EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

Eph receptor (Eph)‐ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4–ephrinB2 regulates cardiovascular development. To assess the role of EphB4–ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5⁺ cardiac progenitor cells and in α‐MHC⁺ cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5‐GFP⁺ cardiac progenitor cells, but not in α‐MHC‐GFP⁺ cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL‐RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4–ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full‐length EphB4 or intracellular domain‐truncated EphB4 in EphB4‐null ES cells was established to investigate the role of EphB4‐forward signaling in ES cells. Interestingly, while full‐length EphB4 was able to restore the cardiac lineage development in EphB4‐null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor‐kinase‐dependent EphB4‐forward signaling plays a crucial role in the development of cardiac progenitor cells. J. Cell. Biochem. 116: 467–475, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

EphB receptors and ephrinB ligands: regulators of vascular assembly and homeostasis

Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding glycosyl-phosphatidyl-inositol-anchored ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, genetic loss of function experiments have identified EphB receptors and ephrinB ligands as crucial regulators of vascular assembly, orchestrating arteriovenous differentiation and boundary formation. Despite these clearly defined rate-limiting roles of the EphB/ephrinB system for developmental angiogenesis, the mechanisms of the functions of EphB receptors and ephrinB ligands in the cells of the vascular system are poorly understood. Moreover, little evidence can be found in the recent literature regarding complementary EphB and ephrinB expression patterns that occur in the vascular system and that may bring cells into juxtapositional contact to allow bi-directional signaling between neighboring cells. This review summarizes the current knowledge of the role of EphB receptors and ephrinB ligands during embryonic vascular assembly and discusses recent findings on EphB/ephrinB-mediated cellular functions pointing to the crucial role of the Eph/ephrin system in controlling vascular homeostasis in the adult.Eph/ephrin work in the laboratory of the authors is supported by a grant from the Deutsche Forschungsgemeinschaft (Au83/3–2 within the SPP1069 "Angiogenesis")     

EphB/ephrinB Signaling in Cell Adhesion and Migration

Eph receptors and their ligands, ephrins, represent the largest group of the receptor tyrosine kinase (RTK) family, and they mediate numerous developmental processes in a variety of organisms. Ephrins are membrane-bound proteins that are mainly divided into two classes: A class ephrins, which are linked to the membrane by a glycosylphosphatidylinositol (GPI) linkage, and B class ephrins, which are transmembrane ligands. Based on their domain structures and affinities for ligand binding, the Eph receptors are also divided into two groups. Trans-dimerization of Eph receptors with their membrane-tethered ligands regulates cell-cell interactions and initiates bidirectional signaling pathways. These pathways are intimately involved in regulating cytoskeleton dynamics, cell migration, and alterations in cellular dynamics and shapes. The EphBs and ephrinBs are specifically localized and modified to promote higher-order clustering and initiate of bidirectional signaling. In this review, we present an in-depth overview of the structure, mechanisms, cell signaling, and functions of EphB/ephrinB in cell adhesion and migration.     

Cupredoxin-cancer interrelationship: azurin binding with EphB2, interference in EphB2 tyrosine phosphorylation, and inhibition of cancer growth

Azurin is a member of a family of metalloproteins called cupredoxins. Although previously thought to be involved in electron transfer, azurin has recently been shown to preferentially enter cancer cells than normal cells and induce apoptosis in such cells. Azurin also demonstrates structural similarity to a ligand known as ephrinB2, which binds its cognate receptor tyrosine kinase EphB2 to initiate cell signaling. Eph/ephrin signaling is known to be involved in cancer progression. We now demonstrate that azurin binds to the EphB2-Fc receptor with high affinity. We have localized a C-terminal domain of azurin (Azu 96-113) that exhibits structural similarity to ephrinB2 at the G-H loop region known to be involved in receptor binding. A synthetic peptide (Azu 96-113) as well as a GST fusion derivative GST-Azu 88-113 interferes with the growth of various human cancer cells. In a prostate cancer cell line DU145 lacking functional EphB2, azurin or its GST-fusion derivatives had little cytotoxic effect. However, in DU145 cells expressing functional EphB2, azurin and GST-Azu 88-113 demonstrated significant cytotoxicity, whereas ephrinB2 promoted cell growth. Azurin inhibited the ephrinB2-mediated autophosphorlyation of the EphB2 tyrosine residue, thus interfering in upstream cell signaling and contributing to cancer cell growth inhibition.     

Bone cell interactions through Eph/ephrin

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.     

EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have a variety of roles in the developing and adult central nervous system that require direct cell–cell interactions; including regulating axon path finding, cell proliferation, migration and synaptic plasticity. Recently, we identified a novel pro-survival role for ephrins in the adult subventricular zone, where ephrinB3 blocks Eph-mediated cell death during adult neurogenesis. Here, we examined whether EphB3 mediates cell death in the adult forebrain following traumatic brain injury and whether ephrinB3 infusion could limit this effect. We show that EphB3 co-labels with microtubule-associated protein 2-positive neurons in the adult cortex and is closely associated with ephrinB3 ligand, which is reduced following controlled cortical impact (CCI) injury. In the complete absence of EphB3 (EphB3^−/−), we observed reduced terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), and functional improvements in motor deficits after CCI injury as compared with wild-type and ephrinB3^−/− mice. We also demonstrated that EphB3 exhibits dependence receptor characteristics as it is cleaved by caspases and induces cell death, which is not observed in the presence of ephrinB3. Following trauma, infusion of pre-clustered ephrinB3-Fc molecules (eB3-Fc) into the contralateral ventricle reduced cortical infarct volume and TUNEL staining in the cortex, dentate gyrus and CA3 hippocampus of wild-type and ephrinB3^−/− mice, but not EphB3^−/− mice. Similarly, application of eB3-Fc improved motor functions after CCI injury. We conclude that EphB3 mediates cell death in the adult cortex through a novel dependence receptor-mediated cell death mechanism in the injured adult cortex and is attenuated following ephrinB3 stimulation.     

EphrinB2/EphB4 在血管生长中的作用及中医药研究展望*

近年来,有关ephrin及其Eph受体的作用研究已从神经系统方面逐步向血管生长扩展。已有研究表明ephrinB2/EphB4及其独特的双向信号转导几乎参与血管生长的每个方面,涉及血管发育过程中的动静脉分化、胚胎后血管新生包括内皮细胞增殖、迁移、粘附和分化等过程,且与VEGF、Notch等血管新生调控因子关系密切。另外,实验表明活血化瘀名方血府逐瘀汤显著的促血管新生作用与ephrinB2/EphB4相关,说明中医药促血管新生中ephrinB2/EphB4具有重要作用。本文部分总结了ephrinB2/EphB4在血管生长中的作用,并提出中医药在这方面的展望。     

Bone cell interactions through Eph/ephrin: Bone modeling,remodeling and associated diseases

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.     

Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.     

EphB receptor tyrosine kinases control morphological development of the ventral midbrain

EphB receptor tyrosine kinases and ephrin-B ligands regulate several types of cell-cell interactions during brain development, generally by modulating the cytoskeleton. EphB/ephrinB genes are expressed in the developing neural tube of early mouse embryos with distinct overlapping expression in the ventral midbrain. To test EphB function in midbrain development, mouse embryos compound homozygous for mutations in the EphB2 and EphB3 receptor genes were examined for early brain phenotypes. These mutants displayed a morphological defect in the ventral midbrain, specifically an expanded ventral midline evident by embryonic day E9.5-10.5, which formed an abnormal protrusion into the cephalic flexure. The affected area was comprised of cells that normally express EphB2 and ephrin-B3. A truncated EphB2 receptor caused a more severe phenotype than a null mutation, implying a dominant negative effect through interference with EphB forward (intracellular) signaling. In mutant embryos, the overall number, size, and identity of the ventral midbrain cells were unaltered. Therefore, the defect in ventral midline morphology in the EphB2;EphB3 compound mutant embryos appears to be caused by cellular changes that thin the tissue, forcing a protrusion of the ventral midline into the cephalic space. Our data suggests a role for EphB signaling in morphological organization of specific regions of the developing neural tube.     

Distinct roles for ephrinB3 in the formation and function of hippocampal synapses

The transmembrane ephrinB ligands and their Eph receptor tyrosine kinases are known to regulate excitatory synaptic functions in the hippocampus. In the CA3-CA1 synapse, ephrinB ligands are localized to the post-synaptic membrane, while their cognate Eph receptors are presumed to be pre-synaptic. Interaction of ephrinB molecules with Eph receptors leads to changes in long-term potentiation (LTP), which has been reported to be mediated by reverse signaling into the post-synaptic membrane. Here, we demonstrate that the cytoplasmic domain of ephrinB3 and hence reverse signaling is not required for ephrinB dependent learning and memory tasks or for LTP of these synapses. Consistent with previous reports, we find that ephrinB3(KO) null mutant mice exhibit a striking reduction in CA3-CA1 LTP that is associated with defective learning and memory tasks. We find the null mutants also show changes in both pre- and post-synaptic proteins including increased levels of synapsin and synaptobrevin and reduced levels of NMDA receptor subunits. These abnormalities are not observed in ephrinB3(lacZ) reverse signaling mutants that specifically delete the ephrinB3 intracellular region, supporting a cytoplasmic domain-independent forward signaling role for ephrinB3 in these processes. We also find that both ephrinB3(KO) and ephrinB3(lacZ) mice show an increased number of excitatory synapses, demonstrating a cytoplasmic-dependent reverse signaling role of ephrinB3 in regulating synapse number. Together, these data suggest that ephrinB3 may act like a receptor to transduce reverse signals to regulate the number of synapses formed in the hippocampus, and that it likely acts to stimulate forward signaling to modulate a number of other proteins involved in synaptic activity and learning/memory.     

EphB4 promotes site-specific metastatic tumor cell dissemination by interacting with endothelial cell-expressed ephrinB2

The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4-expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. AACR.     

Myosin 1b functions as an effector of EphB signaling to control cell repulsion

Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion.     

Ligand binding induces Cbl-dependent EphB1 receptor degradation through the lysosomal pathway

Eph receptor tyrosine kinases play a critical role in embryonic patterning and angiogenesis. In the adult, they are involved in carcinogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1 and then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1 as kinase-dead EphB1-K652R was resistant to Cbl. Glutathione S-transferase binding experiments showed that Cbl bound to EphB1 through its tyrosine kinase-binding domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.     

Eps15R and clathrin regulate EphB2‐mediated cell repulsion

Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans‐endocytosis of the inter‐cellular receptor‐ligand EphB‐ephrinB complex is required. The molecular mechanism underlying trans‐endocytosis is poorly defined. Here we show that the process is clathrin‐ and Eps15R‐mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co‐cultures of EphB2‐ and ephrinB1‐expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2‐mediated cell repulsion as shown in a rescue experiment in the EphB2 co‐culture assay where wild type Eps15R but not the clathrin‐binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans‐endocytosis and thereby cell repulsion.      

Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity.

During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors were recently shown to associate with NMDA receptors at synaptic sites and were suggested to play a role in synaptogenesis. Here we show that Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2(-/-) hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) was impaired, and two forms of synaptic depression were completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescued the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions.     

EphrinB-EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1

Recent studies show that Eph receptors act mainly through the regulation of actin reorganization. Here, we show a novel mode of action for EphB receptors. We identify synaptojanin 1 - a phosphatidylinositol 5'-phosphatase that is involved in clathrin-mediated endocytosis - as a physiological substrate for EphB2. EphB2 causes tyrosine phosphorylation in the proline-rich domain of synaptojanin 1, and inhibits both the interaction with endophilin and the 5'-phosphatase activity of synaptojanin 1. Treatment with the EphB ligand, ephrinB2, elevates the cellular level of phosphatidylinositol 4,5-bisphosphate and promotes transferrin uptake. A kinase inactive mutant of EphB2 and a phosphorylation site mutant of synaptojanin 1 both neutralize the increase of transferrin uptake after ephrinB2 treatment. These mutants also inhibit AMPA glutamate receptor endocytosis in hippocampal neurons. Interestingly, incorporated transferrin does not reach endosomes, suggesting dual effects of EphB signalling on the early and late phases of clathrin-mediated endocytosis. Our results indicate that ephrinB-EphB signalling regulates clathrin-mediated endocytosis in various cellular contexts by influencing protein interactions and phosphoinositide turnover through tyrosine phosphorylation of synaptojanin 1.     

EphB4/ephrinB2逆向信号对RAW264．7破骨细胞分化中PDZ结构域蛋白表达变化的研究

Eph受体是酪氨酸蛋白激酶受体家族中最大的亚家族,ephrin(Eph受体相互作用蛋白)是其配体,它们是膜结合蛋白,相互依赖进行信号转导.内居蛋白(syntenin)与Pick1属于PDZ结构域(PSD-95/Dlg-/Zo-1 domain)蛋白,报道称能与ephrinB配体结合,但是否受Eph受体调控尚未见报道.以RAW264.7细胞株为研究对象,通过蛋白质印迹及/或免疫荧光分析显示RAW264.7细胞经RANKL诱导的破骨细胞表达ephrinB2、内居蛋白(syntenin)和Pick1三个蛋白质.将提前成簇的可溶性EphB4蛋白加入培养液,与ephrinB2配体结合,用来研究EphB4/ephrinB2逆向信号对syntenin和Pick1表达水平变化的影响.免疫印迹及Real-time RT-PCR分析结果显示,在EphB4-Fc实验组中Pick1的蛋白质及mRNA水平都有明显增加,然而在EphB4-Fc实验组与Fc对照组别间syntenin的蛋白质及mRNA水平未见明显变化.免疫共沉淀结果显示,syntenin和Pick1不能与ephrinB2共沉淀.以上结果初步探索了体外破骨细胞分化过程中,EphB4/ephrinB2逆向信号对PDZ结构域蛋白(ephrinB2配体潜在的下游信号分子)表达变化的调控.     

Sizes of interface residues account for cross‐class binding affinity patterns in Eph receptor–ephrin families

Eph receptors comprise the largest known family of receptor tyrosine kinases in mammals. They bind members of a second family, the ephrins. As both Eph receptors and ephrins are membrane bound, interactions permit unusual bidirectional cell–cell signaling. Eph receptors and ephrins each form two classes, A and B, based on sequences, structures, and patterns of affinity: Class A Eph receptors bind class A ephrins, and class B Eph receptors bind class B ephrins. The only known exceptions are the receptor EphA4, which can bind ephrinB2 and ephrinB3 in addition to the ephrin‐As (Bowden et al., Structure 2009;17:1386–1397); and EphB2, which can bind ephrin‐A5 in addition to the ephrin‐Bs (Himanen et al., Nat Neurosci 2004;7:501–509). A crystal structure is available of the interacting domains of the EphA4‐ephrin B2 complex (wwPDB entry 2WO2) (Bowden et al., Structure 2009;17:1386–1397). In this complex, the ligand‐binding domain of EphA4 adopts an EphB‐like conformation. To understand why other cross‐class EphA receptor–ephrinB complexes do not form, we modeled hypothetical complexes between (1) EphA4–ephrinB1, (2) EphA4–ephrinB3, and (3) EphA2–ephrinB2. We identify particular residues in the interface region, the size variations of which cause steric clashes that prevent formation of the unobserved complexes. The sizes of the sidechains of residues at these positions correlate with the pattern of binding affinity. Proteins 2014; 82:349–353. © 2013 Wiley Periodicals, Inc.     

The kinase-null EphB6 receptor undergoes transphosphorylation in a complex with EphB1.

Uniquely for the Eph family of receptor tyrosine kinases, the EphB6 receptor is catalytically inactive due to the alteration of several critical residues in its kinase domain. This has cast doubt upon its ability to participate in cytoplasmic signaling events. We show here that despite its lack of kinase activity, EphB6 undergoes inducible tyrosine phosphorylation upon stimulation with the Eph-B receptor subfamily ligand ephrin-B1. We also demonstrate, for the first time, evidence of cross-talk between Eph receptors. Overexpression of a catalytically active member of the Eph-B subfamily, EphB1, resulted in increased EphB6 phosphorylation. EphB1-induced EphB6 phosphorylation was ligand-dependent and required the functional catalytic activity of EphB1. EphB1 not only transphosphorylated EphB6, but together they also formed a stable hetero-complex. In addition, we identify the proto-oncogene c-Cbl as an EphB6-binding protein. Although EphB6-Cbl association appeared to be constitutive, Cbl required a functional phosphotyrosine binding domain in order to bind the receptor, whereas its RING finger motif ubiquitin-transfer domain was not necessary. Our findings demonstrate that EphB6 is an actively signaling receptor that undergoes transphosphorylation upon ligand binding and that can initiate specific cytoplasmic signaling events.