Characterization of a soluble vascular endothelial growth factor receptor immunoglobulin chimera

To investigate the interaction between vascular endothelial growth factor (VEGF) and its receptor, we have constructed a chimeric protein consisting of the extracellular ligand-binding domain of the human VEGF receptor subtype KDR fused to a human IgG1 Fc domain (KDR-Fc), KDR-Fc was expressed in human 293 kidney epithelial cells as a 300-kDa secreted, dimeric glycoprotein that bound I-125-VEGF(165) with high affinity (K-d = 150 pM), Unlike the full length cellular receptor, KDR-Fc did not require heparin for I-125-VEGF(165) binding, although heparin did stimulate I-125-VEGF(165) binding approximately 50 to 100%, Similar results were observed for KDR-Fc expressed in yeast cells, Since yeast do not synthesize heparan sulfate proteoglycans, we conclude that cellular heparan sulfates do not account for the lack of a heparin requirement for I-125-VEGF(165) binding to KDR-Fc, The polycationic protein protamine, which inhibits (IC50 = 1 mu g/ml) I-125-VEGF(165) binding to bovine aortic endothelial cells and other KDR-expressing cells by blocking heparin interactions, had no effect on the heparin independent component of I-125-VEGF(165) binding to KDR-Fc. Protamine does inhibit (IC50 = 1 mu g/ml) the heparin dependent component of I-125-VEGF(165) binding to KDR-Fc, KDR-Fc bound VEGF(121) with the same affinity as VEGF(165), Heparin had no effect on I-125-VEGF(121) binding to KDR-Fc, indicating that heparin interaction with the 44 amino acids contained in VEGF(165) but not VEGF(121) allow for maximal VEGF(165) binding, Deletion analysis of KDR-Fc demonstrated that the determinants required for high affinity VEGF binding are located in the three aminoterminal Ig-domains of the protein, Heparin had no effect on I-125-VEGF(165) binding to the three Ig-domain receptor, suggesting that there are heparin binding determinants located in KDR Ig-domains 4 to 7.