Furin Processing of Semaphorin 3F Determines Its Anti-Angiogenic Activity by Regulating Direct Binding and Competition for Neuropilin

Abstract

Neuropilin is an essential cell surface receptor that functions in both semaphorin dependent axon guidance and vascular endothelial growth factor (VEGF) dependent angiogenesis. The interplay between these two seemingly distinct pathways is a source of considerable interest. Indeed, several semaphorin family members have been shown to have potent anti-angiogenic activity in vivo. However, reports conflict as to whether semaphorin and VEGF competitively bind to neuropilin. Previous work has demonstrated that all known ligands and inhibitors of neuropilin interact with the b1 domain of neuropilin via a C-terminal arginine. No semaphorin family member possesses a Cterminal arginine, leading to uncertainty as to the physical mechanism of interaction between the Cterminal domain of semaphorin and the b1 domain of neuropilin. Semaphorin 3F (Sema3F) possesses an RXRR furin recognition site in its C-terminus and we demonstrate that it is proteolytically processed. This processing is found to be essential for the interaction of the C-terminus of Sema3F with the b1 domain of neuropilin. We further demonstrate that furin activation of the C-terminus of Sema3F produces a species that potently inhibits the binding of VEGF to neuropilin. These studies provide a mechanistic basis for understanding the anti-angiogenic activity of semaphorin as well as the physical interaction and competition between neuropilin ligands.Vertebrates employ a wide array of secreted growth factors and cell surface receptors to regulate the growth and guidance of axons. The semaphorins represent one of the largest families of cytokines that directly guide axon growth (1,2). There are five recognized families of semaphorins in vertebrates, including the class III semaphorin family, all six members of which are secreted and able to diffuse through tissues (3). Neuropilin directly binds to most class III semaphorins and is essential for axonal guidance (4,5).Neuropilin interacts with members of the semaphorin family of ligands and functions together with plexin family receptors in semaphorin mediated axon guidance (6,7). Neuropilin also interacts with the VEGF family of ligands and functions together with VEGF-R family

Neuropilin-1 biases dendrite polarization in the retina

“…More likely, given that the binding affinity of Sema3f for Nrp1 is similar to that of Sema3a (Chen et al, 1997), is that Sema3f might function through Nrp1 if Nrp2 is not present. Carboxy terminal proteolytic processing of Sema3f could further enhance binding of Sema3f to Nrp1 (Parker et al, 2010). Whether other Sema3s also participate in dendrite polarization is unclear.…”

Structural Basis for Selective Vascular Endothelial Growth Factor-A (VEGF-A) Binding to Neuropilin-1

“…Protein Expression and Purification-Nrp1-b1b2, Nrp2-b1b2, and VEGF-A 165 were expressed in Escherichia coli and purified using established methods (16,19). Nrp point mutants were introduced using the megaprimer method.…”

“…Alkaline phosphatase (AP) fusion proteins were produced from pAPtag-5 (GenHunter, Nashville, TN) via large scale transient transfection of Chinese hamster ovary cells according to previously reported methods (19). VEGF-A point mutants expressed near wild-type levels and were secreted as correctly processed disulfide linked dimers as assessed by Western blotting (data not shown).…”

“…Nrp affinity plates were produced by adsorption of purified Nrp1-b1b2 or Nrp2-b1b2 proteins to high binding plates (Costar, 9018) with ϳ500 ng of bound protein/well (19). For binding experiments, AP-tagged protein concentrations were normalized using AP activity.…”

The role of the semaphorins in cancer

“…100,204 However, cleavage with furin like pro-protein convertases at the sema3F C-terminus was found to result in an increase in its affinity to neuropilin-1 enabling it to compete effectively with VEGF-A for binding to neuropilin-1. 63,205 The down regulation of endogenous sema3F expression in tumor cells which accompanies tumor progression in several types of cancer may enable the transition to the angiogenic phase in tumor development. 128 Indeed, sema3F inhibits the expression of HIF1a and consequently VEGF expression, resulting in the inhibition of hypoxia induced angiogenesis.…”