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NEUROPILIN IN THE VASCULAR SYSTEM: MECHANISTIC BASIS OF ANGIOGENESIS

OF DISSERTATION NEUROPILIN IN THE VASCULAR SYSTEM: MECHANISTIC BASIS OF ANGIOGENESIS The vascular system is critical for maintaining homeostasis in all vertebrates. The development and function of this essential network is tightly regulated by diffusible cytokines. Interestingly, two families of Nrp-1 ligands, Vascular Endothelial Growth Factor A (VEGF-A) and Semaphorin3F (Sema3F), physically compete for binding to the Nrp-1 b1 domain, and have opposite roles in regulating angiogenesis. Indeed, VEGF-A is a potent pro-angiogenic cytokine while Sema3F is an angiogenesis inhibitor. Although the interaction between VEGF and Nrp-1 is well characterized, little is known about the physical basis of Sema3F binding to Nrp-1. By utilizing chemically synthesized peptides and platebased binding assays, we showed that Sema3F C-terminal ligand binding motif (C-furSema) binding to Nrp1 requires engagement of two distinct binding sites on the Nrp-1: a C-terminal arginine binding motif and a helical binding motif. Covalent tethering of these two motifs leads to synergistic binding to Nrp-1. In addition, the helical motif was shown to be conserved in all Sema3 family members, suggesting this tethering mode is widely utilized. By using the natural variants of Sema3A, we further demonstrated that the distance between this helical region and the C-terminal arginine determines binding affinity. Finally, we showed that C-furSema potently inhibits VEGF-A signaling in human umbilical vein endothelial cells (HUVECs). These data provide a basis for the rational design of novel high affinity Nrp-1 inhibitor. VEGF functions via two essential cell surface receptor families: VEGF receptor (VEGFR) and Neuropilin (Nrp). Although Nrp1 is a mandatory co-receptor for VEGF-A-mediated angiogenesis, little is known about the specific role of Nrp-1 and the physical basis of its function. By utilizing biochemical and biophysical tools, we demonstrated that Nrp-1 b1b2 domain directly interacts with the VEGFR-2 membrane proximal domain 7. This suggested that Nrp-1 promotes full VEGFR-2 activation by direct coupling to the active form of VEGFR-2 domain 7. Indeed, we further demonstrated that disrupting the interaction between Nrp-1 b1b2 domain and VEGFR-2 domain 7 decreases VEGFR-2 activation upon VEGF-A stimulation. This defines the molecular mechanism of Nrp-1 in VEGFR-2 activation. This also provides a novel mechanism to inhibit VEGFR-2 signaling.