IDENTIFICATION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR DETERMINANTS FORBINDING KDR AND FLT-1 RECEPTORS - GENERATION OF RECEPTOR-SELECTIVE VEGF VARIANTS BY SITE-DIRECTED MUTAGENESIS

Vascular endothelial growth factor (VEGF) expression in Various cell t ypes is induced by hypoxia and other stimuli. VEGF mediates endothelia l cell proliferation, angiogenesis, vascular growth, and vascular perm eability via the endothelial cell receptors, kinase insert domain-cont aining receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine- scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg(82), Lys(84) and His( 86), located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp(63), Glu(64), and Gl u(67), were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficien t in FLT-1 binding did not affect KDR binding. Endothelial cell mitoge nesis was abolished in mutants lacking KDR affinity; however, FLT-1 de ficient mutants induced normal proliferation. These results suggest du al sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furth ermore, this mutational analysis implicates KDR, but not FLT-1, in VEG F induction of endothelial cell proliferation.