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Concept: Neuropilin

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Neuropilin (Nrp) receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF) family of proangiogenic cytokines and the semaphorin 3 (Sema3) family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5) isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.

Concepts: Signal transduction, Angiogenesis, Hormone, Vascular endothelial growth factor, Cell signaling, Endothelium, Neuropilin, Semaphorin

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Class-3 semaphorin guidance factors bind to receptor complexes containing neuropilin and plexin receptors. A semaphorin may bind to several receptor complexes containing somewhat different constituents, resulting in diverse effects on cell migration. U87MG glioblastoma cells express both neuropilins and the four class-A plexins. They respond by cytoskeletal collapse and cell contraction to sema3A or sema3B but fail to contract in response to Sema3C, Sema3D, Sema3G or sema3E even when class-A plexins are over-expressed in the cells. In-contrast, expression of recombinant plexin-D1 enabled contraction in response to these semaphorins. Surprisingly, unlike sema3D and sema3G, sema3C also induced the contraction and repulsion of plexin-D1 expressing U87MG cells in which both neuropilins were knocked-out using CRISPR/cas9. In the absence of neuropilins the EC-50 of sema3C was 5.5 fold higher, indicating that the neuropilins function as enhancers of plexin-D1 mediated sema3C signaling but are not absolutely required for sema3C signal transduction. Interestingly, in the absence of neuropilins, plexin-A4 formed complexes with plexin-D1, and was required in addition to plexin-D1 to enable sema3C induced signal transduction.

Concepts: DNA, Protein, Signal transduction, Hormone, Signal, Neuropilin, Semaphorin, Plexin

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We investigated the expression of neuropilin-1 (NRP1), neuropilin-2 (NRP2), vascular endothelial growth factor-A (VEGF-A), semaphorin-3A (Sema-3A), and semaphorin-3F (Sema-3F) in normal salivary gland (NSG) tissue, nonmetastatic salivary adenoid cystic carcinoma (SACC), and metastatic SACC to better understand their role in intratumoral angiogenesis and hematogenous metastasis of SACC.

Concepts: Vascular endothelial growth factor, Neuropilin

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All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue (1) , and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs. (2) There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms - that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a - bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not ‘bridge’ VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane ™ and intracellular (IC) domains.

Concepts: Signal transduction, Angiogenesis, Cell membrane, Cell biology, Hormone, Vascular endothelial growth factor, VEGF receptors, Neuropilin

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Neuropilins (NRPs) are single transmembrane receptors with short cytoplasmic tails and are dependent on receptors like VEGF receptors or Plexins for signal transduction. NRPs are known to be important in angiogenesis, lymphangiogenesis, and axon guidance. The Neuropilin-family consists of two members, Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2). They are up to 44 % homologous and conserved in all vertebrates. High levels of NRP2 are found on immune cells. Current research is very limited regarding the functions of NRP2 on these cells. Recent evidence suggests that NRP2 is important for migration, antigen presentation, phagocytosis and cell-cell contact within the immune system. Additionally, posttranslational NRP2 modifications like polysialylation are crucial for the function of some immune cells. This review is an overview about expression and functions of NRP2 in the immune system.

Concepts: Immune system, Antibody, Protein, Cell membrane, Vascular endothelial growth factor, Adaptive immune system, Cell signaling, Neuropilin

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Hedgehog (HH) signaling critically regulates embryonic and postnatal development as well as adult tissue homeostasis, and its perturbation can lead to developmental disorders, birth defects, and cancers. Neuropilins (NRPs), which have well-defined roles in Semaphorin and VEGF signaling, positively regulate HH pathway function, although their mechanism of action in HH signaling remains unclear. Here, using luciferase-based reporter assays, we provide evidence that NRP1 regulates HH signaling, specifically at the level of GLI transcriptional activator function. Moreover, we show that NRP1 localization to the primary cilium, a key platform for HH signal transduction, does not correlate with HH signal promotion. Rather, a structure-function analysis suggested that the NRP1 cytoplasmic and transmembrane domains are necessary and sufficient to regulate HH pathway activity. Furthermore, we identify a previously uncharacterized, 12-amino-acid region within the NRP1 cytoplasmic domain that mediates HH signal promotion. Overall, our results provide mechanistic insight into NRP1 function within and potentially beyond the HH signaling pathway. These insights have implications for the development of novel modulators of HH-driven developmental disorders and diseases.

Concepts: Protein, Gene expression, Enzyme, Cell membrane, Transcription factor, Cytoskeleton, Necessary and sufficient condition, Neuropilin

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Cortical interneurons are generated predominantly in the medial ganglionic eminence of the ventral telencephalon and migrate to the cortex during embryonic development. These cells express neuropilin (Nrp1 and Nrp2) receptors which mediate their response to the chemorepulsive class 3 semaphorin (Sema) ligands. We show here that semaphorins Sema3A and Sema3F are expressed in layers adjacent to cortical interneuron migratory streams as well as in the striatum, suggesting they may have a role in guiding these cells throughout their journey. Analysis of Sema3A (-/-) and Sema3F (-/-) mice during corticogenesis showed that absence of Sema3A, but not Sema3F, leads to aberrant migration of cortical interneurons through the striatum. Reduced number of cortical interneurons was found in the cortex of Sema3A (-/-), Nrp1 (-/-) and Nrp2 (-/-) mice, as well as altered distribution in Sema3F (-/-), Nrp1 (-/-), Nrp2 (-/-) animals and especially in neuropilin double mutants. The observed decrease in interneurons in Sema3A (-/-) and Nrp1 (-/-) mice was due to altered proliferative activity of their progenitors highlighted by changes in their mitotic spindle positioning and angle of cleavage plane during cell division. These findings point to a novel role for Sema3A-Nrp1 signalling in progenitor cell dynamics and in the generation of interneurons in the ventral telencephalon.

Concepts: Eukaryote, Developmental biology, Cell division, Cerebral cortex, Cerebrum, Mitosis, Neuropilin, Semaphorin

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Neuropilin-2 is a transmembrane receptor involved in lymphangiogenesis and neuronal development. In adults, neuropilin-2 and its homologous protein neuropilin-1 have been implicated in cancers and infection. Molecular determinants of ligand selectivity of neuropilins are poorly understood. We have identified and structurally characterised a Zn ion binding site on human neuropilin-2. The neuropilin-2-specific Zn binding site is located near the interface between domains b1 and b2 in the ectopic region of the protein, away from the neuropilin binding site for its physiological ligand - vascular endothelial growth factor. We also present an X-ray crystal structure of neuropilin-2 b1 domain in a complex with the C-terminal sub-domain of VEGF-A. Zn(2+) -binding to neuropilin-2 destabilizes the protein structure but this effect could be counteracted by heparin suggesting that modifications with glycans and zinc in the extracellular matrix could impact functional neuropilin-2 ligand-binding and signalling activity. This article is protected by copyright. All rights reserved.

Concepts: Immune system, Protein, Signal transduction, Vascular endothelial growth factor, Zinc, X-ray crystallography, Neuropilin, Transmembrane receptors

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Polysialic acid is an oncofetal glycopolymer, added to the glycans of a small group of substrates, that controls cell adhesion and signaling. One of these substrates, neuropilin-2, is a VEGF and semaphorin co-receptor that is polysialylated on its O-glycans in mature dendritic cells and macrophages by the polysialyltransferase ST8SiaIV. To understand the biochemical basis of neuropilin-2 polysialylation, we created a series of domain swap chimeras with sequences from neuropilin-1, a protein for which polysialylation had not been previously reported. To our surprise we found that membrane-associated neuropilin-1 is polysialylated at ~50% the level of neuropilin-2, but not polysialylated when it lacks its cytoplasmic tail and transmembrane region and is secreted from the cell. This was not the case for neuropilin-2, which is polysialylated when either membrane-associated or soluble. Evaluation of the soluble chimeric proteins demonstrated that the Meprin-A5 antigen-μ tyrosine phosphatase (MAM) domain and the O-glycan containing linker region of neuropilin-2 are necessary and sufficient for its polysialylation, and serve as better recognition and acceptor sites in the polysialylation process than those regions of neuropilin-1. In addition, specific acidic residues on the surface of the MAM domain are critical for neuropilin-2 polysialylation. Based on these data and pull-down experiments, we propose a model where ST8SiaIV recognizes and docks on an acidic surface of the neuropilin-2 MAM domain to polysialylate O-glycans on the adjacent linker region. These results together with those related to neural cell adhesion molecule polysialylation establish a paradigm for the process of protein specific polysialylation.

Concepts: Protein, Cell, Amino acid, Enzyme, Cell membrane, Cell biology, Cytoplasm, Neuropilin

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Neuropilin 2 (NRP2) isa multi-functional co-receptor to many receptors, including VEGF receptor, c-Met and others. NRP2 has recently been implicated in tumor angiogenesis, growth, and metastasis of many other cancers. However, its role in osteosarcoma remains poorly understood.

Concepts: Cancer, Metastasis, Oncology, Angiogenesis, Vascular endothelial growth factor, Receptor antagonist, Neuropilin, NRP2