title: Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165.
abstract: Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF(165)). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF(165). We constructed Npn-1 variants containing deletions within the "a" and "b" domains of Npn-1. More than 16 variants were expressed in COS-1 cells and tested for alkaline phosphatase-Sema3A as well as alkaline phosphatase-VEGF(165) binding. Our results indicate that each of the two Npn-1 CUB domains and the amino-terminal coagulation factor V/VIII domain (CF V/VIII) are essential for Sema3A binding, but only the amino-terminal Npn-1 CF V/VIII domain is required for binding to VEGF(165). Guided by the structure of the bovine spermadhesin CUB domain, point mutants targeting defined surfaces of the Npn-1 a1 CUB domain were generated and tested for Sema3A and VEGF(165) binding. One Npn-1 variant, Npn-1(2ABC), exhibits complete loss of Sema3A binding while retaining normal VEGF(165) binding. Moreover, co-immunoprecipitation experiments show that Npn-1(2ABC) can form a signaling complex with the VEGF(165) signaling receptor KDR/VEGFR-2. These results establish the identity of contact sites between Npn-1 and its semaphorin ligands, and they provide a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo.
authors.affiliation: Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Chenghua
authors.initials: C
authors.lastname: Gu
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Limberg
authors.forename: G Brian
authors.initials: GB
authors.lastname: Whitaker
authors.forename: Ben
authors.initials: B
authors.lastname: Perman
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Leahy
authors.forename: Jan S
authors.initials: JS
authors.lastname: Rosenbaum
authors.forename: David D
authors.initials: DD
authors.lastname: Ginty
authors.forename: Alex L
authors.initials: AL
authors.lastname: Kolodkin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008222:Lymphokines / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D039942:Neuropilin-1; D010766:Phosphorylation; D017354:Point Mutation; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor; D040121:Semaphorin-3A; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D016228:Endothelial Growth Factors; D006023:Glycoproteins; D008222:Lymphokines; D009419:Nerve Tissue Proteins; D017978:Receptors, Growth Factor; D040121:Semaphorin-3A; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D039942:Neuropilin-1; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor