Identification of the ligand-binding domain of human vascular-endothelial-growth-factor receptor Flt-1.
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).
Ma
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L
Institute of Molecular Immunology, The First Military Medical University, Guangzhou 510515, People's Republic of China.
Wang
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X
Zhang
Z
Z
Zhou
X
X
Chen
A
A
Yao
L
L
D020533:Angiogenesis Inhibitors / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D016228:Endothelial Growth Factors / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008024:Ligands; D008297:Male; D023421:Models, Animal; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D010843:Pichia / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D014471:Umbilical Veins / Q000166:cytology; D040281:Vascular Endothelial Growth Factor Receptor-1; D015003:Yeasts
D020533:Angiogenesis Inhibitors; D016228:Endothelial Growth Factors; D008024:Ligands; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1