Receptor-selective variants of human vascular endothelial growth factor - Generation and characterization

Vascular endothelial growth factor (VEGF) is a pleiotropic factor that exer ts a multitude of biological effects through its interaction with two recep tor tyrosine kinases, fms-like tyrosine kinase (Flt-1) or VEGF receptor 1 a nd kinase insert domain-containing receptor (KDR) or VEGF receptor 2. Where as it is commonly accepted that KDR is responsible for the proliferative ac tivities of VEGF, considerable controversy and uncertainty exist about the role of the individual receptors in eliciting many of the other effects. Ba sed on a comprehensive mutational analysis of the receptor-binding site of VEGF, an Flt-1-selective variant was created containing four substitutions from the wild-type protein. This variant bound with wild-type affinity to F lt-1, was at least 470-fold reduced in binding to KDR, and had no activity in cell-based assays measuring autophosphorylation of KDR or proliferation of primary human vascular endothelial cells. Using a competitive phage disp lay strategy, two KDR-selective variants were discovered with three and fou r changes from wild-type, respectively. Both variants had approximately wil d-type affinity for KDR, were about 2000-fold reduced in binding to Flt-1, and showed activity comparable with the wild-type protein in KDR autophosph orylation and endothelial cell proliferation assays. These variants will se rve as useful reagents in elucidating the roles of Flt-1 and KDR.