title: Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis.
abstract: 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.
authors.affiliation: Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Keyt
authors.forename: H V
authors.initials: HV
authors.lastname: Nguyen
authors.forename: L T
authors.initials: LT
authors.lastname: Berleau
authors.forename: C M
authors.initials: CM
authors.lastname: Duarte
authors.forename: J
authors.initials: J
authors.lastname: Park
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: N
authors.initials: N
authors.lastname: Ferrara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D016228:Endothelial Growth Factors / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D015151:Immunoblotting; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008222:Lymphokines / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010982:Platelet-Derived Growth Factor / Q000737:chemistry; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors
chemical_list: D016228:Endothelial Growth Factors; D008222:Lymphokines; D046911:Macromolecular Substances; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1