Mapping of the Sites for Ligand Binding and Receptor Dimerization at the Extracellular Domain of the 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 Ig-like loops within the extracellular domain. Upon VEGF binding FLT-1 becomes phosphorylated, which has been thought to be preceded by receptor dimerization. To further investigate high affinity binding of VEGF to FLT-1 and ligand-induced receptor dimerization, we expressed in Sf9 cells the entire extracellular domain comprising all seven Ig-like loops: sFLT-1(7) and several truncated mutants consisting of loop one, one and two, one to three, one to four, and one to five. The corresponding proteins, named sFLT-1(1), (2), (3), (4), and (5) were purified. Only mutants sFLT-1(3) to (7) were able to bind 125 I-VEGF with high affinity. No binding of VEGF was observed with sFLT-1(1) and sFLT-1(2), indicating that the first three Ig-like loops are involved in high affinity binding of VEGF. The binding of VEGF to sFLT-1(3) could be competed with placenta growth factor (PlGF), a VEGF-related ligand, suggesting that high affinity binding of VEGF and PlGF is mediated by the same or closely related contact sites on sFLT-1. Deglycosylation of the sFLT-1(3), (4), (5), and (7) did not abolish VEGF binding. Furthermore, unglycosylated sFLT-1(3), expressed in Escherichia coli, was able to bind VEGF with similar affinity as sFLT-1(3) or sFLT-1(7), both expressed in Sf9 cells. This indicates that receptor glycosylation is not essential for high affinity binding. Dimerization of the extracellular domains of FLT-1 upon addition of VEGF was detected with all mutants containing the Ig-like loop four. Although sFLT-1(3) was able to bind VEGF, dimerization of this mutant was inefficient, indicating that sites on Ig-like loop four are essential to stabilize receptor dimers.

Development of a chemiluminescent screening assay for detection of vascular endothelial growth factor receptor 1 ligands

“…The binding capacity of btVEGF 165 reached its maximum when added at a concentration of 10 nM. Based on three independent experiments, the dissociation constant (K d ) was estimated at 750 pM, a value comparable to 4 HAL author manuscript inserm-00168674, version 1 the one found by Barleon et al [11]. Considering that performing the assay with recombinant receptors does not allow to reproduce the favourable effect of membrane microenvironment existing in cell based assays, it is not surprising to obtain a value superior to the K d previously described for VEGF 165 interaction with Cos cells transfected with VEGF-R1 (10-30 pM) [12].…”

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation

“…Compared with D1-3, D1-5 showed 86-fold greater affinity and D1-4 showed 16-fold greater affinity (34). Similarly, both D4 and D4-5 of VEGFR-1 have been shown to enhance ligand-induced receptor dimerization in cross-linking experiments (35). Thus, the results of the VEGFR-1-and VEGFR-2-binding studies support a role for D5 in VEGFR dimerization, although additional interactions in D4 cannot be ruled out.…”

Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization

“…11,12 The VEGF-binding function of Flt-1 has been mapped to the second domain. [13][14][15][16] There have been previous studies with two truncated soluble receptor hybrids, Flt(1-3)-IgG and Flt(1-7)-IgG, consisting of either the first three domains or all seven domains fused to human IgG1-Fc region. 13 The molecule Flt(1-3)-IgG was reported to have the same VEGF-binding affinity as Flt(1-7)-IgG, however Flt(2)-IgG that contains only second domain was not capable of inhibiting VEGF.…”