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• W1974854032 abstract "Vascular endothelial growth factor (VEGF) is an angiogenesis factor for which two signaling protein tyrosine kinase receptors, Flt1 and KDR, have been identified. We describe here a 190-kDa component present on a human glioma cell line that binds VEGF165 with high affinity. In contrast, VEGF121 is bound only with low affinity, suggesting that the C-terminal part of VEGF165 is important for interaction with the 190-kDa component. No internalization or stimulation of tyrosine phosphorylation was recorded after ligand binding to the 190-kDa component, suggesting that it may not be directly involved in signaling; its function may be to present ligand or stabilize ligand binding to signaling receptors. Vascular endothelial growth factor (VEGF) is an angiogenesis factor for which two signaling protein tyrosine kinase receptors, Flt1 and KDR, have been identified. We describe here a 190-kDa component present on a human glioma cell line that binds VEGF165 with high affinity. In contrast, VEGF121 is bound only with low affinity, suggesting that the C-terminal part of VEGF165 is important for interaction with the 190-kDa component. No internalization or stimulation of tyrosine phosphorylation was recorded after ligand binding to the 190-kDa component, suggesting that it may not be directly involved in signaling; its function may be to present ligand or stabilize ligand binding to signaling receptors. Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor/vascular permeability factor; PAE, porcine aortic endothelial. is a potent inducer of angiogenesis and vessel permeability (1Thomas K.A. J. Biol. Chem. 1996; 271: 603-606Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). Structurally, VEGF is a dimeric molecule related to platelet-derived growth factor and occurs as four different isoforms with 121, 165, 189, or 206 amino acid residues, as a result of different splicing of the gene (2Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connolly D.T. Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1811) Google Scholar, 3Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar, 4Houck K.A. Ferrara N. Winer J. Cachianes G. Li B. Leung D.W. Mol. Endocrinol. 1991; 5: 1806-1814Crossref PubMed Scopus (1241) Google Scholar, 5Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar). Whereas VEGF121 does not bind heparin, VEGF165 binds heparin with medium affinity, and VEGF189 and VEGF206 bind heparin with high affinity (6Houck K.A. Leung D.W. Rowland A.M. Winer J. Ferrara N. J. Biol. Chem. 1992; 267: 26031-26037Abstract Full Text PDF PubMed Google Scholar, 7Kim K.J. Li B. Houck K. Winer J. Ferrara N. Growth Factors. 1992; 7: 53-64Crossref PubMed Scopus (272) Google Scholar, 8Park J.E. Keller G.-A. Ferrara N. Mol. Biol. Cell. 1993; 4: 1317-1326Crossref PubMed Scopus (972) Google Scholar). VEGF binds to two structurally similar protein tyrosine kinase receptors, denoted Flt1 and KDR (9Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Bohlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1405) Google Scholar, 10Matthews W. Jordan C.T. Gavin M. Jenkins N.A. Copeland N.G. Lemischka I.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9026-9030Crossref PubMed Scopus (451) Google Scholar, 11Shibuya M. Yamaguchi S. Yamane A. Ikeda T. Tojo A. Matsushime H. Sato M. Oncogene. 1990; 5: 519-524PubMed Google Scholar, 12de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1896) Google Scholar). Activation of the KDR receptor has been shown to lead to mitogenicity, chemotaxis, and morphological changes (13Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 14Millauer B. Wizigmann-Voos S. Schnürch H. Martinez R. M⊘ller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1764) Google Scholar). The role of Flt1 in signal transduction is less well characterized, but Flt1 has been shown to mediate e.g.tissue factor induction in endothelial cells (15Clauss M. Weich H. Breier G. Knies U. Röckl W. Waltenberger J. Risau W. J. Biol. Chem. 1996; 271: 17629-17634Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar). VEGF was originally identified as a growth factor specific for endothelial cells; however, recent observations have suggested that the VEGF receptors KDR and Flt1 are not exclusively expressed on endothelial cells (16Cohen T. Gitay-Goren H. Sharon R. Shibuya M. Halaban R. Levi B.-Z. Neufeld G. J. Biol. Chem. 1995; 270: 11322-11326Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Recently, cell surface proteins of 120–130 kDa were identified on a breast cancer cell line and on endothelial cells that were shown to bind the exon 7-encoded sequence of VEGF165; VEGF121, which lacks this sequence, did not bind the 120–130-kDa components (17Gitay-Goren H. Cohen T. Tessler S. Soker S. Gengrinovitch S. Rockwell P. Klagsbrun M. Levi B.-Z. Neufeld G. J. Biol. Chem. 1996; 271: 5519-5523Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 18Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). In this report, we describe a 190-kDa component present on a human glioma cell line, which binds VEGF165 but not VEGF121 with high affinity. Human VEGF165 and a rabbit antiserum against human VEGF were purchased from Pepro Tech Inc. Human VEGF121 was a kind gift from Dr. Gera Neufeld (16Cohen T. Gitay-Goren H. Sharon R. Shibuya M. Halaban R. Levi B.-Z. Neufeld G. J. Biol. Chem. 1995; 270: 11322-11326Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 19Tessler S. Rockwell P. Hicklin D. Cohen T. Levi B.-Z. Witte L. Lemischka I.R. Neufeld G. J. Biol. Chem. 1994; 269: 12456-12461Abstract Full Text PDF PubMed Google Scholar). A rabbit antiserum raised against the intracellular domain part of KDR has been described previously (13Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). An Flt1 antiserum was purchased from Santa Cruz Biotechnology Inc., and an anti-phosphotyrosine monoclonal antibody (PY20) was purchased from Transduction Laboratories. The anti-phosphotyrosine polyclonal antiserum (PY6) has been described previously (20Rönnstrand L. Beckmann M.P. Faulders B. Östman A. Ek B. Heldin C.-H. J. Biol. Chem. 1987; 262: 2929-2932Abstract Full Text PDF PubMed Google Scholar). Endoglycosidase F/peptideN-glycosidase F was purchased from Boehringer Mannheim. Iodination of human VEGF165 and VEGF121 were performed using the Bolton and Hunter method (22Bolton A.E. Hunter W.M. Biochem. J. 1973; 133: 529-539Crossref PubMed Scopus (2403) Google Scholar);125I-labeled VEGF was separated from free 125I using Sephadex G-25 and kept in phosphate-buffered saline with 5 mg/ml bovine serum albumin at 4 °C. The specific activities of the labeled VEGF165 and VEGF121 were about 6 × 104 cpm/ng and 3 × 104 cpm/ng, respectively. The porcine aortic endothelial (PAE) cell line and PAE cell lines transfected with cDNA for KDR (PAE/KDR) and Flt1 (PAE/Flt1) were cultured in Ham's F-12 medium (13Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). U-178MG human glioma cells (gift of B. Westermark, Department of Pathology, Uppsala, Sweden) (21Nistér M. Claesson-Welsh L. Eriksson A. Heldin C.-H. Westermark B. J. Biol. Chem. 1991; 266: 16755-16763Abstract Full Text PDF PubMed Google Scholar) and MDA MB231 cells (purchased from American Type Culture Collection) were cultured in Eagle's minimum essential medium and Dulbecco's modified essential medium, respectively. All cell lines were cultured in 10% fetal calf serum, 100 units/ml penicillin, 50 mg/ml streptomycin, and 4 mml-glutamine. In some experiments125I-VEGF165 purchased from Amersham Corp. was used. Binding experiments with 1 ng/ml125I-VEGF165 and125I-VEGF121 in the absence or the presence of various concentrations of unlabeled ligands were performed as described before, using U-178MG cells, as well as nontransfected and transfected PAE cell lines in 24-well dishes (13Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). To investigate ligand-dependent internalization of VEGF receptors, cells were preincubated or not with 100 ng/ml VEGF165 for 60 min at 37 °C in culture medium, incubated for 1 min with binding buffer supplemented with 20 mm acetic acid, pH 3.75, to dissociate cell surface receptor-bound VEGF, and then subjected to125I-VEGF165 binding assays (13Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). Cells were metabolically labeled with [35S]methionine for 3 h. After extraction with lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 0.5% deoxycholic acid, 10 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% Trasylol, 1 mm benzamidine, and 5 μg/ml leupeptin) and centrifugation at 10,000 × g for 15 min, the receptors were immunoprecipitated with antisera against KDR or Flt1. After SDS gel electrophoresis using 7% polyacrylamide gels, the labeled proteins were analyzed using a phosphoimager (FUJIX BAS 2000, Fuji). For immunoprecipitation with anti-VEGF antisera, cells labeled for 3 h with [35S]methionine were incubated with 100 ng/ml VEGF165 for 1 h at 4 °C and then analyzed as described above. U-178MG cells, MDA MB231 cells, as well as nontransfected and transfected PAE cell lines (cultured in 10-cm dishes) were washed twice with phosphate-buffered saline supplemented with 1 mg/ml bovine serum albumin and incubated for 90 min on ice with 10 ng/ml 125I-VEGF165. After three washes with phosphate-buffered saline, ligand-receptor complexes were cross-linked by incubation in 0.1 mmbis(sulfosuccinimidyl)suberate for 30 min at room temperature. After incubation in 70 mm methylammonium chloride for 10 min, cell lysates were prepared as described above and subjected to immunoprecipitation with antisera against KDR, Flt1, or VEGF. Some of these samples were heat-denatured in the presence of 0.2% SDS and 2% 2-mercaptoethanol and deglycosylated with endoglycosidase F/peptideN-glycosidase F (0.2 units) overnight at 37 °C in 0.1m potassium phosphate buffer, pH 6.5, containing 10 mm EDTA and 2%n-octyl-β-d-glycoside. Samples were analyzed by SDS gel electrophoresis using 4–12% gradient or 7% homogenous polyacrylamide gels, followed by autoradiography using a phosphoimager. Cells were stimulated with 100 ng/ml VEGF165 for 60 min at 4 °C, washed with Tris-buffered saline containing 0.5 mmNa3VO4, and 1 mm dithiothreitol, and solubilized in 20 mm Tris-HCl pH 7.5, 150 mm NaCl, 1% Triton X-100, 10% glycerol, 0.5 mm Na3VO4, 1 mmdithiothreitol, 1% Trasylol, and 1 mm phenylmethylsulfonyl fluoride. The cell lysates were centrifuged at 10,000 ×g for 15 min, and the supernatants were immunoprecipitated with an anti-phosphotyrosine polyclonal antiserum (PY6). After SDS gel electrophoresis, samples were blotted onto nitrocellulose membranes (Hybond C extra, Amersham Corp.). Membranes were incubated with PY20, and immune complexes were detected using horseradish peroxidase-linked secondary antibodies and enhanced chemoluminescence (ECL, Amersham Corp.). High specific binding of 125I-VEGF165 was found on the human glioma cell line U-178MG. To estimate the binding affinity, the binding of 125I-VEGF165 was determined in the presence of various concentrations of unlabeled VEGF165; half maximal competition of specific binding was observed at approximately 100 ng/ml (Fig. 1). Scatchard analysis revealed about 84000 VEGF binding sites on U-178MG cells. In parallel, similar binding studies were performed on PAE cells stably transfected with KDR and Flt1 (PAE/KDR and PAE/Flt1, respectively). Untransfected PAE cells do not bind VEGF. The affinity of binding of125I-VEGF165 to PAE/KDR cells was similar to the binding to U-178 MG cells with half-maximal competition at about 100 ng/ml, whereas half-maximal competition for the binding to Flt1 cells was observed at approximately 15 ng/ml of VEGF165. The higher binding affinity for VEGF165 to PAE/Flt1 cells as compared with PAE/KDR cells is in agreement with previous studies using the same cell lines (13Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). To investigate if the binding of VEGF165 was mediated by KDR or Flt1 receptors, immunoprecipitation experiments using KDR and Flt1 antisera were performed (Fig. 2). PAE, PAE/KDR, PAE/Flt1, and U-178MG cells were incubated on ice with 125I-VEGF165, and bound ligand was covalently cross-linked to the receptors by incubation with bis(sulfosuccinimidyl)suberate. After lysis of the cells, immunoprecipitations were performed with KDR antisera on PAE, PAE/KDR, and U-178MG cells and with Flt1 antibodies on lysates from PAE, PAE/Flt1, and U-178MG cells. Immune complexes were then analyzed by SDS-polyacrylamide gel electrophoresis and visualized by phosphoimager analysis. As shown in Fig. 2, the expected cross-linked monomeric and dimeric KDR and Flt1 receptor forms were recovered from the transfected cells (lanes 2 and 5). A component of about 130 kDa was also seen in the PAE/Flt1 cells (lane 5); this component was not seen in the untransfected PAE cells (lane 4) and may represent a degraded form of Flt1. However, no KDR (lane 3) or Flt1 (lane 6) receptors were precipitated from U-178MG cells using the respective antisera. A faint component of about 190 kDa was sometimes seen after precipitation with KDR antiserum, but this component was clearly smaller than KDR. The notion that U-178MG cells do not contain any KDR protein was also strengthened by the lack of signal in in vitro kinase assays performed on anti-KDR immune complexes derived from U-178MG cells (data not shown). Neither was any KDR or Flt1 mRNA detected in Northern blot analysis of U-178MG cells (data not shown). We thus conclude that the high affinity VEGF binding to U-178MG cells are not due to the presence of KDR or Flt1 receptors. A VEGF165 binding protein, distinct from Flt1 and KDR, of 120–130 kDa was recently identified on MDA MB231 cells (18Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Interestingly, VEGF121 did not bind to this receptor. A component of similar size and with similar binding specificity was also identified on human umbilical vein-derived endothelial cells (17Gitay-Goren H. Cohen T. Tessler S. Soker S. Gengrinovitch S. Rockwell P. Klagsbrun M. Levi B.-Z. Neufeld G. J. Biol. Chem. 1996; 271: 5519-5523Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). To explore if this pattern of splice form-specific binding was also a property of the VEGF binding protein on U-178MG cells, we performed binding experiments with125I-labeled forms of VEGF165 and VEGF121. For comparison the same type of binding experiments were also performed on PAE/KDR and PAE/Flt1 cells. As shown in Fig. 3 A, the binding of 125I-VEGF165 to PAE/KDR and PAE/Flt1 cells was efficiently competed for with high concentrations of both VEGF165 and VEGF121. In contrast,125I-VEGF165 binding to U-178MG was only inhibited to about 50% of maximum binding by 625 ng/ml by VEGF121, whereas almost complete competition was obtained by the same concentration of VEGF165. When125I-VEGF121 was used, binding was readily detected to PAE/KDR and PAE/Flt1 cells (Fig. 3 B). In contrast, very low specific binding was observed to U-178MG cells. Together these data demonstrate a clear difference with regard to the dependence on the exon 7-coded sequence for high affinity binding between KDR and Flt1 on one hand and the VEGF binding proteins on U-178MG cells (Fig. 3) and the 120–130-kDa proteins detected on MDA MB231 (18Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar) and umbilical vein-derived endothelial cells (17Gitay-Goren H. Cohen T. Tessler S. Soker S. Gengrinovitch S. Rockwell P. Klagsbrun M. Levi B.-Z. Neufeld G. J. Biol. Chem. 1996; 271: 5519-5523Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) on the other. To determine the M r of the VEGF receptor on U-178MG cells conventional affinity labeling techniques were tried but were found to give too high a background. Two different alternative strategies, both involving VEGF antibody-mediated precipitation of the complex between ligand and binding protein, were therefore employed (Fig. 4). In the first approach, PAE, PAE/KDR, and U-178MG cells were metabolically labeled with [35S]methionine and subsequently incubated on ice in the absence or the presence of unlabeled VEGF165 (Fig. 4 A). After lysis of cells, anti-VEGF immunoprecipitations were performed on lysates, and immune complexes were subjected to SDS-polyacrylamide gel electrophoresis. Recovered components were then visualized by phosphoimager analysis. The feasibility of this approach was demonstrated by the VEGF-dependent recovery of the 200-kDa KDR protein from PAE/KDR cells (lane 4). Inspection of the lanes corresponding to U-178MG lysates revealed one single VEGF-dependent component with a molecular mass of 190 kDa (Fig. 4 A, lane 6). In the second approach, 125I-VEGF165 was covalently cross-linked to U-178MG cells, and cell lysates were subsequently immunoprecipitated with VEGF antibodies. As in the previous experiment, PAE/KDR cells and untransfected PAE cells were included as controls. The VEGF antiserum brought down a 220-kDa complex from U-178MG cells, and in addition, a component of 140 kDa (Fig. 4 B, lane 6). No component corresponding to the lower molecular mass component was observed in the experiment using metabolically labeled cells (Fig. 4 A). It may therefore correspond to a component interacting with VEGF165 with lower affinity that only can be seen after covalent cross-linking. A complex of about 220 kDa was recovered from PAE/KDR cells (Fig. 4 B, lane 5). Because these complexes were not recovered as anti-VEGF precipitates in untransfected PAE cells (lane 4) nor when preimmune sera were used on the lysates (lanes 1–3), we conclude that they represent125I-VEGF165 cross-linked to the KDR receptor on PAE/KDR cells and to the novel binding protein on U-178MG cells. Because the contribution of cross-linked VEGF165 to the mass of the complexes should be 23 or 45 kDa, depending on if one or two subunits of the VEGF dimer was present in the cross-linked complex, these experiments suggest a mass of 180–200 kDa for the U-178MG VEGF binding protein, which is in good agreement with the size detected from the immunoprecipitation of metabolically labeled cells (Fig. 4 A). The estimated size of KDR from the cross-linking experiment is consistent with previous results (23Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar). Thus, we conclude that the major VEGF binding protein on U-178MG cells has a size of about 190 kDa. VEGF binding proteins of 120–130 kDa have been described on the breast cancer cell line MDA MB231 and on endothelial cells (17Gitay-Goren H. Cohen T. Tessler S. Soker S. Gengrinovitch S. Rockwell P. Klagsbrun M. Levi B.-Z. Neufeld G. J. Biol. Chem. 1996; 271: 5519-5523Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar,18Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). To explore the relatedness of these components with the VEGF binding proteins on U-178MG cells, anti-VEGF immunoprecipitations of125I-VEGF165 cross-linked to MDA MB231 and U-178MG cells were compared. To investigate the glycoprotein nature of the components, the effect of treatment of the immunoprecipitates withN-glycosidases was also investigated. As shown in Fig. 5, the 190-kDa component of U-178MG cells did not shift in size after glycosidase-treatment, suggesting that this component contains no or low amounts of N-linked carbohydrate. A less abundant component of similar size, which does not either appear to be a glycoprotein, was also seen in MDA MB231 cells. It is unlikely that the 190-kDa VEGF binding protein on MDA MB231 cells corresponds to the tyrosine kinase receptors KDR or Flt1, because these are known to be glycoproteins. In contrast, the 120–130-kDa VEGF binding component of MDA MB231 decreased in size about 10 kDa after glycosidase treatment; the similarly sized component of U-178MG cells also showed a similar shift in size. These observations suggest that the 120–130-kDa components of MDA MB231 cells and U-178MG cells may be related and distinct from the 190-kDa component of U-178MG cells, which may be related to a component of similar size present on MDA MB231 cells. Exposure of high concentration of ligand to cells carrying signaling growth factors receptors is in most cases followed by loss of binding sites as a consequence of ligand-induced receptor internalization and down-regulation (24Lai W.H. Cameron P.H. Wada I. Doherty J.J.d. Kay D.G. Posner B.I. Bergeron J.J. J. Cell Biol. 1989; 109: 2741-2749Crossref PubMed Scopus (82) Google Scholar, 25Mascarelli F. Tassin J. Courtois Y. Growth Factors. 1991; 4: 81-95Crossref PubMed Scopus (50) Google Scholar, 26Sorkin A. Westermark B. Heldin C.-H. Claesson-Welsh L. J. Cell Biol. 1991; 112: 469-478Crossref PubMed Scopus (127) Google Scholar). We therefore investigated if the U-178MG VEGF binding protein was down-regulated after exposure to high concentrations of VEGF165. Identical experiments were performed in parallel on PAE/KDR cells. Cells were first incubated for 1 h at 37 °C in the absence or the presence of 100 ng/ml of VEGF165 and thereafter transferred to ice. To remove ligand bound to receptors or to binding proteins remaining on the cell surface, cells were incubated for 1 min in a buffer of pH 3.75 before being subjected to a 125I-VEGF165 binding experiment. As shown in Fig. 6, pre-exposure of U-178MG cells to 100 ng/ml of VEGF165 at 37 °C did not affect the binding of125I-VEGF165 (Fig. 6, right part). As expected, pretreatment of PAE/KDR cells reduced binding to the background levels (Fig. 6, left part). Because all known signaling molecules for VEGF165 are receptor tyrosine kinases, we also investigated if an alteration in the pattern of tyrosine phosphorylated proteins could be detected in U-178MG cells after stimulation with VEGF165. PAE, PAE/KDR, and U-178MG cells were incubated on ice for 1 h in the absence or the presence of 100 ng/ml of VEGF165. Cell lysates were immunoprecipitated with phosphotyrosine antibodies; after SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose filters, tyrosine-phosphorylated proteins were detected by immunoblotting using phosphotyrosine antibodies. As shown in Fig. 7 (lanes 5 and 6), no ligand-dependent alteration in the pattern of tyrosine-phosphorylated proteins was detected in U-178MG cells. In contrast, VEGF stimulation of PAE/KDR led to the phosphorylation of a 200-kDa protein, most likely representing autophosphorylation of KDR, as well as to the phosphorylation of some other components with masses of 140–160 kDa. To characterize the interaction between VEGF165 and the binding protein on U-178MG cells, a modified binding experiment was performed; after binding of ligand, cells were washed with binding buffer containing 0.5 m NaCl. As shown in Fig. 8, this change in washing conditions did not affect VEGF binding to Flt1 and had only a small effect on VEGF165 binding to KDR. In contrast, almost no binding of VEGF to the binding protein on U-178MG cells was detected after this treatment. To exclude that the loss of binding to U-178MG cells after the high salt wash reflected dissociation of the binding protein from the cells rather than dissociation of the ligand from the receptor, we also investigated the effect of a high salt wash of the cells prior to the binding experiment. This treatment did not affect the binding of VEGF to the U-178MG cells (Fig. 8). We therefore conclude that the VEGF binding to the 190-kDa binding protein differs from the binding to KDR and Flt1 with regard to sensitivity to increased ionic strength. Furthermore, the fact that the binding capacity of the U-178MG cells was not affected by pretreatment of the cells with 0.5 mNaCl suggests that the binding protein is tightly associated with the cell, possibly as an integral membrane protein rather than as a protein loosely associated with the cell surface. We describe in the present communication a 190-kDa VEGF165 binding protein present on a human glioma cell line. Because the 190-kDa component does not appear to have any intrinsic tyrosine kinase activity or to stimulate tyrosine phosphorylation in the cells, it is possible that it is not directly involved in signaling. This conclusion is supported by the lack of observable effect of VEGF on growth or shape of U-178MG cells, which have the 190-kDa component but lack both KDR and Flt1 receptors (data not shown). It is possible, however, that the 190-kDa protein serves an accessory role in VEGF stimulation of cells with Flt1 or KDR receptors,e.g. by presenting ligand to signaling receptors or forming a complex with signaling receptors with increased ligand binding affinity. Such roles have been suggested, e.g. for betaglycan in transforming growth factor-β signaling (27Miyazono K. ten Dijke P. Ichijo H. Heldin C.-H. Adv. Immunol. 1994; 55: 181-220Crossref PubMed Scopus (182) Google Scholar) and for heparan sulfate proteoglycan in fibroblast growth factor signaling (28Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Abstract Full Text PDF PubMed Scopus (453) Google Scholar). VEGF165, but not VEGF121, bound to the 190-kDa component on U-178MG cells with high affinity. This observation suggests that the sequence encoded by exon 7 in the VEGF gene is important for the binding. In contrast, the N-terminal region with homology to platelet-derived growth factor appears to be mainly responsible for the binding to the signaling receptors KDR and Flt1. The splice form-specific binding of VEGF to the 190-kDa protein suggests one mechanism whereby the different isoforms of VEGF may have different effects on different cells. The notion that interactions mediated by the exon 7-encoded sequence are functionally important is supported by the observation that full mitogenic effect of VEGF was not obtained by VEGF molecules lacking the exon 7-encoded sequence (29Keyt B.A. Berleau L.T. Nguyen H.V. Chen H. Heinsohn H. Vandlen R. Ferrara N. J. Biol. Chem. 1996; 271: 7788-7795Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). Interestingly, a similar splice form-specific binding of VEGF was recently reported for VEGF binding proteins on a breast cancer cell line (18Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar) and on endothelial cells (17Gitay-Goren H. Cohen T. Tessler S. Soker S. Gengrinovitch S. Rockwell P. Klagsbrun M. Levi B.-Z. Neufeld G. J. Biol. Chem. 1996; 271: 5519-5523Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The VEGF binding protein on glioma cells is clearly larger (190 kDa) than these proteins (120–130 kDa). Furthermore, glycosidase treatment did not affect the size of the 190-kDa component, whereas the size of the 120–130-kDa component of MDA MB231 cells decreased about 10 kDa. The 190-kDa component is thus clearly different from the VEGF binding protein of MDA MB231 cells. However, the smaller VEGF binding component we observed on U-178MG cells may be related to the 120–130-kDa component of MDA MB231 cells, because their sizes and susceptibility to glycosidase treatment are the same. The 190-kDa VEGF binding protein was observed on a human glioma cell line lacking KDR and Flt1 receptors. It should be noted that on cells having KDR or Flt1 receptors the 190-kDa protein may be difficult to discern in conventional cross-linking experiments due to its similarity in size with the tyrosine kinase receptors. The VEGF binding protein described in the present report was observed on a human glioma cell line. Investigations of the breast carcinoma cell line MDA MB231 (Fig. 5) and three other human glioma cell lines 2T. Omura, K. Miyazawa, A. Östman, and C.-H. Heldin, unpublished observations. revealed high molecular mass VEGF binding proteins, which may be related to the VEGF binding protein on U-178MG cells. Purification, cDNA cloning, and the establishment of specific antisera will make it possible to further characterize the 190-kDa component and its role in VEGF signaling. We thank Dr. G. Neufeld for VEGF121, Dr. B. Westermark for the U-178MG cells, Dr. M. Klagsbrun for MDA MB231 cells, and Drs. M. Klagsbrun, G. Neufeld, and L. Claesson-Welsh for critical comments on the manuscript." @default.
• W1974854032 created "2016-06-24" @default.
• W1974854032 creator A5041272737 @default.
• W1974854032 creator A5066010228 @default.
• W1974854032 creator A5079512529 @default.
• W1974854032 creator A5084535456 @default.
• W1974854032 date "1997-09-01" @default.
• W1974854032 modified "2023-10-17" @default.
• W1974854032 title "Identification of a 190-kDa Vascular Endothelial Growth Factor 165 Cell Surface Binding Protein on a Human Glioma Cell Line" @default.
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