Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis

• Published in: The Journal of biological chemistry 1996-03, Vol.271 (10), p.5638-5646
• Main Authors: Keyt, B A, Nguyen, H V, Berleau, L T, Duarte, C M, Park, J, Chen, H, Ferrara, N
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; CHO Cells; Cricetinae; Endothelial Growth Factors - chemistry; Endothelial Growth Factors - metabolism; Endothelial Growth Factors - pharmacology; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Enzyme-Linked Immunosorbent Assay; Genetic Variation; Immunoblotting; Kinetics; Liver - metabolism; Lymphokines - chemistry; Lymphokines - metabolism; Lymphokines - pharmacology; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Platelet-Derived Growth Factor - chemistry; Point Mutation; Protein Structure, Secondary; Proto-Oncogene Proteins - metabolism; Receptor Protein-Tyrosine Kinases - metabolism; Receptors, Growth Factor - metabolism; Receptors, Vascular Endothelial Growth Factor; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Sequence Homology, Amino Acid; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.10.5638

Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing 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. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, 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 deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.