Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1(TK-/-)) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1(TK-/-) mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.     

Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells

The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp63, Asp64, and Glu67 to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B186 dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.     

Mapping the charged residues in the second immunoglobulin-like domain of the vascular endothelial growth factor/placenta growth factor receptor Flt-1 required for binding and structural stability

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).     

Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, induces endothelial proliferation in vitro and vascular permeability in vivo. The human transmembrane c-fms-like tyrosine kinase Flt-1 has recently been identified as a VEGF receptor. Flt-1 kinase has seven immunoglobulin-like extracellular domains and a kinase insert sequence, features shared by two other human gene-encoded proteins, kinase insert domain-containing receptor (KDR) and FLT-4. In this study we show that the mouse homologue of KDR, Flk-1, is a second functional VEGF receptor. Flk-1 binds VEGF with high affinity, undergoes autophosphorylation, and mediates VEGF-dependent Ca2+ efflux in Xenopus oocytes injected with Flk-1 mRNA. We also demonstrate by in situ hybridization that Flk-1 protein expression in the mouse embryo is restricted to the vascular endothelium and the umbilical cord stroma. VEGF and its receptors Flk-1/KDR and Flt-1 may play a role in vascular development and regulation of vascular permeability.     

Identification of the extracellular domains of Flt-1 that mediate ligand interactions

Vascular Endothelial Growth Factor (VEGF) mediates its actions through the Flt-1 and KDR(Flk-1) receptor tyrosine kinases. To localize the extracellular region of Flt-1 that is involved in ligand interactions, we prepared secreted fusion proteins between various combinations of its seven extracellular IgG-like folds. Ligand binding studies show that in combination, domains one and two (amino acids 1-234) are sufficient to achieve VEGF165 interactions. Either domain alone is insufficient to achieve this effect. However, Scatchard analysis reveals that despite the binding capabilities of this construct, the Kd is five fold lower than ligand binding to the full extracellular domain. We find that addition of domain three to this minimal site restores high affinity receptor binding. Further, we show that domains one and two are sufficient to achieve interactions of Flt-1 with Placental Growth Factor (PIGF-1).     

Flt-1, a receptor for vascular endothelial growth factor, has transforming and morphogenic potentials

A paradox of Flt-1, a tyrosine kinase receptor for vascular endothelial growth factor (VEGF), is that the ligand cannot activate the receptor to stimulate growth of cells that exogenously overexpress the receptor. In order to find Flt-1 kinase-dependent biological systems, we obtained for the first time activated forms of the Flt-1 kinase in a ligand-independent manner. Replacement of the ABL sequences in the human leukemia oncoprotein BCR-ABL with the cytoplasmic domain of Flt-1 (BCR-FLT) followed by a retroviral random mutagenesis scheme gave constitutively active artificial chimera BCR-FLTm with mutations within the Flt-1 sequence. Like BCR-ABL it could, but not the original BCR-FLT, transform Rat1 fibroblasts, abrogate cytokine dependence in Ba/F3 cells, and induce neurite-like structures in neuronal PC12 cells. Interestingly, Rat1 cells transformed by BCR-FLTm formed tube-like structures in basement membrane matrix. BCR-FLTm retroviruses may be a very useful tool to investigate an as yet uncovered functions of the Flt-1 kinase.     

A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain

Vascular endothelial growth factor (VEGF) mediates endothelial cell proliferation, angiogenesis, and vascular permeability via the endothelial cell receptors, KDR/Flk-1 and Flt-1. Recently, a gene encoding a polypeptide with about 25% amino acid identity to mammalian VEGF was identified in the genome of Orf virus (OV), a parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with angiogenesis. In this study, we examined the biological activities and receptor of OV-derived NZ-7 VEGF (VEGF-E). VEGF-E was found to be a dimer of about 20 kDa with no basic domain nor affinity for heparin column, similar to VEGF121 subtype. VEGF121 has 10-100-fold less endothelial cell mitotic activity than VEGF165 due to lack of a heparin-binding basic region. Interestingly, however, VEGF-E showed almost equal levels of mitotic activity on primary endothelial cells and vascular permeability activity as VEGF165. Furthermore, VEGF-E bound KDR/Flk-1 (VEGFR-2) and induced its autophosphorylation to almost the same extent as VEGF165, but did not bind Flt-1 (VEGFR-1) nor induce autophosphorylation of Flt-1. These results indicate that VEGF-E is a novel type of endothelial growth factor, utilizing only one of the VEGF receptors, and carrying a potent mitogenic activity without affinity to heparin.     

Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.     

Regulation of Schizosaccharomyces pombe Wee1 tyrosine kinase

Wee1 tyrosine kinase regulates mitosis by carrying out the inhibitory tyrosine 15 phosphorylation of Cdc2 M-phase inducing kinase. Schizosaccharomyces pombe Wee1 is a large protein, consisting of a C-terminal catalytic domain of approximately 350 amino acids preceded by a N-terminal domain of approximately 550 residues. The functional properties of the Wee1 N-terminal domain were investigated by expressing truncated forms of Wee1 in S. pombe. Both positive and negative regulatory domains were identified. Sequences important for Wee1 function were mapped to a central region (residues 363-408). This region is not required for kinase activity or nuclear localization, suggesting it may be involved in substrate recognition. The negative regulatory domain resides in the N-terminal third of Wee1, Wee1 constructs lacking this domain are more effective at delaying mitosis than wild-type Wee1. The negative regulatory domain contains clusters of potential Cdc2 phosphorylation sites. Investigations to monitor the abundance of Wee1 mRNA and protein during the cell cycle were also carried out.     

Role of VEGF receptor-1 (Flt-1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells

Vascular endothelial growth factor (VEGF) receptor KDR (kinase-insert-domain-containing receptor) is linked to endothelial cell proliferation, and VEGF receptor Flt-1 (fms-like tyrosine kinase) is essential for the organization of embryonic vasculature. Flt-1 is also known to be expressed on adult endothelial and trophoblast cells, although its function has not yet been established. Herein we report that human trophoblast and endothelial cells contain functional Flt-1 receptors for VEGF that trigger the synthesis and release of nitric oxide (NO) by the activation of constitutive NO synthase (cNOS). In first-trimester human trophoblast cells isolated by chorionic villous sampling, VEGF165 stimulated NO release in a concentration- and time-dependent manner, with a maximal increase of 60% (in comparison to basal release levels) occurring within 30 minutes (basal: 1342 pmol/ml; VEGF (10 ng/ml): 2162 pmol/ml; p < 0.001), as measured by an NO chemiluminescence analyzer. VEGF20, a peptide fragment that is composed of the first 20 amino acids at N-terminus, displayed properties of a partial agonist. VEGF165- and VEGF20-mediated NO biosynthesis was attenuated by NG-nitro-L-arginine in a concentration-dependent fashion, indicating NOS activation. VEGF-neutralizing anti-VEGF monoclonal antibody significantly inhibited VEGF-mediated NO release (p < 0.001), and the addition of a neutralizing anti-Flt-1 antibody inhibited the response by 79.6% +/- 7.59%, an effect found to be reversible with higher concentrations of VEGF. In contrast, anti-KDR antibody had no significant inhibitory effect. RT-PCR confirmed the presence of mRNA encoding the Flt-1 and KDR receptors as well as the endothelial form of cNOS in trophoblast cells. VEGF165-stimulated NO release was inhibited by genistein (5 microM; p < 0.001) as well as by the removal of calcium from the extracellular environment (p < 0.001), which suggests the contingency of this process on tyrosine phosphorylation and extracellular calcium, respectively. Addition of sodium nitroprusside, an NO donor, inhibited trophoblast DNA synthesis in a concentration-dependent manner, as measured by [3H]thymidine incorporation, without affecting cell viability. VEGF under maximal NO production had no mitogenic activity, suggesting that trophoblast-derived NO may limit trophoblast proliferation. Endogenous trophoblast DNA synthesis increased 3-fold in the presence of anti-Flt-1 antibody but not in the presence of anti-KDR antibody, suggesting that Flt-1 functions as a growth suppressive receptor to counteract the proliferative actions of KDR. Levels of immunoreactive endothelial cNOS were markedly increased in growth-restricted placentae (n = 4) in comparison to those of normal (n = 5) placentae, which may account for the relatively small-sized placentae associated with intrauterine growth restriction. VEGF165 stimulated NO release via phosphorylation of the Flt-1 receptor, indicating that VEGF may be an autocrine regulator of NO biosynthesis by aiding trophoblast penetration into spinal arterioles during the first trimester and preventing platelet aggregation within the placenta. Finally, the activation of Flt-1 receptor suppressed trophoblast DNA synthesis within the placenta via NO.     

Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor

The kinase insert domain-containing receptor (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed various deletion mutants of the extracellular region fused with the crystallizable fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH2-terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule. The first Ig-like domain may regulate the ligand binding.     

The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS: We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS: A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.     

A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.     

Eicosapentaenoic acid enhances nitric oxide production by cultured human endothelial cells

Flt-1, a tyrosine kinase receptor for vascular endothelial growth factor (VEGF), plays important roles in the angiogenesis required for embryogenesis and in monocyte/macrophage migration. However, the signal transduction of Flt-1 is poorly understood due to its very weak tyrosine kinase activity. Therefore, we overexpressed Flt-1 in insect cells using the Baculovirus system in order to examine for autophosphorylation sites and association with adapter molecules such as phospholipase Cgamma-1 (PLCgamma). Tyr-1169 and Tyr-1213 on Flt-1 were found to be auto-phosphorylated, but only a phenylalanine mutant of Tyr-1169 strongly suppressed its association with PLCgamma. In Flt-1 overexpressing NIH3T3 cells, VEGF induced autophosphorylation of Flt-1, tyrosine-phosphorylation of PLCgamma and protein kinase C-dependent activation of MAP kinase. These results strongly suggest that Tyr-1169 on Flt-1 is a major binding site for PLCgamma and important for Flt-1 signal transduction within the cell.     

The amino-terminal region of Tyk2 sustains the level of interferon alpha receptor 1, a component of the interferon alpha/beta receptor

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-alpha (IFN-alpha) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-alpha. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-alpha receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-alpha. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.     

Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human

Blood supply is essential for the maintenance of epididymal function. Since there is no considerable neovascularization in the epididymis, this tissue could represent a suitable model to study the vascular endothelial growth factor (VEGF) effect for vascular permeability. We studied the expression and function of VEGF and its receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase (designated as kinase insert domain-containing receptor, KDR in the human) in the human epididymis. VEGF and VEGF receptors mRNA were detected in the human epididymal tissue. VEGF protein was localized in peritubular and in ciliated cells of efferent ducts as well as in peritubular and basal cells of the epididymal duct. Vascular endothelial cells did not express VEGF. Flt-1 protein was localized in ciliated cells of efferent ducts and in lymphatic vessels. Vascular endothelial cells were negative for Flt-1 but positive for KDR. In vitro VEGF165 treatment of epididymal tissue induced endothelial fenestrations and opening of interendothelial junctions. Additionally, we observed for the first time that VEGF could induce transendothelial gaps. We conclude that these gaps might be of importance not only for molecular transport but also for cell passage across the vessel wall, which may be significant for tumor metastasis. VEGF may act as a paracrine effector to influence the permeability of lymphatic vessels via Flt-1, and of blood vessels via KDR.     

Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind approximately 100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.     

Leucine zipper-mediated dimerization is essential for the PTC1 oncogenic activity

The PTC1 chimeric oncogene is generated by the fusion of the tyrosine kinase domain of the RET proto-oncogene to the 5'-terminal region of another gene named H4 (D10S170). This oncogene has been detected only in human papillary thyroid carcinomas. We have previously demonstrated that the putative leucine zipper in the N-terminal region of H4 can mediate oligomerization of the PTC1 oncoprotein in vitro. In this study, we further demonstrated that the PTC1 oncoprotein forms a dimer in vivo, and the leucine zipper is responsible for this dimerization. The H4 leucine zipper-mediated dimerization is essential for tyrosine hyperphosphorylation and the transforming activity of the PTC1 oncoprotein. Introducing a loss-of-function PTC1 mutant into PTC1-transformed NIH3T3 cells suppressed the transforming activity of PTC1 and reversed the transformed phenotype of these cells, presumably by forming inactive heterodimers between the two forms of PTC1. Taken together, these data indicate that constitutive dimerization of the PTC1 oncoprotein is essential for PTC1 transforming activity and suggest that constitutive oligomerization acquired by rearrangement or by point mutations may be a general mechanism for the activation of receptor tyrosine kinase oncogenes.     

SCCA1 and SCCA2 are proteinase inhibitors that map to the serpin cluster at 18q21.3

Vascular endothelial growth factor (VEGF) is an essential molecule in the development and formation of mammalian blood vessels in health and disease. VEGF is also increasingly implicated in other biological processes including renal development and pathophysiology. The biological activities of VEGF in vivo and in its target cells in culture are mediated through two receptor protein tyrosine kinases, KDR/Flk-1 and Flt-1. KDR/Flk-1 is able to mediate the tyrosine phosphorylation of several cellular components as well as the generation of second messengers. Recent findings have revealed novel signaling mechanisms which may mediate the biological actions of VEGF. In contrast, the signal transduction mechanisms triggered by Flt-1 remain largely unknown.