Identification of the ligand-binding domain of human vascular-endothelial-growth-factor receptor Flt-1

Biotechnol Appl Biochem. 2001 Dec;34(3):199-204. doi: 10.1042/ba20010043.

Abstract

The vascular-endothelial-growth-factor (VEGF) receptor Flt-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven immunoglobulin-like loops within the extracellular domain and the first three N-terminal immunoglobulin-like loops are involved in high-affinity binding of VEGF. The minimal extracellular domains of Flt-1 to achieve VEGF binding were screened using the yeast two-hybrid system. The result showed that the binding capacity of loop2-3 was close to that of loop1-3. The two truncated mutants consisting of loop2-3 and loop1-3 were expressed in the methylotrophic yeast Pichia pastoris at high levels (0.3 g[corrected]/litre). The corresponding proteins, named soluble (s)Flt-1(2-3) and sFlt-1(1-3), were purified. An in vitro biological activity assay showed that the binding capacity of sFlt-1(2-3) to human VEGF(165) and the inhibiting effect of it on human umbilical-vein endothelial cell proliferation stimulated by human VEGF(165) were close to those of sFlt-1(1-3). Animal tests showed that sFlt-1(2-3) could significantly inhibit the formation of regenerated blood vessels stimulated by hVEGF(165).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiogenesis Inhibitors / metabolism
  • Angiogenesis Inhibitors / pharmacology
  • Animals
  • Binding, Competitive
  • Cell Division / drug effects
  • Cell Line
  • Endothelial Growth Factors / metabolism*
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects
  • Humans
  • Ligands
  • Male
  • Models, Animal
  • Neovascularization, Physiologic / drug effects
  • Peptides / metabolism
  • Peptides / pharmacology
  • Pichia / metabolism
  • Protein Conformation
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins / pharmacology
  • Rabbits
  • Receptor Protein-Tyrosine Kinases / chemistry
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Receptor Protein-Tyrosine Kinases / pharmacology
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Two-Hybrid System Techniques
  • Umbilical Veins / cytology
  • Vascular Endothelial Growth Factor Receptor-1
  • Yeasts

Substances

  • Angiogenesis Inhibitors
  • Endothelial Growth Factors
  • Ligands
  • Peptides
  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • Receptor Protein-Tyrosine Kinases
  • Vascular Endothelial Growth Factor Receptor-1