In recent years a role for EphB receptor tyrosine kinases and their ephrinB ligands in activity-dependent synaptic plasticity in the CNS has been identified. The aim of the present study was to test the hypothesis that EphB receptor activation in the adult rat spinal cord is involved in synaptic plasticity and processing of nociceptive inputs, through modulation of the function of the glutamate ionotropic receptor NMDA (N-methyl-D-aspartate). In particular, EphB receptor activation would induce phosphorylation of the NR2B subunit of the NMDA receptor by a Src family non-receptor tyrosine kinase. Intrathecal administration of ephrinB2-Fc in adult rats, which can bind to and activate EphB receptors and induce behavioral thermal hyperalgesia, led to NR2B tyrosine phosphorylation, which could be blocked by the Src family kinase inhibitor PP2. Furthermore animals pre-treated with PP2 did not develop behavioral thermal hyperalgesia following EphrinB2-Fc administration, suggesting that this pathway is functionally significant. Indeed, EphB1-Fc administration, which competes with the endogenous receptor for ephrinB2 binding and prevents behavioral allodynia and hyperalgesia in the carrageenan model of inflammation, also inhibited NR2B phosphorylation in this model. Taken together these findings support the hypothesis that EphB–ephrinB interactions play an important role in NMDA-dependent, activity-dependent synaptic plasticity in the adult spinal cord, inducing the phosphorylation of the NR2B subunit of the receptor via Src family kinases, thus contributing to chronic pain states.
EphB receptor; pain; plasticity; NMDA; Src; rat; CaMKII, calcium-calmodulin kinase II; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; pY-NR2B, NR2B phosphorylated in tyrosine; SDS, sodium dodecyl sulfate; TBST, TBS containing 0.1% Tween-20
Bones cannot properly form or be maintained without cell-cell interactions through ephrin ligands and Eph receptors. Cell culture analysis and evaluation of genetic mouse models and human diseases reveal various ephrins and Eph functions in the skeletal system. Migration, attachment and spreading of mesenchymal stem cells are regulated by ephrinB ligands and EphB receptors. ephrinB1 loss-of-function is associated with craniofrontonasal syndrome (CFNS) in humans and mice. In bone remodeling, ephrinB2 is postulated to act as a “coupling stimulator.” In that case, bidirectional signaling between osteoclastic ephrinB2 and osteoblastic EphB4 suppresses osteoclastic bone resorption and enhances osteoblastic bone formation, facilitating the transition between these two states. Parathyroid hormone (PTH) induces ephrinB2 in osteoblasts and enhances osteoblastic bone formation. In contrast to ephrinB2, ephrinA2 acts as a “coupling inhibitor,” since ephrinA2 reverse signaling into osteoclasts enhances osteoclastogenesis and EphA2 forward signaling into osteoblasts suppresses osteoblastic bone formation and mineralization. Furthermore, ephrins and Ephs likely modulate pathological conditions such as osteoarthritis, rheumatoid arthritis, multiple myeloma and osteosarcoma. This review focuses on ephrin/Eph-mediated cell-cell interactions in bone biology.
chondrocyte; osteoclast; osteoblast; coupling factor; coupling inhibitor; cell-cell interaction; osteoarthritis; rheumatoid arthritis; multiple myeloma; osteosarcoma
EphB4 and ephrinB2 expressions in ovarian cancers were studied to analyse EphB4/ephrinB2 functions against clinical backgrounds. EphB4 and ephrinB2 were dominantly localised in ovarian cancer cells of all cases studied. Both the histoscores and mRNA levels of EphB4 and ephrinB2 significantly increased with clinical stages (I
EphB4; ephrinB2; prognostic indicator; tumour advancement; ovarian cancers
EphB4 and its cognitive ligand ephrinB2 play an important role in embryonic vessel development and vascular remodeling. In addition, several reports suggest that this receptor ligand pair is also involved in pathologic vessel formation in adults including tumor angiogenesis. Eph/ephrin signaling is a complex phenomena characterized by receptor forward signaling through the tyrosine kinase of the receptor and ephrin reverse signaling through various protein–protein interaction domains and phosphorylation motifs of the ephrin ligands. Therefore, interfering with EphR/ephrin signaling by the means of targeted gene ablation, soluble receptors, dominant negative mutants or antisense molecules often does not allow to discriminate between inhibition of Eph/ephrin forward and reverse signaling. We developed a specific small molecular weight kinase inhibitor of the EphB4 kinase, NVP-BHG712, which inhibits EphB4 kinase activity in the low nanomolar range in cellular assays showed high selectivity for targeting the EphB4 kinase when profiled against other kinases in biochemical as well as in cell based assays. Furthermore, NVP-BHG712 shows excellent pharmacokinetic properties and potently inhibits EphB4 autophosphorylation in tissues after oral administration. In vivo, NVP-BHG712 inhibits VEGF driven vessel formation, while it has only little effects on VEGF receptor (VEGFR) activity in vitro or in cellular assays. The data shown here suggest a close cross talk between the VEGFR and EphR signaling during vessel formation. In addition to its established function in vascular remodeling and endothelial arterio-venous differentiation, EphB4 forward signaling appears to be an important mediator of VEGF induced angiogenesis since inhibition of EphB4 forward signaling is sufficient to inhibit VEGF induced angiogenesis.
Angiogenesis; EphB4; EphrinB2; VEGF; Protein kinase inhibitor
EphB4 receptor (EphB4) and its ligand (EphrinB2) play an important role in the regulation of cell adhesion, growth and migration. The purpose of this study was to determine the effects of EphB4 blockade by soluble EphB4 (sEphB4) on retinal pigment epithelial (RPE) cell migration and proliferation, induced by platelet-derived growth factor-BB (PDGF), and establish its relevance to proliferative vitreoretinopathy (PVR).
The expression of EphB4 and EphrinB2 in early passage human RPE cells and in human PVR membranes was evaluated by confocal microscopy. The effect of sEphB4 (0.1–3 ug/ml) on PDGF (20 ng/ml)-induced RPE migration and proliferation was evaluated using a modified Boyden chamber assay, and MTT assay, respectively. Attachment onto basement membrane matrix and fibronectin was assayed by MTT. Phosphorylation of FAK and p42/44 MAP Kinase in RPE was determined by Western blot after exposure to sEphB4. The effect of sEphB4 on phosphorylation of EphB4/EphrinB2 was demonstrated using immunoprecipitation assays.
EphrinB2 and EphB4 were expressed on human RPE cells in vitro, and in cells within human PVR membranes. sEphB4 blocked EphB4 and EphrinB2 phosphorylation in RPE cells in vitro. sEphB4 reduced RPE migration in response to PDGF stimulation (P<0.01). Similarly, sEphB4 inhibited RPE attachment and proliferation in a dose-dependent manner (P<0.05). PDGF-induced phosphorylation of FAK and MAP Kinase was inhibited by sEphB4.
EphB4 and EphrinB2 are expressed in RPE cells and PVR membranes. sEphB4 inhibits PDGF induced RPE cell attachment, proliferation and migration. This effect may result from inhibition of FAK and MAP Kinase phosphorylation.
The mechanisms that promote excitatory synapse formation and maturation have been extensively studied. However, the molecular events that limit excitatory synapse development so that synapses form at the right time and place and in the correct numbers are less well understood. We have identified a RhoA guanine nucleotide exchange factor, Ephexin5, which negatively regulates excitatory synapse development until EphrinB binding to the EphB receptor tyrosine kinase triggers Ephexin5 phosphorylation, ubiquitination, and degradation. The degradation of Ephexin5 promotes EphB-dependent excitatory synapse development and is mediated by Ube3A, a ubiquitin ligase that is mutated in the human cognitive disorder Angelman syndrome and duplicated in some forms of Autism Spectrum Disorders (ASDs). These findings suggest that aberrant EphB/Ephexin5 signaling during the development of synapses may contribute to the abnormal cognitive function that occurs in Angelman syndrome and, possibly, ASDs.
Bisphosphonates are therapeutic agents in the treatment of post-menopausal osteoporosis. Although they have been associated with delayed healing in injured tissues, inappropriate femoral fractures, and osteonecrosis of the jaw (ONJ), the pathophysiological mechanisms involved are not clear. Our hypothesis is that alendronate, a member of the N-containing bisphosphonates, indirectly inhibits osteoblast function through the coupling of osteoclasts to osteoblasts by ephrinB-EphB interaction. We found that alendronate increased gene and protein expression of ephrinB1 and EphB1, as well as B3, in femurs of adult mice injected with alendronate (10 µg/100 g/wk) for 8 weeks. Alendronate suppressed the expression of bone sialoprotein (BSP) and osteonectin in both femurs and bone marrow osteoblastic cells of mice. After elimination of pre-osteoclasts from bone marrow cells, alendronate did not affect osteoblast differentiation, indicating the need for pre-osteoclasts for alendronate’s effects. Alendronate stimulated EphB1 and EphB3 protein expression in osteoblasts, whereas it enhanced ephrinB1 protein in pre-osteoclasts. In addition, a reverse signal by ephrinB1 inhibited osteoblast differentiation and suppressed BSP gene expression. Thus, alendronate, through its direct effects on the pre-osteoclast, appears to regulate expression of ephrinB1, which regulates and acts through the EphB1, B3 receptors on the osteoblast to suppress osteoblast differentiation.
bone biology; cell biology; gene expression; osteoblast(s); osteoclast(s); osteonecrosis
Eph-related receptor tyrosine kinases (RTK) have been implicated in several biological functions including synaptic plasticity, axon guidance and morphogenesis, yet the details of the signal transduction pathways that produce these specific biological functions after ligand-receptor interaction remain unclear. We used Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS to characterize cellular signaling following stimulation by ephrinB1-Fc of NG-108 cells that overexpress EphB2 receptors. Because tyrosine phosphorylation functions as a key regulatory event in RTK signaling, we used anti-phosphotyrosine immunoprecipitation (pY IP) of cell lysates to isolate potential participants in the EphB2 pathway. Our SILAC experiments identified 127 unique proteins, 40 of which demonstrated increased abundance in pY IPs from ephrinB1-Fc stimulated cells as compared with unstimulated cells. Six proteins demonstrated decreased abundance, and 81 did not change significantly in relative abundance. Western blotting analysis of five proteins after pY IP verified their SILAC results. Based on previously published work and use of PathwayAssist™ software, we proposed an interaction network downstream of EphB2 for the proteins with changed ratios.
Phosphoproteomics; SILAC; Eph Signaling; Mass Spectrometry
The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. The transmembrane ephrinB1 protein is a bidirectional signaling molecule that signals through its cytoplasmic domain to promote cellular movements into the eye field, whereas activation of the fibroblast growth factor receptor (FGFR) represses these movements and retinal fate. In Xenopus embryos, ephrinB1 plays a role in retinal progenitor cell movement into the eye field through an interaction with the scaffold protein Dishevelled (Dsh). However, the mechanism by which the FGFR may regulate this cell movement is unknown. Here, we present evidence that FGFR-induced repression of retinal fate is dependent upon phosphorylation within the intracellular domain of ephrinB1. We demonstrate that phosphorylation of tyrosines 324 and 325 disrupts the ephrinB1/Dsh interaction, thus modulating retinal progenitor movement that is dependent on the planar cell polarity pathway. These results provide mechanistic insight into how fibroblast growth factor signaling modulates ephrinB1 control of retinal progenitor movement within the eye field.
Ephrins and Eph receptor(s) have recently been implicated in regulating neurogenesis in the adult subventricular zone (SVZ) and rostral migratory stream (RMS). Here, we examined the role of ephrinB3-EphB3 signaling in mediating the SVZ response to traumatic brain injury (TBI). Analysis of EphB3 expression showed co-localization with glial fibrillary acidic protein (GFAP)-positive neural stem progenitor cells (NSPCs) and doublecortin-positive neuroblasts, while ephrinB3 was expressed outside the neurogenic region. TBI resulted in a significant reduction in EphB3 expression, which coincided with enhanced NSPC survival and proliferation at 3 and 7 days post-injury. Analysis of mice lacking either ephrinB3 (ephrinB3−/−) or EphB3 (EphB3−/−) showed a significant increase in bromodeoxyuridine (BrdU) incorporation and Ki67 immunoreactivity in the SVZ. Interestingly, cell death was dissimilar between knockout mice, where cell death was reduced in EphB3−/− but increased in ephrinB3−/− mice. Lateral ventricle infusion of soluble pre-clustered ephrinB3-Fc reversed the proliferative and cell death defects in ephrinB3−/− but not EphB3−/− mice and prevented TBI-induced proliferation in wild type NSPCs. Coincidently, tumor suppressor p53 expression was increased following EphB3 stimulation and is reduced in the absence of either EphB3 or ephrinB3. Furthermore, pharmacological inhibition and siRNA knockdown of p53 attenuated ephrinB3-Fc mediated growth suppression while having no effect on cell death in cultured NSPCs. These data demonstrate that EphB3 signaling suppresses NSPC proliferation in a p53-dependent manner, induces cell death in the absence of ligand stimulation and is transiently reduced in the SVZ to initiate the expansion and survival of endogenous adult NSPCs following TBI.
traumatic brain injury; ephrin; Eph; subventricular zone; p53
Cell-cell adhesion is a critical process for the formation and maintenance of tissue patterns during development, as well as invasion and metastasis of cancer cells. Although great strides have been made regarding our understanding of the processes that play a role in cell-cell adhesion, the precise mechanisms by which diverse signaling events regulate cell and tissue architecture is poorly understood. In this commentary we will focus on the Eph/ephrin signaling system, and specifically how the ephrinB1 transmembrane ligand for Eph receptor tyrosine kinases sends signals affecting cell-cell junctions. In a recent study using the epithelial cells of early stage Xenopus embryos, we have shown that loss- or gain-of function of ephrinB1 can disrupt cell-cell contacts and tight junctions. This study reveals a mechanism where ephrinB1 competes with active Cdc42 for binding to Par-6, a scaffold protein central to the Par polarity complex (Par-3/Par-6/Cdc42/aPKC) and disrupts the localization of tight junction-associated proteins (ZO-1, Cingulin) at tight junctions. This competition reduces aPKC activity critical to maintaining and/or forming tight junctions. Finally, phosphorylation of ephrinB1 on specific tyrosine residues can block the interaction between ephrinB1 and Par-6 at tight junctions, and restore tight junction formation. Recent evidence indicates that de-regulation of forward signaling through EphB receptors may play a role in metastatic progression in colon cancer. In light of the new data showing an effect of ephrinB reverse signaling on tight junctions, an additional mechanism can be hypothesized where de-regulation of ephrinB1 expression or phosphorylation may also impact metastatic progression.
ephrin; Eph; cell-cell adhesion; Par-6; tight junctions
EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis.
C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5μls of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC).
Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 hr stimulation with sEphrinB2 ± VEGF compared to IgG control ± VEGF (p<0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures.
These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.
Angiogenesis; Apoptosis; EphrinB2; EphB4; Neovascularization; Retinopathy of Prematurity
EphB receptors tyrosine kinases and ephrinB ligands were first identified as guidance molecules involved in the establishment of topographical mapping and connectivity in the nervous system during development. Later in development and into adulthood their primary role would switch from guidance to activity-dependent modulation of synaptic efficacy. In sensory systems, they play a role in both the onset of inflammatory and neuropathic pain, and in the establishment of central sensitisation, an NMDA-mediated form of synaptic plasticity thought to underlie most forms of chronic pain. We studied wild type and EphB1 knockout mice in a range of inflammatory and neuropathic pain models to determine 1), whether EphB1 expression is necessary for the onset and/or maintenance of persistent pain, regardless of origin; 2), whether in these models cellular and molecular changes, e.g. phosphorylation of the NR2B subunit of the NMDA receptor, increased c-fos expression or microglial activation, associated with the onset of pain, are affected by the lack of functional EphB1 receptors. Differences in phenotype were examined behaviourally, anatomically, biochemically and electrophysiologically. Our results establish firstly, that functional EphB1 receptors are not essential for the development of normal nociception, thermal or mechanical sensitivity. Secondly, they demonstrate a widespread involvement of EphB1 receptors in chronic pain. NR2B phosphorylation, c-fos expression and microglial activation are all reduced in EphB1 knockout mice. This last finding is intriguing, since microglial activation is supposedly triggered directly by primary afferents, therefore it was not expected to be affected. Interestingly, in some models of long-term pain (days), mechanical and thermal hyperalgesia develop both in wild type and EphB1 knockout mice, but recovery is faster in the latter, indicating that in particular models these receptors are required for the maintenance, rather than the onset of, thermal and mechanical hypersensitivity. This potentially makes them an attractive target for analgesic strategies.
EphB receptors and their ephrinB ligands play a key role in the formation of a regular vascular system. Recent studies have also shown the involvement of Eph/ephrin interactions in malignant tumor progression and angiogenesis. We have generated soluble monomeric EphB4 (sEphB4)-expressing A375 melanoma cells to study the effect of dominant negatively acting sEphB4 on tumor growth and angiogenesis. Soluble EphB4-expressing A375 tumors grown subcutaneously in nude mice show dramatically reduced tumor growth compared to control tumors. The proliferative capacity of sEphB4-expressing cells in monolayer culture is not altered. Yet, sEphB4-expressing A375 cells cannot establish proper cell-cell contacts in three-dimensional spheroids. However, sEphB4 transfectants have reduced proliferation and apoptosis rates when grown in three-dimensional culture in vitro or in subcutaneous tumors in vivo. Analysis of the vascular phenotype of the tumors revealed a reduction of intratumoral microvessel density in sEphB4-expressing tumors. Corresponding to these mouse experiments, a matched pair analysis of EphB4 and ephrinB2 expression in human colon carcinomas revealed significantly upregulated levels of EphB4 expression compared to adjacent normal tissue. Taken together, the data identify dual effects of sEphB4 on the tumor and the vascular compartment that collectively inhibit tumor growth.
Tumor; angiogenesis; endothelial cells; EphB4; EphrinB2
EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1–A5 and ephrinB1–B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (Koff = 1.94 × 10−3 s−1 versus 1.06 × 10−4 s−1 for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu–Trp residues in the solvent exposed G–H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr–Met residues in the homologous positions in ephrinB1 with Leu–Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G–H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.
Nipah virus is a deadly virus that can cause death in up to 70% of infected patients, mostly from fatal inflammation of the brain. Nipah virus is considered a “priority pathogen” for bioterrorism purposes, and it has the potential for widespread economic devastation as it can spread rapidly among susceptible livestock. The authors had previously identified the receptor that mediates Nipah virus entry into cells. This receptor, ephrinB2, is a critical molecule for the development of the vascular and nervous system and is highly expressed on endothelial cells and neurons, which are also the two cell types preferentially infected by Nipah virus in vivo.
EphrinB2 belongs to a large family of related molecules that are variably conserved in structure and function. Thus, the authors screened all known ephrins, and found that a closely related molecule, ephrinB3, also can function as an entry receptor for Nipah virus. In addition, the authors established that while ephrinB2 was better used than ephrinB3 as an entry receptor, the same two critical amino acids in ephrinB2 and B3 were responsible for the viral receptor activity of these molecules. The discovery of a more comprehensive set of NiV receptors will aid our understanding of the pathology underlying NiV disease.
Eph receptors play important roles in development, neural plasticity and cancer. We used an Orbitrap mass spectrometer and Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) to identify and quantify 204 proteins with significantly changed abundance in anti-phosphotyrosine immunoprecipitates after ephrinB1-Fc stimulation. More than half of all known effectors downstream of EphB receptors were identified in this study, as well as numerous novel candidates for EphB signaling.
EphB; SILAC; mass spectrometry; phosphoproteomics; Orbitrap; IP
The groundbreaking discovery about arterial and venous expression of ephrinB2 and EphB4, respectively, in early embryonic development has led to a new paradigm for vascular research, providing compelling evidence that arterial and venous endothelial cells are established by genetic mechanisms before circulation begins. For arterial specification, vascular endothelial growth factor (VEGF) induces expression of Notch signaling genes, including Notch1 and its ligand, Delta-like 4 (Dll4), and Foxc1 and Foxc2 transcription factors directly regulate Dll4 expression. Upon activation of Notch signaling, the Notch downstream genes, Hey1/2 in mice or gridlock in zebrafish, further promote arterial differentiation. On the other hand, the orphan nuclear receptor COUP-TFII is a determinant factor for venous specification by inhibiting expression of arterial specific genes, including Nrp1 and Notch. After arterial and venous endothelial cells differentiate, a subpopulation of venous endothelial cells is thought to become competent to acquire lymphatic endothelial cell fate by progressively expressing the transcription factors Sox18 and Prox1 to differentiate into lymphatic endothelial cells. Therefore, it has now evident that arterial-venous cell fate determination and subsequent lymphatic development are regulated by the multi-step regulatory system associated with the key signaling pathways and transcription factors. Furthermore, new signaling molecules as additional regulators in these processes have recently been identified. As the mechanistic basis for a link between signaling pathways and transcriptional networks in arterial, venous and lymphatic endothelial cells begins to be uncovered, it is now time to summarize the literature on this exciting topic and provide perspectives for future research in the field.
Arterial-venous specification; lymphatic specification; VEGF; Notch; Fox
Erythropoietin-producing hepatocyte (Eph) kinases represent the largest receptor tyrosine kinase family. Some of them are expressed in the T cell compartment, but their function in T cells is unknown. In peripheral blood, EphB6 was predominantly expressed on T cells, and was upregulated after culture. EphB6 crosslinking by anti-EphB6 mAb or ephrinB2 in the presence of suboptimal T cell receptor (TCR) stimulation led to drastic T cell proliferation, suggesting that EphB6 can co-stimulate T cells. The proliferation was accompanied by enhanced production of several lymphokines, such as IFN-γ, IL-6, IL-10, TGF-β, TNF-α, and GM-CSF, but not IL-2 and IL-4. Sorted EphB6+ T cells had significantly stronger response to anti-CD3 and anti-CD28 stimulation than EphB6– T cells had. Taken together, these data suggest an important role of EphB6 in normal T cell activation. Within two minutes of anti-CD3 and anti-CD28 stimulation, EphB6 aggregated and colocalized with TCR, and this provides a morphological basis for EphB6 to enhance TCR signaling. The capping was followed by p38 MAPK activation, showing that EphB6 is capable of signaling, in spite of its lack of intrinsic kinase activity. This study demonstrates that interaction between EphB6 and its ligands facilitates T cell responses to antigen.
Eph receptors and their cell membrane–bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5–coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph–Eph interactions.
fluorescence resonance energy transfer microscopy; EphA3 receptor; receptor protein tyrosine kinase; receptor aggregation; signal transduction