Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165

Chenghua Gu, Brian J. Limberg, G. Brian Whitaker, Ben Perman, Daniel J. Leahy, Jan S. Rosenbaum, David D. Ginty, Alex L. Kolodkin

Research output: Contribution to journalArticle

Abstract

Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF165). 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 VEGF165. 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-VEGF165 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 VEGF165. 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 VEGF165 binding. One Npn-1 variant, Npn-12ABC, exhibits complete loss of Sema3A binding while retaining normal VEGF165 binding. Moreover, co-immunoprecipitation experiments show that Npn-12ABC can form a signaling complex with the VEGF165 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.

Original languageEnglish (US)
Pages (from-to)18069-18076
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number20
DOIs
StatePublished - May 17 2002

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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