Abstract
The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000-1,600,000 m(-1) s(-1)). These results suggest that Npn-1 possesses a "heparin" mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.
Highlights
Neuropilin-1 (Npn-1)1 was first described as a transmembrane co-receptor for semaphorin-3A axon repellent factor, one of the six members of the semaphorin-3/collapsin family [1]
The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGFbinding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface
The results show that Npn-1 interacts with a subset of the heparinbinding proteins tested, notably, fibroblast growth factor (FGF)-1, FGF-2, FGF-4, FGF-7, FGF receptor 1 (FGFR1), and hepatocyte growth factor/scatter factor (HGF/SF)
Summary
Npn-1, neuropilin-1; sNpn-1, soluble isoform of Npn-1; CyPB, cyclophilin B; FGF, fibroblast growth factor; FGFBP, FGF-binding protein; FGFR1, FGF receptor 1; FGFR1-ST, extracellular domain of FGFR1 with a C-terminal Strep-tag II; HGF/ SF, hepatocyte growth factor/scatter factor; HUVEC, human umbilical vein endothelial cell(s); PBS, phosphate-buffered saline; PBST, PBS supplemented with 0.02% (v/v) Tween 20; PrPc, prion protein; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; HS, heparan sulfate. The presence of Npn-1 apparently enhances the interaction of VEGF165 with its VEGFR2 signaling receptor and potentiates both VEGF-stimulated mitogenesis and chemotaxis in cultured endothelial cells. Overexpression of sNpn-1 by prostate carcinoma cells inhibited tumor growth and reduced both the number and integrity of tumor blood vessels, suggesting that sNpn-1 inhibits VEGF165 binding to Npn-1 or VEGFR2. This is supported by the recent finding that soluble monomeric Npn-1 suppressed angiogenesis in vitro [11]. Because Npn-1 potentiated the growth stimulatory activity of FGF-2 in human umbilical vein endothelial cells (HUVEC), the possibility is raised that Npn-1 may have a broad modulatory activity on a subset of growth factors, morphogens. and other heparin-binding proteins
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