT-1 receptors as indicated by
IC values of 15 and 13 pM, respectively.
Figure 5:
Competitive displacement of I-labeled VEGF
from KDR or FLT-1 with
triple alanine scan or glycosylation mutants. Displacement curves with
KDR-IgG (panel A) or FLT-1 IgG (panel B) binding
labeled VEGF in competition with wild-type VEGF (filled
circles), D63A/E64A/E67A VEGF (open boxes), D64N/L66S
VEGF (filled boxes), R82A/K84A/H86A VEGF (open triangles),
R82N/I83L/K84S VEGF (filled triangles). These experiments were
performed in duplicate in the absence of
heparin.
Figure 6: Endothelial cell proliferation in response to VEGF and VEGF mutants in the 63-67 region and 82-86 region. VEGF-induced growth of endothelial cells was assessed as the percent of maximal growth observed with basic fibroblast growth factor at 5 ng/ml. The results with CHO-derived VEGF (open circles), 293 cell-derived VEGF (open boxes), D63A/E64A/E67A VEGF (filled circles), R82A/K84A/H86A VEGF (open triangles), and R82N/I83L/K84S (filled triangles) were plotted as a function of growth factor concentration. These experiments were done in triplicate. The error bars indicate (±) standard deviation.
KDR and FLT-1 receptor binding determinants on VEGF, identified by site-directed mutagenesis, were localized on a model of VEGF based on the crystal structure of a homologous protein, PDGFb. VEGF and PDGFb display 28% amino acid sequence homology with conserved alignment of eight cysteines within the receptor binding domain (i.e. 1-110 of VEGF). The crystal structure of PDGFb dimer has been elucidated at a 3.0-Å resolution(50) . A model of the three-dimensional structure of PDGFb dimer is shown (see Fig. 7), with the appropriate amino acids of VEGF highlighted that are involved in receptor binding as identified by mutational analysis.
Figure 7:
Three dimensional model of
the(1-110) domain of VEGF dimer and the positions of two types of
mutations. The model is based on the crystal structure of PDGFb
dimer(50) . Shown as an oval ribbon diagram is the
polypeptide backbone in green, and disulfides in yellow. The ribbon begins with residue 17 (VEGF numbering) and
extends to residue 109 with a gap in sequence due to unresolved
structure (from 37 to 45). The side chains of acidic amino acids
involved in FLT-1 receptor binding are shown in red for
Asp, Glu
, and Glu
. The side
chains of basic amino acids involved in KDR receptor binding are shown
in blue for Arg
, Lys
, and
His
. PDGFb has three additional amino acids; Arg, Lys, Lys
(shown in gray) that are conserved in PDGFa, but not VEGF or
PLGF (see Fig. 1).
The polypeptide chain of each monomer is folded into two
twisted anti-parallel pairs of -strands (shown as green
ribbons, Fig. 7). The amino termini of the PDGFb dimer were
not resolved, and the structure begins at residue 17 in VEGF numbering.
Three intramolecular disulfide bonds are arranged in a knotted
configuration, shown in yellow for
Cys
-Cys
,
Cys
-Cys
, and
Cys
-Cys
(VEGF numbering, see (48) for PDGF disulfides). Two intermolecular disulfide bonds
form the covalent linkage of the homodimeric structure via cysteines at
positions 51 and 60. Dimerization leads to an elongated structure with
three surface loops that cluster at each end of the molecule. Oefner
and co-workers (50) suggest that these loops would probably
form receptor recognition sites for PDGF. The surface loops, denoted as
I, II, and III in PDGFb, include the corresponding regions of VEGF from
36 to 46, 61 to 68, and 84 to 87, respectively (VEGF numbering). Loop I
is not resolved in the PDGFb structure and is denoted by a gap in the
peptide strand from residues 37 to 45 (VEGF numbering). Loops II and
III in VEGF are involved in receptor recognition as indicated by the
binding studies with mutants D63A/E64A/E67A VEGF and R82A/K84A/H86A
VEGF, respectively. Interestingly, the loop II residues
Asp
, Glu
, and Glu
that mediate
VEGF binding predominantly to FLT-1, form a negatively charged surface
at one end of each monomer (residues 63, 64, and 67 shown in red, see Fig. 7). In contrast, the loop III associated
residues, Arg
, Lys
, and His
,
which predominantly mediate binding to KDR, cluster to form a
positively charged surface at the other end of the VEGF monomer
(residues 82, 84, and 86 shown in blue). These oppositely
charged surface loops are at distal ends of the monomer, but they are
in close proximity in the dimeric form of VEGF. Although the receptor
binding regions of loop II and III predominantly mediate binding of
FLT-1 and KDR, respectively, there is indication that KDR interacts
with residues in both loops. For example, the VEGF mutant with a
potential extraglycosylation site at position 64 exhibits reduced
affinity for KDR as well as for FLT-1 receptors (
6- and 40-fold,
respectively). Altogether, the functional analysis of VEGF by
site-directed mutagenesis indicates that the VEGF dimer exhibits
receptor binding regions at both ends of the protein.
Two sets of
receptor binding sites on VEGF interact predominantly with FLT-1 or
KDR. These sites, shown schematically as A and B in Fig. 8, display binding to KDR and FLT-1, respectively. Site A,
composed of basic amino acids (including Arg,
Lys
, and His
) predominantly mediates the
interaction of VEGF with KDR. It is interesting to note that in recent
studies, a cationic series of amino acids in a pentapeptide sequence of
PDGF
(Val
-Arg
-Lys
-Lys
-Pro
,
PDGF numbering) has been demonstrated to play a critical role in ligand
binding to the PDGF
receptor(52) . This sequence is
perfectly conserved between PDGFa and PDGFb, but is not found in either
VEGF or PLGF (see Fig. 1). The cationic sequence is located in
the loop III region of the PDGF structure, which corresponds to amino
acids 80-90 of VEGF. PDGF and VEGF appear to share a requirement
for basic amino acids in the loop III region (site A) in binding to
PDGF
receptor and KDR, respectively. These data do not exclude
significant contributions by noncharged amino acids, such
Gln
, Ile
, Met
, Ile
,
and/or Pro
.
Figure 8: VEGF displays different receptor binding sites for KDR and FLT-1. This schematic diagram provides a functional description of the two receptor epitopes identified by mutational analysis. The A site, composed of basic residues (region 82-86), mediates the binding to KDR. The B site, composed of acidic residues (region 63-67), mediates the interaction with FLT-1 receptor. This diagram illustrates bivalent epitopes at opposite ends of VEGF, suggesting a possible mechanism for ligand-induced dimerization of receptors.
The B site of VEGF, composed of acidic
residues (including Asp, Glu
, and
Glu
) is shown in Fig. 8as weakly interacting with
the KDR receptor. This second site, however, appears to be directly
involved with the binding of VEGF to FLT-1. LaRochelle et
al.(54) , utilizing site-directed mutagenesis of PDGF,
have described similar findings with respect to mapping the
/
receptor binding determinants. Mutations of Asn
and
Arg
in PDGF, which correspond to Asp
and
Glu
of VEGF (see Fig. 1), have been shown to be
essential for the
/
receptor specificity of PDGFa and PDGFb.
In the present study, the effect of mutations within the loop II of
VEGF (site B) did not strongly affect the binding of VEGF
to FLT-1 as compared with the effect of loop III mutations (site
A) on KDR binding. The alanine replacement of Asp
,
Glu
, and Glu
resulted in 30-fold decreased
affinity with FLT-1, whereas the R82A/K84A/H86A mutant exhibited
1000-fold decreased binding to KDR (see Table 1). Noncharged
amino acids such as Asn
and Leu
may also
contribute to the interaction of VEGF with FLT-1. These data suggest
additional site(s) of FLT-1 interaction, possibly in the
carboxyl-terminal domain, residues 111-165 of VEGF. Amino acids
in site A appear not to interact with the FLT-1 receptor, as indicated
by the wild-type phenotype associated with R82A/K84A/H86A and
R82A/I83A/K84A mutations.
The 2-fold symmetry of PDGFbb localizes
the three loop regions on either end of the molecule(50) . The
evidence of receptor binding sites at both ends of the dimer is
consistent with the growth factors of the PDGF/VEGF family exhibiting
bivalency. Fretto et al.(55) have established that
PDGF is bivalent with respect to PDGF receptor binding(55) .
Bivalent binding of VEGF provides a mechanism for dimerization of the
KDR and/or FLT-1 tyrosine kinase receptors on endothelial cells.
Consistent with cross-linking of tyrosine kinase receptors followed by
tyrosine phosphorylation, VEGF has been demonstrated to stimulate
tyrosine phosphorylation of a 200-kDa membrane protein from endothelial
cells(56, 57) . It remains to be seen if KDR and FLT-1
can form heterodimeric receptors on endothelial cells, as has been
postulated by Waltenberger et al.(57) . The concept of
heterodimerization of receptor tyrosine kinases is well documented,
particularly in the case of /
heterodimers of PDGF receptors,
which differentially bind PDGFa and PDGFb(58) . The recently
reported finding of VEGF/PLGF heterodimers adds another layer of
complexity with respect to various forms of VEGF and PLGF, which have
differential specificity for FLT-1 and/or KDR(59) .
In the present study, we describe VEGF mutants that are relatively selective for KDR or FLT-1 receptors. These mutants, with differential receptor specificity, will provide molecular tools to probe the relative functional significance of KDR and FLT-1 receptors. The role of KDR and FLT-1 in stimulating endothelial cell proliferation has been addressed using the VEGF mutants. R82A/K84A/H86A and R82N/I83L/K84S mutants of VEGF are significantly decreased in both KDR binding and endothelial cell growth assays ( Fig. 5and Fig. 6and Table 1). Furthermore, we have observed that the FLT-1 deficient mutants (D63A, E64A, E67A, and E64N/L66S forms of VEGF) display full activity with respect to endothelial cell proliferation. These results are consistent with the lack of endothelial growth in response to PLGF, which is specific for FLT-1 but does not bind KDR(34) . These data, with the present study, correlate the mitogenic and proliferative effects of VEGF with KDR binding and subsequent receptor phosphorylation. In another study, it was shown that KDR-expressing cells respond to VEGF stimulation with marked changes in cell morphology, actin organization, chemotaxis, and mitogenicity, whereas FLT-1 expressing cells lacked these responses to VEGF(57) . The significance of the regulation of endothelial function by receptor tyrosine kinases is unclear, especially with respect to the role of FLT-1 in angiogenesis. The pivotal role of both FLT-1 and KDR/Flk-1 in development has been clearly demonstrated by the recent reports on mice deficient in these endothelial-specific receptor kinases. Homozygous mutations that inactivate the FLK gene result in failure of blood island formation and vasculogenesis, whereas homozygous mutations that inactivate the FLT-1 gene result in defective assembly of endothelial cells into tubes(41, 60) . Both mutations result in intrauterine death at day 8.5. Clearly, both FLT-1 and KDR/Flk-1 are critical for growth and development of normal vascular endothelium. We anticipate further analysis of the biological significance of KDR and FLT-1-mediated responses to VEGF in vitro and in vivo through the use of receptor-selective VEGF mutants.