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 VEGF homologs, suggesting that NP-1 may also function in angiogenesis. Here we report in vitro binding experiments that shed light on the interaction between 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. BIAcore analysis demonstrated that, with the extracellular domain (ECD) of NP-1 immobilized at low density, VEGF165 bound with low affinity (K(d) = 2 microm) and fast kinetics. The interaction was dependent on the heparin-binding domain of VEGF165 and increased the affinity of VEGF165 for its signaling receptor VEGFR2 or kinase insert domain-containing receptor. The affinity of VEGF165 for the NP-1 ECD was greatly enhanced either by increasing the density of immobilized NP-1 (K(d) = 113 nm) or by the addition of heparin (K(d) = 25 nm). We attribute these affinity enhancements to avidity effects mediated by the bivalent VEGF165 homodimer or multivalent heparin. We also show that 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 various ligands and that these functions are dependent on the density of NP-1 on the cell membrane. Furthermore, Flt-1 may function as a negative regulator of angiogenesis by competing for NP-1.
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Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080, USA.
Garcia
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de Vos
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D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D016228:Endothelial Growth Factors / Q000378:metabolism*; D006493:Heparin / Q000502:physiology; D006801:Humans; D008222:Lymphokines / Q000378:metabolism*; D051379:Mice; D018919:Neovascularization, Physiologic / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000502:physiology; D039942:Neuropilin-1; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D039942:Neuropilin-1; D006493:Heparin; D020794:Receptor Protein-Tyrosine Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1