Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165.
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.
Mesh Terms:
Amino Acid Sequence, Amino Acid Substitution, Animals, COS Cells, Electrophoresis, Polyacrylamide Gel, Endothelial Growth Factors, Glycoproteins, Humans, Lymphokines, Molecular Sequence Data, Mutagenesis, Site-Directed, Nerve Tissue Proteins, Neuropilin-1, Phosphorylation, Point Mutation, Rats, Receptor Protein-Tyrosine Kinases, Receptors, Growth Factor, Receptors, Vascular Endothelial Growth Factor, Semaphorin-3A, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors
Amino Acid Sequence, Amino Acid Substitution, Animals, COS Cells, Electrophoresis, Polyacrylamide Gel, Endothelial Growth Factors, Glycoproteins, Humans, Lymphokines, Molecular Sequence Data, Mutagenesis, Site-Directed, Nerve Tissue Proteins, Neuropilin-1, Phosphorylation, Point Mutation, Rats, Receptor Protein-Tyrosine Kinases, Receptors, Growth Factor, Receptors, Vascular Endothelial Growth Factor, Semaphorin-3A, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors
J. Biol. Chem.
Date: May. 17, 2002
PubMed ID: 11886873
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