The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor Flt-1

Neuropilin-1 (NP-1) was first identified as a semaphorin receptor involved in neuron guidance. Subsequent studies demonstrated that NP-1 also binds an isoform of vascular endothelial growth factor (VEGF) as well as several VE GF homologs, suggesting that NP-1 may also function in angiogenesis. Here w e report in vitro binding experiments that shed light on the interaction be tween VEGF165 and NP-1, as well as a previously unknown interaction between NP-1 and one of the VEGF receptor tyrosine kinases, VEGFR1 or Flt-1. BIA-c ore analysis demonstrated that, with the extracellular domain (ECD) of NP-1 immobilized at low density, VEGF165 bound with low affinity (K-d = 2 mu M) and fast kinetics. The interaction was dependent on the heparin-binding do main of VEGF165 and increased the affinity of VEGF165 for its signaling rec eptor VEGFR2 or kinase insert domain-containing receptor. The affinity of V EGF165 for the NP-1 ECD was greatly enhanced either by increasing the densi ty of immobilized NP-1 (K-d = 113 aw) or by the addition of heparin (K-d = 25 nM). We attribute these affinity enhancements to avidity effects mediate d by the bivalent VEGF165 homodimer or multivalent heparin. We also show th at the NP-1 ECD binds with high affinity (K-d = 1.8 nM) to domains 3 and 4 of Flt-1 and that this interaction inhibits the binding of NP-1 to VEGF165. Based on these results, we propose that NP-1 acts as a coreceptor for vari ous ligands and that these functions are dependent on the density of NP-1 o n the cell membrane. Furthermore, Flt-1 may function as a negative regulato r of angiogenesis by competing for NP-1.