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

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).