Site-directed Mutagenesis in the B-Neuropilin-2 Domain Selectively Enhances Its Affinity to VEGF165, but Not to Semaphorin 3F

Class three semaphorins (SEMAs) were originally shown to be mediators of axon guidance that repelled axons and collapsed growth cones, but it is now evident that SEMA3F, for example, has similar effects on tumor cells and endothelial cells (EC). In both human U87MG glioma cells and human umbilical vein EC, SEMA3F induced rapid cytoskeletal collapse, suppressed cell contractility, decreased phosphorylation of cofilin, and inhibited cell migration in culture. Analysis of the signaling pathways showed that SEMA3F formed a complex with NRP2 (neuropilin-2) and plexin A1. These interactions eventually led to inactivation of the small GTPase, RhoA, which is necessary for stress fiber formation and cytoskeleton integrity. A novel upstream RhoA mediator was shown to be ABL2, also known as ARG, a membrane-anchored nonreceptor tyrosine kinase. Within minutes after the addition of SEMA3F, ABL2 directly bound plexin A1 but not to a plexin A1 mutant lacking the cytoplasmic domain. In addition, ABL2 phosphorylated and thereby activated p190RhoGAP, which inactivated RhoA (GTP to GDP), resulting in cytoskeleton collapse and inhibition of cell migration. On the other hand, cells overexpressing an ABL2 inactive kinase mutant or treated with ABL2 small interfering RNA did not inactivate RhoA. Cells treated with p190RhoGAP small interfering RNA also did not inactivate RhoA. Together, these results suggested that ABL2/ARG is a novel mediator of SEMA3F-induced RhoA inactivation and collapsing activity.

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“…The cofilin construct was purchased from Open Biosystems (Huntsville, AL). The human NRP2 construct was described previously (23).…”

Section: Methodsmentioning

confidence: 99%

“…PAE/ NRP1 and PAE/NRP2 were described previously (23). HUVEC purchased from Lonza Inc. (Allendale, NJ) were cultured in EBM-2 (Lonza), supplemented with EGM-2 Single Quote.…”

Section: Cell Culturementioning

confidence: 99%

ABL2/ARG Tyrosine Kinase Mediates SEMA3F-induced RhoA Inactivation and Cytoskeleton Collapse in Human Glioma Cells

Shimizu

Mammoto

Italiano

et al. 2008

Journal of Biological Chemistry

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116

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“…The extracellular region of neuropilins contains 5 different structural domains-2 CUB motifs, a1 and a2, homologous to complement components C1r/C1s, 2 coagulation factor V/VIII homology domains b1 and b2, and one c domain (MAM, homologous to meprin, A5, ). 11 The high-affinity binding site for VEGF-A 165 has been localized to the b1 and b2 domains of NRP1 12,13 and NRP2, 14 whereas the binding of semaphorins requires both the a1a2 and b1b2 repeats. 12 NRP1 and NRP2 interact selectively with different members of the VEGF and semaphorin families and have nonoverlapping expression patterns.…”

Section: Introductionmentioning

confidence: 99%

Neuropilin-1 and neuropilin-2 act as coreceptors, potentiating proangiogenic activity

Sulpice

Plouët

Bergé

et al. 2008

Blood

148

130

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Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins interacting with 2 types of ligands: class III semaphorins and several members of the VEGF family, the main regulators of blood and lymphatic vessel growth. We show here that both NRP1 and NRP2 can also bind hepatocyte growth factor (HGF). HGF is a pleiotropic cytokine and potent proangiogenic molecule that acts on its target cells by binding to the c-met receptor. We IntroductionNeuropilins (NRPs) are transmembrane glycoproteins that play an important role in various biological processes, including axonal guidance, angiogenesis, tumorigenesis, and the immunologic response. [1][2][3][4] NRPs have been characterized as coreceptors for 2 unrelated families of extracellular secreted ligands-class III semaphorins and several members of the vascular endothelial growth factor (VEGF) family, the main regulators of blood and lymphatic vessel growth. 5 NRP acts in conjunction with membraneassociated signal transducers, such as the VEGF receptor tyrosine kinases (VEGFR-1, -2, and -3) 6,7 and plexins, the transmembrane receptors of the semaphorin family. 8 In higher eukaryotes, 2 neuropilin genes, NRP1 and NRP2, have been identified. 9 They code for proteins displaying about 44% amino-acid sequence identity, with a similar domain structure. 10 Both NRP1 and NRP2 contain a large extracellular region and a short cytoplasmic tail of about 40 amino acids, lacking any enzymatic activity. The extracellular region of neuropilins contains 5 different structural domains-2 CUB motifs, a1 and a2, homologous to complement components C1r/C1s, 2 coagulation factor V/VIII homology domains b1 and b2, and one c domain (MAM, homologous to meprin, A5, ). 11 The high-affinity binding site for VEGF-A 165 has been localized to the b1 and b2 domains of NRP1 12,13 and NRP2, 14 whereas the binding of semaphorins requires both the a1a2 and b1b2 repeats. 12 NRP1 and NRP2 interact selectively with different members of the VEGF and semaphorin families and have nonoverlapping expression patterns. Thus, among the VEGF members, NRP1 binds VEGF-A 165 , VEGF-B, VEGF-E, and placental growth factor (PlGF), whereas NRP2 binds VEGF-A 165 ,VEGF-A 145 , VEGF-C, and PlGF. 15 The non-heparin-binding isoforms of VEGF, such as VEGF-A 121 , have long been considered unable to interact with NRPs. Current evidence suggests, however, that VEGF-A 121 does bind NRP1 via the C-terminal sequence of 6 amino acids encoded by exon 8. [16][17][18][19] During the development of the cardiovascular system, NRP1 is detected primarily in the arterial endothelial cells, and NRP2 is detected in the venous and lymphatic endothelial cells. 20 Genetic studies in mice have shown that both the overexpression of NRP1 21 and the targeted inactivation of the NRP1 gene 22,23 are lethal, provoking, in addition to neuronal defects, disorganization of the vascular network and defects in heart development. Inactivation of the NRP2 gene has less severe consequences, limited to defects in the formation of small lymphatic vessel...

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“…38 Our Sema3F immunostaining data (Supplemental Figure S1E) are in agreement with those from previous mRNA staining 39 and suggest that Sema3F is constitutively produced in the epidermis and acts, in a paracrine fashion, as an endogenous damper of inflammation and edema. SEMA3F protein has been shown to compete with VEGFA on NRP2 35 and to inhibit EC functions. 33,40,41 Loss of the functional Sema3F receptor, Nrp2, increased edema and lymphedema after inflammation.…”

Section: Discussionmentioning

confidence: 99%

“…34,35 We hypothesized that the edema seen in inflamed Nrp2 À/À mice may have been due to the lack of endogenous Sema3F signaling. Immunostaining and immunoblot analysis results demonstrated endogenous Sema3F expression in normal mouse skin epithelium but not in endothelium or fibroblasts (Supplemental Figure S1, D and E).…”

Section: Nrp2 Regulates Vascular Permeability and Edemamentioning

confidence: 99%

Inflammation and Lymphedema Are Exacerbated and Prolonged by Neuropilin 2 Deficiency

Mucka

Levonyak

Geretti

et al. 2016

The American Journal of Pathology

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The vasculature influences the progression and resolution of tissue inflammation. Capillaries express vascular endothelial growth factor (VEGF) receptors, including neuropilins (NRPs), which regulate interstitial fluid flow. NRP2, a receptor of VEGFA and semaphorin (SEMA) 3F ligands, is expressed in the vascular and lymphatic endothelia. Previous studies have demonstrated that blocking VEGF receptor 2 attenuates VEGFA-induced vascular permeability. The inhibition of NRP2 was hypothesized to decrease vascular permeability as well. Unexpectedly, massive tissue swelling and edema were observed in Nrp2 À/À mice compared with wild-type littermates after delayed-type hypersensitivity reactions. Vascular permeability was twofold greater in inflamed blood vessels in Nrp2-deficient mice compared to those in Nrp2-intact littermates. The addition of exogenous SEMA3F protein inhibited vascular permeability in Balb/cJ mice, suggesting that the loss of endogenous Sema3F activity in the Nrp2-deficient mice was responsible for the enhanced vessel leakage. Functional lymphatic capillaries are necessary for draining excess fluid after inflammation; however, Nrp2-mutant mice lacked superficial lymphatic capillaries, leading to 2.5-fold greater fluid retention and severe lymphedema after inflammation. In conclusion, Nrp2 deficiency increased blood vessel permeability and decreased lymphatic vessel drainage during inflammation, highlighting the importance of the NRP2/SEMA3F pathway in the modulation of tissue swelling and resolution of postinflammatory edema. (Am J Pathol 2016 http://dx

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Site-directed Mutagenesis in the B-Neuropilin-2 Domain Selectively Enhances Its Affinity to VEGF165, but Not to Semaphorin 3F

Cited by 40 publications

References 58 publications

ABL2/ARG Tyrosine Kinase Mediates SEMA3F-induced RhoA Inactivation and Cytoskeleton Collapse in Human Glioma Cells

ABL2/ARG Tyrosine Kinase Mediates SEMA3F-induced RhoA Inactivation and Cytoskeleton Collapse in Human Glioma Cells

Neuropilin-1 and neuropilin-2 act as coreceptors, potentiating proangiogenic activity

Inflammation and Lymphedema Are Exacerbated and Prolonged by Neuropilin 2 Deficiency

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