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. 1996 Sep 16;15(18):4919–4927.

The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade.

T Davis-Smyth 1, H Chen 1, J Park 1, L G Presta 1, N Ferrara 1
PMCID: PMC452229  PMID: 8890165

Abstract

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aiello 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. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994 Dec 1;331(22):1480–1487. doi: 10.1056/NEJM199412013312203. [DOI] [PubMed] [Google Scholar]
  2. Aiello L. P., Pierce E. A., Foley E. D., Takagi H., Chen H., Riddle L., Ferrara N., King G. L., Smith L. E. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10457–10461. doi: 10.1073/pnas.92.23.10457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barleon B., Sozzani S., Zhou D., Weich H. A., Mantovani A., Marmé D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood. 1996 Apr 15;87(8):3336–3343. [PubMed] [Google Scholar]
  4. Blechman J. M., Lev S., Barg J., Eisenstein M., Vaks B., Vogel Z., Givol D., Yarden Y. The fourth immunoglobulin domain of the stem cell factor receptor couples ligand binding to signal transduction. Cell. 1995 Jan 13;80(1):103–113. doi: 10.1016/0092-8674(95)90455-7. [DOI] [PubMed] [Google Scholar]
  5. Capon D. J., Chamow S. M., Mordenti J., Marsters S. A., Gregory T., Mitsuya H., Byrn R. A., Lucas C., Wurm F. M., Groopman J. E. Designing CD4 immunoadhesins for AIDS therapy. Nature. 1989 Feb 9;337(6207):525–531. doi: 10.1038/337525a0. [DOI] [PubMed] [Google Scholar]
  6. Carmeliet P., Ferreira V., Breier G., Pollefeyt S., Kieckens L., Gertsenstein M., Fahrig M., Vandenhoeck A., Harpal K., Eberhardt C. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996 Apr 4;380(6573):435–439. doi: 10.1038/380435a0. [DOI] [PubMed] [Google Scholar]
  7. Fantl W. J., Johnson D. E., Williams L. T. Signalling by receptor tyrosine kinases. Annu Rev Biochem. 1993;62:453–481. doi: 10.1146/annurev.bi.62.070193.002321. [DOI] [PubMed] [Google Scholar]
  8. Ferrara N., Carver-Moore K., Chen H., Dowd M., Lu L., O'Shea K. S., Powell-Braxton L., Hillan K. J., Moore M. W. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996 Apr 4;380(6573):439–442. doi: 10.1038/380439a0. [DOI] [PubMed] [Google Scholar]
  9. Finnerty H., Kelleher K., Morris G. E., Bean K., Merberg D. M., Kriz R., Morris J. C., Sookdeo H., Turner K. J., Wood C. R. Molecular cloning of murine FLT and FLT4. Oncogene. 1993 Aug;8(8):2293–2298. [PubMed] [Google Scholar]
  10. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995 Jan;1(1):27–31. doi: 10.1038/nm0195-27. [DOI] [PubMed] [Google Scholar]
  11. Fong G. H., Rossant J., Gertsenstein M., Breitman M. L. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature. 1995 Jul 6;376(6535):66–70. doi: 10.1038/376066a0. [DOI] [PubMed] [Google Scholar]
  12. Galland F., Karamysheva A., Pebusque M. J., Borg J. P., Rottapel R., Dubreuil P., Rosnet O., Birnbaum D. The FLT4 gene encodes a transmembrane tyrosine kinase related to the vascular endothelial growth factor receptor. Oncogene. 1993 May;8(5):1233–1240. [PubMed] [Google Scholar]
  13. Heidaran M. A., Mahadevan D., Larochelle W. J. Beta PDGFR-IgG chimera demonstrates that human beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding. FASEB J. 1995 Jan;9(1):140–145. doi: 10.1096/fasebj.9.1.7821753. [DOI] [PubMed] [Google Scholar]
  14. Heidaran M. A., Pierce J. H., Jensen R. A., Matsui T., Aaronson S. A. Chimeric alpha- and beta-platelet-derived growth factor (PDGF) receptors define three immunoglobulin-like domains of the alpha-PDGF receptor that determine PDGF-AA binding specificity. J Biol Chem. 1990 Nov 5;265(31):18741–18744. [PubMed] [Google Scholar]
  15. Heldin C. H. Dimerization of cell surface receptors in signal transduction. Cell. 1995 Jan 27;80(2):213–223. doi: 10.1016/0092-8674(95)90404-2. [DOI] [PubMed] [Google Scholar]
  16. Jakeman L. B., Armanini M., Phillips H. S., Ferrara N. Developmental expression of binding sites and messenger ribonucleic acid for vascular endothelial growth factor suggests a role for this protein in vasculogenesis and angiogenesis. Endocrinology. 1993 Aug;133(2):848–859. doi: 10.1210/endo.133.2.7688292. [DOI] [PubMed] [Google Scholar]
  17. Jakeman L. B., Winer J., Bennett G. L., Altar C. A., Ferrara N. Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. J Clin Invest. 1992 Jan;89(1):244–253. doi: 10.1172/JCI115568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Joukov V., Pajusola K., Kaipainen A., Chilov D., Lahtinen I., Kukk E., Saksela O., Kalkkinen N., Alitalo K. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J. 1996 Jan 15;15(2):290–298. [PMC free article] [PubMed] [Google Scholar]
  19. Keyt B. A., Nguyen H. V., Berleau L. T., Duarte C. M., Park J., Chen H., Ferrara N. Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis. J Biol Chem. 1996 Mar 8;271(10):5638–5646. doi: 10.1074/jbc.271.10.5638. [DOI] [PubMed] [Google Scholar]
  20. Kim K. J., Li B., Winer J., Armanini M., Gillett N., Phillips H. S., Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature. 1993 Apr 29;362(6423):841–844. doi: 10.1038/362841a0. [DOI] [PubMed] [Google Scholar]
  21. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee J., Gray A., Yuan J., Luoh S. M., Avraham H., Wood W. I. Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1988–1992. doi: 10.1073/pnas.93.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maglione D., Guerriero V., Viglietto G., Delli-Bovi P., Persico M. G. Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9267–9271. doi: 10.1073/pnas.88.20.9267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mahadevan D., Yu J. C., Saldanha J. W., Thanki N., McPhie P., Uren A., LaRochelle W. J., Heidaran M. A. Structural role of extracellular domain 1 of alpha-platelet-derived growth factor (PDGF) receptor for PDGF-AA and PDGF-BB binding. J Biol Chem. 1995 Nov 17;270(46):27595–27600. doi: 10.1074/jbc.270.46.27595. [DOI] [PubMed] [Google Scholar]
  25. Matthews W., Jordan C. T., Gavin M., Jenkins N. A., Copeland N. G., 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 Sci U S A. 1991 Oct 15;88(20):9026–9030. doi: 10.1073/pnas.88.20.9026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Millauer B., Shawver L. K., Plate K. H., Risau W., Ullrich A. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature. 1994 Feb 10;367(6463):576–579. doi: 10.1038/367576a0. [DOI] [PubMed] [Google Scholar]
  27. Millauer B., Wizigmann-Voos S., Schnürch H., Martinez R., Møller N. P., Risau W., Ullrich A. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell. 1993 Mar 26;72(6):835–846. doi: 10.1016/0092-8674(93)90573-9. [DOI] [PubMed] [Google Scholar]
  28. Mustonen T., Alitalo K. Endothelial receptor tyrosine kinases involved in angiogenesis. J Cell Biol. 1995 May;129(4):895–898. doi: 10.1083/jcb.129.4.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pajusola K., Aprelikova O., Korhonen J., Kaipainen A., Pertovaara L., Alitalo R., Alitalo K. FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines. Cancer Res. 1992 Oct 15;52(20):5738–5743. [PubMed] [Google Scholar]
  30. Pajusola K., Aprelikova O., Pelicci G., Weich H., Claesson-Welsh L., Alitalo K. Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors. Oncogene. 1994 Dec;9(12):3545–3555. [PubMed] [Google Scholar]
  31. Park J. E., Chen H. H., Winer J., Houck K. A., 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. 1994 Oct 14;269(41):25646–25654. [PubMed] [Google Scholar]
  32. Pearlman J. D., Hibberd M. G., Chuang M. L., Harada K., Lopez J. J., Gladstone S. R., Friedman M., Sellke F. W., Simons M. Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis. Nat Med. 1995 Oct;1(10):1085–1089. doi: 10.1038/nm1095-1085. [DOI] [PubMed] [Google Scholar]
  33. Peters K. G., De Vries C., Williams L. T. Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8915–8919. doi: 10.1073/pnas.90.19.8915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Quinn T. P., Peters K. G., De Vries C., Ferrara N., Williams L. T. Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7533–7537. doi: 10.1073/pnas.90.16.7533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schneider R., Schweiger M. A novel modular mosaic of cell adhesion motifs in the extracellular domains of the neurogenic trk and trkB tyrosine kinase receptors. Oncogene. 1991 Oct;6(10):1807–1811. [PubMed] [Google Scholar]
  36. Seetharam L., Gotoh N., Maru Y., Neufeld G., Yamaguchi S., Shibuya M. A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF. Oncogene. 1995 Jan 5;10(1):135–147. [PubMed] [Google Scholar]
  37. Shalaby F., Rossant J., Yamaguchi T. P., Gertsenstein M., Wu X. F., Breitman M. L., Schuh A. C. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature. 1995 Jul 6;376(6535):62–66. doi: 10.1038/376062a0. [DOI] [PubMed] [Google Scholar]
  38. Shibuya M. Role of VEGF-flt receptor system in normal and tumor angiogenesis. Adv Cancer Res. 1995;67:281–316. doi: 10.1016/s0065-230x(08)60716-2. [DOI] [PubMed] [Google Scholar]
  39. Shibuya M., Yamaguchi S., Yamane A., Ikeda T., Tojo A., Matsushime H., Sato M. Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene. 1990 Apr;5(4):519–524. [PubMed] [Google Scholar]
  40. Su W., Middleton T., Sugden B., Echols H. DNA looping between the origin of replication of Epstein-Barr virus and its enhancer site: stabilization of an origin complex with Epstein-Barr nuclear antigen 1. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10870–10874. doi: 10.1073/pnas.88.23.10870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Takeshita S., Zheng L. P., Brogi E., Kearney M., Pu L. Q., Bunting S., Ferrara N., Symes J. F., Isner J. M. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest. 1994 Feb;93(2):662–670. doi: 10.1172/JCI117018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Terman B. I., Carrion M. E., Kovacs E., Rasmussen B. A., Eddy R. L., Shows T. B. Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene. 1991 Sep;6(9):1677–1683. [PubMed] [Google Scholar]
  43. Terman B. I., Dougher-Vermazen M., Carrion M. E., Dimitrov D., Armellino D. C., Gospodarowicz D., Böhlen P. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1579–1586. doi: 10.1016/0006-291x(92)90483-2. [DOI] [PubMed] [Google Scholar]
  44. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  45. Urfer R., Tsoulfas P., O'Connell L., Shelton D. L., Parada L. F., Presta L. G. An immunoglobulin-like domain determines the specificity of neurotrophin receptors. EMBO J. 1995 Jun 15;14(12):2795–2805. doi: 10.1002/j.1460-2075.1995.tb07279.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Waltenberger J., Claesson-Welsh L., Siegbahn A., Shibuya M., Heldin C. H. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem. 1994 Oct 28;269(43):26988–26995. [PubMed] [Google Scholar]
  47. Williams A. F., Barclay A. N. The immunoglobulin superfamily--domains for cell surface recognition. Annu Rev Immunol. 1988;6:381–405. doi: 10.1146/annurev.iy.06.040188.002121. [DOI] [PubMed] [Google Scholar]
  48. Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]
  49. de Vries C., Escobedo J. A., Ueno H., Houck K., Ferrara N., Williams L. T. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science. 1992 Feb 21;255(5047):989–991. doi: 10.1126/science.1312256. [DOI] [PubMed] [Google Scholar]

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