Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2, and heparin via its b1b2 domain

Neuroplin-1 (NRP1), a receptor for vascular endothelial growth factor (VEGF) family members, has three distinct extracellular domains, a1a2, b1b2, and c. To determine the VEGF(165) and placenta growth factor 2 (PIGF-2) -binding sites of NRP1, recombinant NRP1 domains were expressed in mammalian cells as Myc-tagged, soluble proteins, and used in co-precipitation experiments with I-125-VEGF(165) and I-125-PIGF-2. Anti-Myc antibodies immunoprecipitated I-125-VEGF(165) and I-125-PIGF-2 in the presence of the b1b2 but not of the a1a2 and c domains. Neither b1 nor b2 alone was capable of binding I-125-VEGF(165). In competition experiments, VEGF(165) competed PIGF-2 binding to the NRP1 b1b2 domain, suggesting that the binding sites of VEGF(165) and PIGF-2 overlap. The presence of the a1a2 domain greatly enhanced VEGF(165), but not PIGF-2 binding to b1b2. Heparin enhanced the binding of both I-125-VEGF(165) and I-125-PIGF-2 to the b1b2 domain by 20- and 4-fold, respectively. A heparin chain of at least 20-24 monosaccharides was necessary for binding. In addition, the b1b2 domain of NRP1 could bind heparin directly, requiring heparin oligomers of at least 8 monosaccharide units. It was concluded that an intact b1b2 domain serves as the VEGF(165)-, PIGF-2-, and heparin-binding sites in NRP1, and that heparin is a critical component for regulating VEGF(165) and PIGF-2 interactions with NRP1 by physically interacting with both receptor and ligands.