Abstract
Flt‐1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor‐1, binds VEGF and a new VEGF‐related ligand, placenta growth factor, but KDR/Flk‐1 (VEGF receptor‐2) binds only VEGF. To characterize the functional regions in the Flt‐1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt‐1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N‐terminal 1st to 3rd immunoglobulin (Ig)‐like domains of Flt‐1 binds both ligands with high affinity. However, for dimer formation of soluble Flt‐1, a region further downstream in the Flt‐1 extracellular domain was required. Mutant Flt‐1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt‐1 tyrosine kinase. Soluble Flt‐1 carrying the N‐terminal 1st to 3rd Ig region suppressed VEGF‐dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt‐1, suggesting that the binding of one soluble Flt‐1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.
Keywords: VEGF, Angiogenesis, Tyrosine kinase, Flt‐1
Full Text
The Full Text of this article is available as a PDF (647.6 KB).
REFERENCES
- 1. ) Risau , W. , Sariola , H. , Zerwes , H.‐G. , Sasse , J. , Ekblom , P. , Kemler , R. and Doetschman , T.Vasculogenesis and angiogenesis in embryonic‐stem‐cell‐derived embryoid bodies . Development , 102 , 471 – 478 ( 1988. ). [DOI] [PubMed] [Google Scholar]
- 2. ) Folkman , J.What is the evidence that tumors are angiogenesis dependent ? J. Natl. Cancer Inst. , 82 , 4 – 6 ( 1990. ). [DOI] [PubMed] [Google Scholar]
- 3. ) Ferrara , N. , Houck , K. A. , Jakeman , L. B. , Winer , J. and Leung , D. W.The vascular endothelial growth factor family of polypeptides . J. Cell. Biochem. , 47 , 211 – 218 ( 1991. ). [DOI] [PubMed] [Google Scholar]
- 4. ) Shibuya , M.Role of VEGF‐Flt receptor system in normal and tumor angiogenesis . Adv. Cancer Res. , 67 , 281 – 316 ( 1995. ). [DOI] [PubMed] [Google Scholar]
- 5. ) Mustonen , T. and Alitalo , K.Endothelial receptor tyro‐sine kinases involved in angiogenesis . J. Cell Biol. , 129 , 895 – 898 ( 1995. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. ) Shibuya , M. , Yamaguchi , S‐ , Yamane , A. , Ikeda , T. , Tojo , A. , Matsushime , H. and Sato , M.Nucleotide sequence and expression of a novel human receptor‐type tyrosine kinase gene (fit) closely related to the fins family . Oncogene , 5 , 519 – 524 ( 1990. ). [PubMed] [Google Scholar]
- 7. ) Matthews , W. , Jordan , C. T. , Gavin , M. , Jenkins , N. A. , Copeland , N. G. and Lemischka , I. R.A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c‐kit . Proc. Natl Acad. Set. USA , 88 , 9026 – 9030 ( 1991. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. ) de Vries , C. , Escobedo , J. A. , Ueno , H. , Houck , K. , Ferrara , N. and Williams , L. T.The fins‐like tyrosine kinase, a receptor for vascular endothelial growth factor . Science , 255 , 989 – 991 ( 1992. ). [DOI] [PubMed] [Google Scholar]
- 9. ) Terman , B. I. , Dougher‐Vermazen , M. , Carrion , M. E. , Dimitrov , D. , Armellino , D. C. , Gospodarowicz , D. and Bohlen , P.Identification of the KDR tyrosine kinase as a receptor for vascular endothelial growth factor . Biochem. Biophys. Res. Commun. , 187 , 1579 – 1586 ( 1992. ). [DOI] [PubMed] [Google Scholar]
- 10. ) Millauer , B. , Wizigmann‐Voos , S. , Shuruch , H. , Martinez , R. , Moller , N. P. H. , Risau , W. and Ullrich , A.High affinity VEGF binding and developmental expression suggest Flk‐1 as a major regulator of vasculogenesis and angiogenesis . Cell , 72 , 835 – 846 ( 1993. ). [DOI] [PubMed] [Google Scholar]
- 11. ) Maglione , D. , Guerriero , V. , Viglietto , G. , Delli‐Bovi , P. and Persico , M. G.Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor . Proc. Natl. Acad. Sci. USA , 88 , 9267 – 9271 ( 1991. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. ) Park , J. E. , Chen , H. H. , Winer , J. , Houck , K. A. and Ferrara , N.Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to FLT‐1 but not to Flk‐1/ KDR . J. Biol Chem. , 269 , 25646 – 25654 ( 1994. ). [PubMed] [Google Scholar]
- 13. ) Kendall , R. L. , Wang , G. , DiSalvo , J. and Thomas , A.Specificity of vascular endothelial growth factor receptor ligand domains . Biochem, Biophys, Res. Commun , 201 , 326 – 330 ( 1994. ). [DOI] [PubMed] [Google Scholar]
- 14. ) Sawano , A. , Takahashi , T. , Yamaguchi , S. , Aonuma , T. and Shibuya , M.Flt‐1 but not KDR/Flk‐1 tyrosine kinase is a receptor for placenta growth factor (P1GF), which is related to vascular endothelial growth factor (VEGF) . Cell Growth Differ. , 7 , 213 – 221 ( 1996. ). [PubMed] [Google Scholar]
- 15. ) Seetharam , L. , Gotoh , N. , Maru , Y. , Neufeld , G. , Yamaguchi , S. and Shibuya , M.A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF . Oncogens , 10 , 135 – 147 ( 1995. ). [PubMed] [Google Scholar]
- 16. ) Barleon , B. , Sozzani , S. , Zhou , D. , Welch , H. A. , Mantovani , A. and Marme , D.Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt‐1 . Blood , 87 , 3336 – 3343 ( 1996. ). [PubMed] [Google Scholar]
- 17. ) Clauss , M. , Weich , H. , Breier , G. , Knies , U. , Rock , W. , Waltenberger , J. and Risau , W.The vascular endothelial growth factor receptor Flt‐1 mediates biological activities . J. Biol Chem. , 271 , 17629 – 17634 ( 1996. ). [DOI] [PubMed] [Google Scholar]
- 18. ) Keyt , B. A. , Nguyen , H. V. , Berleau , L. T. , Duarte , C. M. , Park , J. , Chen , H. and Ferrara , N.Identification of vascular endothelial growth factor determinants for binding KDR and FLT‐1 receptors . J. Biol Chem. , 271 , 5638 – 5646 ( 1996. ). [DOI] [PubMed] [Google Scholar]
- 19. ) Davis‐Smyth , T. , Chen , H. , Park , J. , Presta , L. G. and Ferrara , N.The second immunoglobulin‐like domain of the VEGF tyrosine kinase receptor Flt‐1 determines ligand binding and may initiate a signal transduction cascade . EMBO J. , 15 , 4919 – 4927 ( 1996. ). [PMC free article] [PubMed] [Google Scholar]
- 20. ) Takebe , Y. , Seiki , M. , Fujisawa , J. , Hoy , P. , Yokota , T. , Arai , K. , Yoshida , M. and Arai , N.SRa promoter: an efficient and versatile mammalian cDNA expression system of human T‐cell leukemia virus type 1 long terminal repeat . Mol Cell. Biol. , 8 , 466 – 472 ( 1988. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. ) Kendall , R. L. and Thomas , K. A.Inhibition of endothelial cell growth factor activity by an endogenously encoded soluble receptor . Proc. Natl. Acad. Sci. USA , 90 , 10705 – 10709 ( 1993. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. ) Fraker , P. J. and Speck , J. C.Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6‐tetrachloro‐3a,6a‐diphenylglycoluril . Biochem. Biophys. Res. Commun. , 80 , 849 – 857 ( 1978. ). [DOI] [PubMed] [Google Scholar]
- 23. ) Duan , D. R. , Pazin , M. J. , Fretto , L. J. and Williams , L. T.A functional soluble extracellular region of the platelet‐derived growth factor (PDGF) β‐receptor antagonizes PDGF‐stimulated responses . J. Biol Chem , 266 , 413 – 418 ( 1991. ). [PubMed] [Google Scholar]
- 24. ) Scatchard , G.The attraction of proteins for small molecules and ions . Ann, N, Y. Acad. Sci. , 51 , 660 – 672 ( 1949. ). [Google Scholar]
- 25. ) Tanaka , K. , Sato , M. , Tomita , Y. and Ichihara , A.Biochemical studies on liver functions in primary cultured hepatocytes of adult rats . J. Biol Chem. , 84 , 937 – 946 ( 1978. ). [DOI] [PubMed] [Google Scholar]
- 26. ) Shimaoka , S. , Nakamura , T. and Ichihara , A.Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells . Exp. Cell Res. , 172 , 228 – 242 ( 1987. ). [DOI] [PubMed] [Google Scholar]
- 27. ) Yamane , A. , Seetharam , L. , Yamaguchi , S. , Gotoh , N. , Takahashi , T. , Neufeld , G. and Shibuya , M.A new communication system between hepatocytes and sinusoidal endothelial cells in liver through vascular endothelial growth factor and Fit tyrosine kinase receptor family (Flt‐1 and KDR/Flk‐1) . Oncogens , 9 , 2683 – 2690 ( 1994. ). [PubMed] [Google Scholar]
- 28. ) Schlessinger , J. and Ullrich , A. , Growth factor signaling by receptor tyrosine kinases . Neuron , 9 , 383 – 391 ( 1992. ). [DOI] [PubMed] [Google Scholar]
- 29. ) Pajusola , K. , Aprelikova , O. , Korhonen , J. , Kaipainen , A. , Pertovaara , L. , Alitalo , R. and Alitalo , K.FLT4 receptor tyrosine kinase contains seven immunoglobulin‐like loops and is expressed in multiple human tissues and cell lines . Cancer Res. , 52 , 5738 – 5743 ( 1992. ). [PubMed] [Google Scholar]
- 30. ) Galland , F. , Karamysheva , A. , Mattei , M. G. , Rosnet , O. , Marchetto , S. and Birnbaum , D.Chromosomal localization of FLT4, a novel receptor‐type tyrosine kinase gene . Genomics , 13 , 475 – 478 ( 1992. ). [DOI] [PubMed] [Google Scholar]
- 31. ) Blechman , J. M. , Lev , S. , Barg , J. , Eisenstein , M. , Baruch , V. , Vogel , Z. , Givol , D. and Yarden , Y.The fourth immunoglobulin domain of the stem cell factor receptor couples ligand binding to signal transduction . Cell , 80 , 103 – 113 ( 1995. ). [DOI] [PubMed] [Google Scholar]