Neuropilin-1 and neuropilin-2 enhance VEGF121 stimulated signal transduction by the VEGFR-2 receptor

The neuropilin-1 (np1) receptor binds the 165 amino-acid form of vascular endothelial growth factor(165) (VEGF(165)) and functions as an enhancer that potentiates VEGF(165) signaling via the VEGFR-2 tyrosine-kinase receptor. To study the mechanism by which neuropilins potentiate VEGF activity we produced a VEGF(165) mutant (VEGF(165KF)) that binds to neuropilins but displays a much lower affinity toward VEGFR-1 and VEGFR-2. VEGF(165KF) failed to induce VEGFR-2 phosphorylation in cells lacking neuropilins. However, in the presence of np1, VEGF(165KF) bound weakly to VEGFR-2, induced VEGFR-2 phosphorylation, and activated ERK1/2. Interestingly, VEGF(165KF) did not promote formation of VEGFR-2/np1 complexes nor did high concentrations of VEGF(165KF) inhibit VEGF165 induced formation of such complexes, suggesting that VEGF(165) does not stabilize VEGFR-2/np1 complexes by forming bridges spanning VEGFR-2 and np1. VEGF(121) is a VEGF form that does not bind to neuropilins. Surprisingly, both np1 and neuropilin-2 (np2) enhanced VEGF(121)-induced phosphorylation of VEGFR-2 and VEGF121-induced proliferation of endothelial cells. The enhancement of VEGF(121) activity by np1 was accompanied by a 10-fold increase in binding affinity. to, VEGFR-2 and was not associated with the formation of new VEGFR-2/np1 complexes. These observations suggest that neuropilins enhance the activity of VEGF forms that do not bind to neuropilins, indicate that np2 is a functional VEGF receptor, and imply that spontaneously formed VEGFR-2/np1 complexes suffice for efficient neuropilin mediated enhancement of VEGF activity.