FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2065860196> ?p ?o ?g. }

• W2065860196 endingPage "11120" @default.
• W2065860196 startingPage "11115" @default.
• W2065860196 abstract "Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations such as tumor angiogenesis, diabetic retinopathy, or psoriasis. By amino-terminal deletion analysis and by site-directed mutagenesis we have identified a new domain within the amino-terminal α-helix that is essential for dimerization of VEGF. VEGF121 variants containing amino acids 8 to 121 or 14 to 121, respectively, either expressed in Escherichia coli and refolded in vitro, or expressed in Chinese hamster ovary cells, were in a dimeric conformation and showed full binding activity to VEGF receptors and stimulation of endothelial cell proliferation as compared with wild-type VEGF. In contrast, a VEGF121 variant covering amino acids 18 to 121, as well as a variant in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 within the amphipathic α-helix near the amino terminus were replaced by serine, failed to form biological active VEGF dimers. From these data we conclude that a domain between amino acids His12 and Asp19 within the amino-terminal α-helix is essential for formation of VEGF dimers, and we propose hydrophobic interactions between VEGF monomers to stabilize or favor dimerization. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations such as tumor angiogenesis, diabetic retinopathy, or psoriasis. By amino-terminal deletion analysis and by site-directed mutagenesis we have identified a new domain within the amino-terminal α-helix that is essential for dimerization of VEGF. VEGF121 variants containing amino acids 8 to 121 or 14 to 121, respectively, either expressed in Escherichia coli and refolded in vitro, or expressed in Chinese hamster ovary cells, were in a dimeric conformation and showed full binding activity to VEGF receptors and stimulation of endothelial cell proliferation as compared with wild-type VEGF. In contrast, a VEGF121 variant covering amino acids 18 to 121, as well as a variant in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 within the amphipathic α-helix near the amino terminus were replaced by serine, failed to form biological active VEGF dimers. From these data we conclude that a domain between amino acids His12 and Asp19 within the amino-terminal α-helix is essential for formation of VEGF dimers, and we propose hydrophobic interactions between VEGF monomers to stabilize or favor dimerization. Vascular endothelial growth factor (VEGF), 1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; CHO, chinese hamster ovary; Flt-1, fms-like tyrosine kinase-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVE, human umbilical vein endothelial; KDR, kinase domain receptor; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; SEAP, secreted placental alkaline phosphatase; TGF, transforming growth factor. 1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; CHO, chinese hamster ovary; Flt-1, fms-like tyrosine kinase-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVE, human umbilical vein endothelial; KDR, kinase domain receptor; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; SEAP, secreted placental alkaline phosphatase; TGF, transforming growth factor. also known as vascular permeability factor, is a mitogen that specifically regulates endothelial cell function. The biological activities of VEGF (for recent reviews, see Refs. 1Martiny-Baron G. Marmé D. Curr. Opin. Biotechnol. 1995; 6: 675-680Crossref PubMed Scopus (107) Google Scholar, 2Thomas K.A. J. Biol. Chem. 1996; 271: 603-606Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 3Breier G. Risau W. Trends Cell Biol. 1996; 6: 454-456Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 4Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4805) Google Scholar) include stimulation of endothelial cell growth and migration, rapid enhancement of microvascular permeabilityin vivo, promotion of vasculogenesis and angiogenesis, and induction of differentiation of embryonic stem cells to hematopoietic precursors (5Kennedy M. Firpo M. Choi K. Wall C. Robertson S. Kabrun N. Keller G. Nature. 1997; 386: 488-493Crossref PubMed Scopus (495) Google Scholar). Recent experiments of targeted disruption of the VEGF gene have demonstrated its essential role for vascular development in the embryo (6Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3430) Google Scholar, 7Ferrara N. Carver-Moore Chen H. Dowd M. Lu L. O'Shea K.S. Powell-Braxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3026) Google Scholar). Even inactivation of a single VEGF allele results in defective development of large vessels, defective capillary sprouting, and embryonic lethality. Aberrant elevated expression of VEGF has been observed in a variety of human pathological situations such as tumor angiogenesis (8Plate K.H. Breier G. Weich H.A. Risau W. Nature. 1992; 359: 845-848Crossref PubMed Scopus (2110) Google Scholar), diabetic retinopathy (9Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3374) Google Scholar), rheumatoid arthritis (10Fava R.A. Olsen N.J. Spencer-Green G. Yeo K.T. Yeo T.K. Berse B. Jackman R.W. Senger D.R. Dvorak H.F. Brown L.F. J. Exp. Med. 1994; 180: 341-346Crossref PubMed Scopus (492) Google Scholar), or psoriasis (11Detmar M. Brown L.F. Claffey K.P. Yeo K.T. Kocher O. Jackman R.W. Berse B. Dvorak H.F. J. Exp. Med. 1994; 180: 1141-1146Crossref PubMed Scopus (645) Google Scholar). Neutralizing of VEGF by antibodies or recombinant soluble receptor domains have shown therapeutic potential as agents capable of suppressing tumor growth (12Kim K.J. Li B. Winer J. Armanini M. Gillett N. Phillips H.S. Ferrara N. Nature. 1993; 362: 841-844Crossref PubMed Scopus (3337) Google Scholar) and retinal neovascularization (13Aiello L.P. Pierce E.A. Foley E.D. Takagi H. Chen H. Riddle L. Ferrara N. King G.L. Smith L.E.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10457-10461Crossref PubMed Scopus (1159) Google Scholar). Two homologous cell-surface receptors of the tyrosine kinase family, Flt-1 (VEGFR-1) and KDR (VEGFR-2), bind VEGF with high affinity (14de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1884) Google Scholar, 15Terman 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 (1393) Google Scholar). VEGF is a homodimeric glycosylated protein that exists in five different isoforms of 121, 145, 165, 189, and 206 amino acids of which the amino-terminal 114 amino acids are identical. Together with placenta growth factor (PlGF) (16Maglione D. Guerriero V. Vigliette G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (831) Google Scholar) and the recently described VEGF-B (17Olofsson B. Pajusola K. Kaipainen A. Von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (620) Google Scholar) and VEGF-C (18Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1144) Google Scholar) VEGF builds a family of related growth factors which show structural homology to PDGF. In particular, the cysteines building up the structural fold of the proteins consisting of three intramolecular disulfide brigdes, and two intermolecular disulfide brigdes cross-linking the polypeptide chains, are conserved for these growth factors (19Pötgens A.J.G. Lubsen N.H. van Altena M.C. Vermeulen R. Bakker A. Schoenmakers J.G.G. Ruiter D.J. de Waal R.M.W. J. Biol. Chem. 1994; 269: 32879-32885Abstract Full Text PDF PubMed Google Scholar, 20Claffey K.P. Senger D.R. Spiegelman B.M. Biochim. Biophys. Acta. 1995; 1246: 1-9Crossref PubMed Scopus (74) Google Scholar). The very recently solved crystal structure of VEGF (21Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (365) Google Scholar) confirmed the overall structural similarity of VEGF and PDGF. Alignment of VEGF121 and PDGF-B/v-sis amino acid sequences showed a 25% identity of the region Cys26 to Cys104 of VEGF and Cys16 to Cys99of mature PDGF-B. This region of PDGF-B/v-sis, the minimal v-sis transforming domain, was described to retain biological activity of the growth factor (22Hannink M. Donoghue D.J. Science. 1984; 226: 1197-1199Crossref PubMed Scopus (29) Google Scholar, 23King C.R. Giese N.A. Robbins K.C. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5295-5299Crossref PubMed Scopus (23) Google Scholar). In case of VEGF, it had previously been shown that a plasmin digested 110-amino acid amino-terminal fragment, which was cleaved between Arg110and Ala111, retains full biological activity as compared with the VEGF121 isoform (24Keyt 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 (530) Google Scholar). To analyze the contribution of amino acids Ala1 to Tyr25, which are located amino-terminal with respect to the homology region of VEGF to the minimal v-sis transforming domain, to structure and/or biological activity of VEGF, we generated amino-terminal truncated VEGF121 variants. Here were show that an amino-terminal domain between amino acids His12 and Asp19 is essential for in vitro dimerization of VEGF and for functional expression of VEGF in vivo. As the conversion of hydrophobic amino acids within this domain to serine impairs formation of VEGF dimers we propose hydrophobic interactions between VEGF monomers to stabilize or favor formation of dimers. cDNAs encoding amino-terminal truncated VEGF proteins were generated by PCR technique using the 5′-primers VEGF-8 (5′-GAGCCATGGAGAATCATCACGAAGTGGTGAAG-3′), VEGF-14 (5′-CACGCCATGGTGAAGTTCATGGATGTCT-3′), and VEGF-18 (5′-AAGTCCATGGATGTCTATCAGCGCAGC-3′), respectively, the 3′-primer VEGF-Bam (5′-AGTGGATCCCCCCCTGGTGA-3′), and cloned human VEGF121 cDNA (25Weindel K. Marmé D. Weich H.A. Biochem. Biophys. Res. Commun. 1992; 183: 1167-1174Crossref PubMed Scopus (159) Google Scholar) as template. Mutant VEGF-V14S/V15S/F17S/M18S was generated by PCR using 5′-primer 207 (5′-TCGGGCCTCCGAAACCATGA-3′) and 3′-primer VEGF-N1 (5′-GGCTCCTGAAGCTGCTAAGCACTACTAAGACGGGAGG-3′) for amplification of the 5′-fragment and 5′-primer VEGF-N2 (5′-AATCGTCGAAGTCCTCGGATGTCTATCAGCGCAGCTA-3′) and 3′-primer VEGF-Bam for amplification of the 3′-fragment. The fragments were purified by agarose gel electrophoresis and used in an equimolar ratio as template for fusion-PCR using 5′-primer 207 and 3′-primer VEGF-Bam. VEGF variant C61S was generated by amplification of the VEGF121 cDNA cloned in pBluescript vector using the 5′-primer C61S (5′-GGCTGCTCCAATGACGAGGGC-3′) and the 3′-primer 252c (5′-CCCGCATCGCATCAGGGGCAC-3′). The resulting PCR fragment containing mutant VEGF sequence and vector sequence was phosphorylated by T4 polynucelotide kinase (Pharmacia, Freiburg, Germany), gel purified, religated, and transformed into Escherichia coli XL1-blue. The mutant VEGF cDNAs were cloned into the His-pET vector (26Siemeister G. Schnurr B. Mohrs K. Schächtele C. Marmé D. Martiny-Baron G. Biochem. Biophys. Res. Commun. 1996; 222: 249-255Crossref PubMed Scopus (54) Google Scholar) viaNcoI and BamHI sites, and transformed intoE. coli BL21DE3 (27Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorf J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5998) Google Scholar). All constructs were verified by DNA sequencing. Solubilization of VEGF proteins from inclusion bodies, refolding, and purification was performed essentially as described for wild-type VEGF121 (26Siemeister G. Schnurr B. Mohrs K. Schächtele C. Marmé D. Martiny-Baron G. Biochem. Biophys. Res. Commun. 1996; 222: 249-255Crossref PubMed Scopus (54) Google Scholar). For the construction of eukaryotic expression plasmids NcoI/BamHI fragments encoding mutant VEGF variants were fused to the VEGF signal sequence by substitution of the NcoI/BamHI restriction fragment of wild-type VEGF121 in a plasmid, which contains the entire VEGF121 coding region cloned into theSmaI site of pBluescript (25Weindel K. Marmé D. Weich H.A. Biochem. Biophys. Res. Commun. 1992; 183: 1167-1174Crossref PubMed Scopus (159) Google Scholar). The cDNA fragments were released by EcoRI/XbaI restriction endonuclease digestion and ligated into pCI-neo expression vector (Promega, Heidelberg, Germany) which provides a cytomegalovirus promoter and enhancer. Transient transfection of chinese hamster ovary (CHO) cells was performed in six-well plates containing approximately 2 × 105 cells/well, which were incubated at 37 °C overnight in the presence of 2 μg/well of calcium phosphate-precipitated VEGF-variant/pCI-neo DNA. For determination of transfection efficiency and protein secretion 1 μg/well pSBC-2/SEAP expression vector DNA (28Dirks W. Wirth M. Hauser H. Gene (Amst .). 1993; 128: 247-249Crossref PubMed Scopus (153) Google Scholar) encoding secreted placental alkaline phosphatase (SEAP) was cotransfected. Cell culture supernatant was replaced with serum-free medium, and cells were incubated for 48 h at 37 °C. Conditioned media (2 ml) were harvested, centrifuged, and stored at −80 °C. Total RNA was prepared from transfected cells using an RNeasy kit (Quiagen, Hilden, Germany). Relative SEAP activity was determined as optical density at 405 nm of heat-inactivated (5 min at 65 °C) aliquots (100 μl) of conditioned media, which were incubated for 30–60 min at room temperature with 100 μl of SEAP-buffer (1m diethanolamine, 10 mm homoarginine, 1.5 mm MgCl2, 23 mm p-nitrophenyl phosphate). Aliquots (30 μl) of conditioned media were electrophoresed on 15% SDS-polyacrylamide gels under nonreducing conditions, electrotransferred to nitrocellulose Hybond-N membranes (Amersham, Braunschweig, Germany), probed with the polyclonal antiserum K7.16 (29Grugel S. Finkenzeller G. Weindel K. Barleon B. Marmé D. J. Biol. Chem. 1995; 270: 25915-25919Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) raised against human VEGF, and detected using the ECL detection system (Amersham, Braunschweig, Germany). Semiquantitative reverse-transcriptase PCR for determination of VEGF RNA in transfected cells was performed by converting 1 μg of total RNA to cDNA using a first-strand cDNA synthesis kit (Pharmacia, Freiburg, Germany) with random hexanucleotide primers followed by PCR amplification of a 247-base pair VEGF cDNA fragment and a 397-base pair GAPDH cDNA fragment simultaneously using the primers VEGF-E3 (5′-GGTGGACATCTTCCAGGAGTACCC-3′), VEGF-E5R (5′-TTCTTGTCTTGCTCTATCTTTCTTTG-3′), GAPDH1 (5′-AGCGAGACCCCACTAACATCAAA-3′), and GAPDH2 (5′-GTGGATGCAGGGATGATGTTCTG-3′). Recombinant extracellular domain of human VEGF receptor Flt-1 (30Barleon B. Totzke F. Herzog C. Blanke S. Kremmer E. Siemeister G. Marmé D. Martiny-Baron G. J. Biol. Chem. 1997; 272: 10382-10388Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and KDR, 2G. Martiny-Baron, B. Barleon, F. Totzke, D. Marmé, and G. Siemeister, unpublished result. respectively, were coated onto Maxisorb plates (1 μg/well) and were incubated with biotinylated VEGF165 (10 ng/ml) in the presence of increased concentrations of VEGF121 proteins as described previously (30Barleon B. Totzke F. Herzog C. Blanke S. Kremmer E. Siemeister G. Marmé D. Martiny-Baron G. J. Biol. Chem. 1997; 272: 10382-10388Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Quiescent HUVE cells were stimulated with increased concentrations of VEGF121proteins. After 18 h of VEGF-incubation [3H]thymidine (0.5 μCi) was added and the incubation was continued for additional 6 h. The cells were washed and the incorporation of radioactivity was determined by scintillation counting. Amino-terminal truncated VEGF121variants were expressed in E. coli using the T7 RNA-polymerase driven pET system (27Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorf J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5998) Google Scholar). The proteins were solubilized from inclusion body material, refolded, and finally purified by Ni2+-affinity chromatography essentially as described previously for wild-type VEGF121 (26Siemeister G. Schnurr B. Mohrs K. Schächtele C. Marmé D. Martiny-Baron G. Biochem. Biophys. Res. Commun. 1996; 222: 249-255Crossref PubMed Scopus (54) Google Scholar). The E. coli derived “wild-type” VEGF121 in fact covered amino acids Met3 to Arg121 of mature human VEGF121 and showed virtually identical biological activity as compared with human VEGF121 covering amino acids 1 to 121 produced by recombinant techniques in Sf9 insect cells (26Siemeister G. Schnurr B. Mohrs K. Schächtele C. Marmé D. Martiny-Baron G. Biochem. Biophys. Res. Commun. 1996; 222: 249-255Crossref PubMed Scopus (54) Google Scholar). The VEGF variants VEGF121Δ1–7 contained amino acids 8 to 121 of mature human VEGF121 with Gly8 changed to Met, and Gln9 changed to Glu due to insertion of anNcoI restriction endonuclease site, VEGF121Δ1–13 contained amino acids 14–121 with Val14 changed to Met, and VEGF121Δ1–17 contained amino acids 18–121 (Fig. 1 A). For control, VEGF121-C61S was prepared, a monomeric VEGF mutant with Cys61 replaced by serine which did not bind to endothelial cells and showed no significant biological activity (19Pötgens A.J.G. Lubsen N.H. van Altena M.C. Vermeulen R. Bakker A. Schoenmakers J.G.G. Ruiter D.J. de Waal R.M.W. J. Biol. Chem. 1994; 269: 32879-32885Abstract Full Text PDF PubMed Google Scholar). An additional amino-terminal His6 tag facilitated affinity purification of the recombinant proteins. On nonreducing SDS gels VEGF121, VEGF121Δ1–7, and VEGF121Δ1–13 migrated with apparent molecular masses of approximately 34, 32, and 30 kDa, respectively, which correspond to the molecular mass of VEGF121 dimers (Fig. 1 B,lanes 1, 3, and 4). In contrast, VEGF121Δ1–17 (Fig. 1 B, lane 5), as well as the control, VEGF121-C61S (Fig. 1 B,lane 2), migrated as monomers of approximately 16 kDa. Upon electrophoresis on reducing SDS gels all of the VEGF variants migrated as monomeric forms of approximately 16–17 kDa (not shown). Inspection of the amino-terminal amino acid sequence using a structure predicting software (31Geourjon C. Deleage G. J. Mol. Graph. 1995; 13: 209-212Crossref PubMed Scopus (50) Google Scholar) revealed an interspersed sequence of charged (His12, Glu13, Lys16, and Asp19) and hydrophobic (Val14, Val15, Phe17, and Met18) residues which might anticipate an amphipathic α-helical conformation (Fig. 2). Deletion of this domain apparently prevents dimerization of VEGF, at least in vitro. The interaction of the hydrophobic interface of the proposed amphipathic α-helix of one VEGF monomer either with the corresponding domain or with another hydrophobic stretch of the other VEGF monomer might stabilize or favor dimerization of VEGF. To further support this model we constructed a VEGF121 variant (VEGF121-Φ/S) covering amino acids Met3 to Arg121 in which we replaced the hydrophobic amino acids Val14, Val15, Phe17, and Met18 by serine. On nonreducing SDS gels this variant migrated most predominantly as a monomer (Fig. 1C, lane 4) as the deletion mutant VEGF121Δ1–17 (lane 3) did, and in contrast to dimeric VEGF121 and VEGF121Δ1–13 (lanes 1 and 2). From these data we concluded that the amino-terminal domain between His12 and Asp19 is essential at least forin vitro dimerization of VEGF. To analyze whether the amino-terminal domain between His12 and Asp19 is also essential for in vivo dimerization of VEGF the variant VEGF121 cDNAs were fused to the VEGF signal sequence, ligated into pCI-neo expression vector providing cytomegalovirus promoter/enhancer sequences, and transiently transfected into CHO cells. Due to fusion to the signal sequence the resulting expression plasmid pCI-neo/VEGF121 encoded the entire human VEGF121 coding region, whereas in construct pCI-neo/VEGF121Δ3–13 amino acids Met3 to Glu13 had been deleted and Val14 was replaced by Met, and in construct pCI-neo/VEGF121Δ3–18 amino acids Met3 to Phe17 had been deleted. Construct pCI-neo/VEGF121-Φ/S encoded the entire VEGF121 coding sequence in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 had been replaced by serine. Immunoblot analysis of conditioned media of the transfected cells using the polyclonal antiserum K7.16 raised against human VEGF protein showed that VEGF121 and VEGF121Δ3–13 migrated under nonreducing conditions as dimers (Fig. 3 A, lanes 1 and2). In contrast, in conditioned media of pCI-neo/VEGF121Δ3–18 and pCI-neo/VEGF121-Φ/S, as well as in pCI-neo vector control transfected CHO cells, no VEGF protein was detectable by Western blot analysis (Fig. 3 A, lanes 3–5). Western blot analysis of VEGF variants expressed in E. coli showed that the antiserum used was able to recognize all of the various VEGF variants including the monomeric ones (data not shown). Measurement of SEAP activity in conditioned media of the transfected cells revealed efficient transfection of the cells and efficient secretion of proteins even in transfections in which VEGF protein was not detectable in the conditioned medium (Fig. 3 B). Using lysates of transfected cells for Western blot analysis neither wild-type VEGF nor mutant VEGF variants were detectable demonstrating that wild-type VEGF and VEGF121Δ3–13 were secreted efficiently by the cells, and that the VEGF variants VEGF121Δ3–18 and VEGF121-Φ/S were not accumulated within the cells (not shown). Expression of transfected constructs for the VEGF variants at the level of mRNA was shown by semiquantitative reverse-transcriptase PCR (Fig. 3 C). Transfection of human “293” embryonic kidney cells gave similar results (not shown). Taken together these results show that the amino-terminal His12 to Asp19 domain is essential for functional expression of VEGF at least in transfected CHO and 293 cells. Truncation or mutation of this domain either impairs synthesis of VEGF or the cells recognize these variants as aberrant proteins which were apparently degraded. A PDGF-B variant in which the two cysteines involved in interchain disulfide bonds had been converted to serine, migrated as a monomer on non-reducing SDS gels, but exists as a noncovalent dimer at pH 4–7 in solution and shows similar mitogenic activity as compared with wild-type PDGF-BB (31Geourjon C. Deleage G. J. Mol. Graph. 1995; 13: 209-212Crossref PubMed Scopus (50) Google Scholar, 32Kenney W.C. Haniu M. Herman A.C. Arakawa T. Costigan V.J. Lary J. Yphantis D.A. Thomason A.R. J. Biol. Chem. 1994; 269: 12351-12359Abstract Full Text PDF PubMed Google Scholar). To analyze the biological activity of the amino-terminal VEGF121 variants receptor binding assays and proliferation assays were performed. Binding of the VEGF variants expressed in E. coli to recombinant extracellular domain of human VEGF receptors Flt-1 (Fig. 4 A) and KDR (Fig. 4 B), respectively, was studied by competition assays with biotinylated VEGF165. The dimeric VEGF variants VEGF121Δ1–7 and VEGF121Δ1–13 competed with biotinylated VEGF for binding of both of the VEGF receptors in an almost undistinguishable manner as compared with wild-type VEGF121. Binding of the monomeric variant VEGF121Δ1–17, as well as the monomeric control VEGF121-C61S, to the receptors was strongly impaired. Growth of HUVE cells was stimulated by the VEGF variants VEGF121Δ1–7 and VEGF121Δ1–13 in a dose dependent manner which was almost undistinguishable from wild-type VEGF121 stimulated growth of the cells. The monomeric variants VEGF121Δ1–17 and VEGF121-C61S failed to induce proliferation of HUVE cells (Fig. 4 C). Taken together these results show that truncation or mutation of the VEGF amino-terminal α-helical domain prevents the formation of stable VEGF dimers although the cysteines involved in formation of the core structure of VEGF had not been affected. Dimerization of VEGF had previously been shown to be a prerequisite for biological activity (19Pötgens A.J.G. Lubsen N.H. van Altena M.C. Vermeulen R. Bakker A. Schoenmakers J.G.G. Ruiter D.J. de Waal R.M.W. J. Biol. Chem. 1994; 269: 32879-32885Abstract Full Text PDF PubMed Google Scholar). The analysis of amino-terminal truncated VEGF121variants revealed a domain between amino acids His12 and Asp19 that is essential for in vitrodimerization of VEGF and for functional expression of VEGF in vivo. This domain showed an interspersed sequence of charged and hydrophobic amino acids which may anticipate an amphipathic α-helical conformation. We postulate that the interaction of hydrophobic interfaces stabilize or favor dimerization of VEGF. Conversion of the hydrophobic amino acids to serine by site-directed mutagenesis, which results in VEGF monomers, supports this model. The very recently by Muller et al. (21Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (365) Google Scholar) solved crystal structure of VEGF indeed revealed that the amino acids near the amino terminus anticipate an α-helical conformation. The crystal structure shows that hydrophobic amino acids from this α-helix together with residues from helix α2, loop regions β1–β3, and β5–β6, and sheet β6 of the other monomer form a small hydrophobic core that presumably stabilizes the central structure of the VEGF dimer. In addition, they found that a Phe17 to alanine mutant VEGF displayed on a phage surface lost KDR receptor binding implicating a contribution of Phe17 to receptor binding. As the conformation of the Phe17 → Ala mutant displayed on the phage surface was not investigated, our results implicate that loss of KDR binding is more likely due to impaired dimerization of the mutant VEGF. The His12 to Asp19 domain is highly conserved between human, sheep (34Redmer D.A. Dai Y. Li J. Charnock-Jones D.S. Smith S.K. Reynolds L.P. Moor R.M. J. Reprod. Fertil. 1996; 108: 157-165Crossref PubMed Scopus (68) Google Scholar), porcine (35Sharma H.S. Tang Z.H. Gho B.C.H. Verdouw P.D. Biochim. Biophys. Acta. 1995; 1260: 235-238Crossref PubMed Scopus (32) Google Scholar), bovine (36Tischer E. Gospodarowicz D. Mitchell R. Silva M. Schilling J. Lau K. Crisp T. Fiddes J.C. Abraham J.A. Biochem. Biophys. Res. Commun. 1989; 165: 1198-1206Crossref PubMed Scopus (255) Google Scholar), and mouse (37Claffey K.P. Wilkison W.O. Spiegelman B.M. J. Biol. Chem. 1992; 267: 16317-16322Abstract Full Text PDF PubMed Google Scholar) VEGF (Fig. 5). Similar interspersed sequences of charged and hydrophobic amino acids are located amino-terminal with respect to the v-sis homology regions of the VEGF-related growth factors PlGF (16Maglione D. Guerriero V. Vigliette G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (831) Google Scholar), VEGF-B (17Olofsson B. Pajusola K. Kaipainen A. Von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (620) Google Scholar), and VEGF-C (18Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1144) Google Scholar,38Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (640) Google Scholar). Heterodimerization of VEGF and PlGF has shown to occur in vitro (39Birkenhäger R. Schneppe B. Röckl W. Wilting J. Weich H.A. McCarthy J.E.G. Biochem. J. 1996; 316: 703-707Crossref PubMed Scopus (47) Google Scholar, 40Cao Y. Chen H. Zhou L. Chiang M.K. Anand-Apte B. Weatherbee J.A. Wang Y. Fang F. Flanagan J.G. Tsang M.L.S. J. Biol. Chem. 1996; 271: 3154-3162Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) and in vivo (41Cao Y. Linden P. Shima D. Browne F. Folkman J. J. Clin. Invest. 1996; 98: 2507-2511Crossref PubMed Scopus (183) Google Scholar), as well as heterodimerization of VEGF and VEGF-B was reported for cells expressing both growth factors (17Olofsson B. Pajusola K. Kaipainen A. Von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (620) Google Scholar). Heterodimerization of the members of the family of VEGF-related growth factors is thought to contribute to a fine tuning of angiogenic stimuli (41Cao Y. Linden P. Shima D. Browne F. Folkman J. J. Clin. Invest. 1996; 98: 2507-2511Crossref PubMed Scopus (183) Google Scholar). Although the involvement of the amino-terminal domain in the heterodimerization between various VEGF-related growth factor monomers has not been investigated so far, the similarity of the amino-terminal domains of the VEGF-related proteins suggest that heterodimerization would be supported by these domains. Heterodimerization of VEGF and PDGF-B has not been observed so far although the eight cysteines building the core structure are perfectly conserved. One of the most significant differences in the crystal structure of VEGF (21Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (365) Google Scholar) and PDGF-B (42Oefner C. D'Arcy A. Winkler F.K. Eggimann B. Hosang M. EMBO J. 1992; 11: 3921-3926Crossref PubMed Scopus (185) Google Scholar) is the structure of the amino terminus which is extended in PDGF rather than alpha-helical as in VEGF, whereas the monomer topology and side-by-side dimer association is highly similar for both proteins. In the PDGF-BB dimer the extended amino terminus of one chain makes contact to the other PDGF chain, but in contrast to VEGF, the amino terminus is not essential for dimerization of PDGF as it was shown by mutagenic analyses which resulted in the identification of the minimal v-sis transforming domain (22Hannink M. Donoghue D.J. Science. 1984; 226: 1197-1199Crossref PubMed Scopus (29) Google Scholar, 23King C.R. Giese N.A. Robbins K.C. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5295-5299Crossref PubMed Scopus (23) Google Scholar). Analysis of the crystal structure of transforming growth factor (TGF)-β2 (43Schlunegger M.P. Grütter M.G. Nature. 1992; 358: 430-434Crossref PubMed Scopus (284) Google Scholar), which is besides PDGF, VEGF, and nerve growth factor, another member of the cystine knot family of growth factors, revealed a close contact of α-helix H3 of one TGF-β2 chain to β-strand structures of the other chain in the TGF-β2dimer, which presumably stabilizes the dimeric structure. α-Helix H3 is located within TGF-β2 at a position that correlates to loop/turn II in PDGF-B and VEGF. Interference with the VEGF/VEGF-receptor system is generally viewed as an attractive target for therapeutical intervention in a variety of human pathological situations which involve elevated VEGF expression and aberrant endothelial cell proliferation such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. Enhanced VEGF expression is induced by various factors and growth conditions including growth factors (see Finkenzeller et al. (44Finkenzeller G. Sparacio A. Technau A. Marmé D. Siemeister G. Oncogene. 1997; 15: 669-676Crossref PubMed Scopus (184) Google Scholar), and references therein), activated oncogenes (28Dirks W. Wirth M. Hauser H. Gene (Amst .). 1993; 128: 247-249Crossref PubMed Scopus (153) Google Scholar, 45Rak J. Mitsuhashi Y. Bayko L. Filmus J. Shirasawa S. Sasazuki T. Kerbel R.S. Cancer Res. 1995; 55: 4575-4580PubMed Google Scholar, 46Mukhopadhyay D. Tsiokas L. Sukhatme V.P. Cancer Res. 1995; 55: 6161-6165PubMed Google Scholar), inactivated tumor suppressor genes (47Kieser A. Weich H.A. Brandner G. Marme D. Kölch W. Oncogene. 1994; 9: 963-969PubMed Google Scholar, 48Siemeister G. Weindel K. Mohrs K. Barleon B. Martiny-Baron G. Marmé D. Cancer Res. 1996; 56: 2299-2301PubMed Google Scholar), and hypoxia (49Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Crossref PubMed Scopus (4144) Google Scholar). Diverse mechanisms acting at the level of promoter activation (44Finkenzeller G. Sparacio A. Technau A. Marmé D. Siemeister G. Oncogene. 1997; 15: 669-676Crossref PubMed Scopus (184) Google Scholar), mRNA stabilization (50Ikeda E. Achen M.G. Breier G. Risau W. J. Biol. Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 51Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 25492-25497Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and translational regulation (52Kevil C.G. De Benedetti A. Payne D.K. Coe L.L. Laroux F.S. Alexander J.S. Int. J. Cancer. 1996; 65: 785-790Crossref PubMed Scopus (173) Google Scholar) have been shown to be involved in up-regulation of VEGF expression. The multiplicity of factors and mechanisms involved in VEGF expression hamper the development of therapeutical approaches directed to reduce enhanced expression of the growth factor in pathological settings. As VEGF dimerization is an event that is necessary for biological activity of the growth factor, but is independent from the diverse mechanisms of regulation of gene expression and translation, prevention of dimerization by interference with the amino-terminal domain may be a promising strategy for therapeutical down-regulation of VEGF expression. We thank Steffi Koidl, Gabi Bader, and Katja Mohrs for excellent technical assistance." @default.
• W2065860196 created "2016-06-24" @default.
• W2065860196 creator A5023028041 @default.
• W2065860196 creator A5025153159 @default.
• W2065860196 creator A5040107084 @default.
• W2065860196 date "1998-05-01" @default.
• W2065860196 modified "2023-10-16" @default.
• W2065860196 title "The α-Helical Domain Near the Amino Terminus Is Essential for Dimerization of Vascular Endothelial Growth Factor" @default.
• W2065860196 cites W1482104159 @default.
• W2065860196 cites W1489873347 @default.
• W2065860196 cites W1565340691 @default.
• W2065860196 cites W1595789730 @default.
• W2065860196 cites W1608077902 @default.
• W2065860196 cites W1679229092 @default.
• W2065860196 cites W1873199501 @default.
• W2065860196 cites W1964059120 @default.
• W2065860196 cites W1967216944 @default.
• W2065860196 cites W1969328947 @default.
• W2065860196 cites W1970918239 @default.
• W2065860196 cites W1974837569 @default.
• W2065860196 cites W1975505830 @default.
• W2065860196 cites W1981612052 @default.
• W2065860196 cites W1988688040 @default.
• W2065860196 cites W1990467912 @default.
• W2065860196 cites W1990494380 @default.
• W2065860196 cites W1993011693 @default.
• W2065860196 cites W1999647970 @default.
• W2065860196 cites W1999759354 @default.
• W2065860196 cites W2009306131 @default.
• W2065860196 cites W2011995063 @default.
• W2065860196 cites W2012925808 @default.
• W2065860196 cites W2013141629 @default.
• W2065860196 cites W2013906072 @default.
• W2065860196 cites W2015344488 @default.
• W2065860196 cites W2017205210 @default.
• W2065860196 cites W2019273955 @default.
• W2065860196 cites W2027713993 @default.
• W2065860196 cites W2029195137 @default.
• W2065860196 cites W2029521699 @default.
• W2065860196 cites W2029763523 @default.
• W2065860196 cites W2030998830 @default.
• W2065860196 cites W2034393061 @default.
• W2065860196 cites W2035205184 @default.
• W2065860196 cites W2037141117 @default.
• W2065860196 cites W2041140715 @default.
• W2065860196 cites W2045247659 @default.
• W2065860196 cites W2061834490 @default.
• W2065860196 cites W2075086348 @default.
• W2065860196 cites W2084506573 @default.
• W2065860196 cites W2093498435 @default.
• W2065860196 cites W2114098415 @default.
• W2065860196 cites W2123840402 @default.
• W2065860196 cites W2131519105 @default.
• W2065860196 cites W2150732175 @default.
• W2065860196 cites W2171543828 @default.
• W2065860196 cites W2227416752 @default.
• W2065860196 cites W2324588265 @default.
• W2065860196 doi "https://doi.org/10.1074/jbc.273.18.11115" @default.
• W2065860196 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9556597" @default.
• W2065860196 hasPublicationYear "1998" @default.
• W2065860196 type Work @default.
• W2065860196 sameAs 2065860196 @default.
• W2065860196 citedByCount "21" @default.
• W2065860196 countsByYear W20658601962014 @default.
• W2065860196 countsByYear W20658601962015 @default.
• W2065860196 countsByYear W20658601962017 @default.
• W2065860196 countsByYear W20658601962019 @default.
• W2065860196 countsByYear W20658601962020 @default.
• W2065860196 crossrefType "journal-article" @default.
• W2065860196 hasAuthorship W2065860196A5023028041 @default.
• W2065860196 hasAuthorship W2065860196A5025153159 @default.
• W2065860196 hasAuthorship W2065860196A5040107084 @default.
• W2065860196 hasBestOaLocation W20658601961 @default.
• W2065860196 hasConcept C103041312 @default.
• W2065860196 hasConcept C12554922 @default.
• W2065860196 hasConcept C134306372 @default.
• W2065860196 hasConcept C146285616 @default.
• W2065860196 hasConcept C167734588 @default.
• W2065860196 hasConcept C170493617 @default.
• W2065860196 hasConcept C185592680 @default.
• W2065860196 hasConcept C2775960820 @default.
• W2065860196 hasConcept C2777025900 @default.
• W2065860196 hasConcept C33923547 @default.
• W2065860196 hasConcept C36503486 @default.
• W2065860196 hasConcept C502942594 @default.
• W2065860196 hasConcept C55493867 @default.
• W2065860196 hasConcept C86803240 @default.
• W2065860196 hasConcept C95444343 @default.
• W2065860196 hasConceptScore W2065860196C103041312 @default.
• W2065860196 hasConceptScore W2065860196C12554922 @default.
• W2065860196 hasConceptScore W2065860196C134306372 @default.
• W2065860196 hasConceptScore W2065860196C146285616 @default.
• W2065860196 hasConceptScore W2065860196C167734588 @default.
• W2065860196 hasConceptScore W2065860196C170493617 @default.
• W2065860196 hasConceptScore W2065860196C185592680 @default.
• W2065860196 hasConceptScore W2065860196C2775960820 @default.
• W2065860196 hasConceptScore W2065860196C2777025900 @default.
• W2065860196 hasConceptScore W2065860196C33923547 @default.

• next