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At least 19 recordsLinked to original sources

PubMed2006

A critical role for the EphA3 receptor tyrosine kinase in heart development.

Eph proteins are receptor tyrosine kinases that control changes in cell shape and migration during development. We now describe a critical role for EphA3 receptor signaling in heart development as revealed by the phenotype of EphA3 null mice. During heart development mesenchymal outgrowths, the atrioventricular endocardial cushions, form in the atrioventricular canal. This morphogenetic event requires endocardial cushion cells to undergo an epithelial to mesenchymal transformation (EMT), and results in the formation of the atrioventricular valves and membranous portions of the atrial and ventricular septa. We show that EphA3 knockouts have significant defects in the development of their atrial septa and atrioventricular endocardial cushions, and that these cardiac abnormalities lead to the death of approximately 75% of homozygous EphA3(-/-) mutants. We demonstrate that EphA3 and its ligand, ephrin-A1, are expressed in adjacent cells in the developing endocardial cushions. We further demonstrate that EphA3(-/-) atrioventricular endocardial cushions are hypoplastic compared to wildtype and that EphA3(-/-) endocardial cushion explants give rise to fewer migrating mesenchymal cells than wildtype explants. Thus our results indicate that EphA3 plays a crucial role in the development and morphogenesis of the cells that give rise to the atrioventricular valves and septa.

Animals↗

PubMed1998

Distinct subdomains of the EphA3 receptor mediate ligand binding and receptor dimerization.

Eph receptor tyrosine kinases and their ligands (ephrins) are highly conserved protein families implicated in patterning events during development, particularly in the nervous system. In a number of functional studies, strict conservation of structure and function across distantly related vertebrate species has been confirmed. In this study we make use of the observation that soluble human EphA3 (HEK) exerts a dominant negative effect on somite formation and axial organization during zebrafish embryogenesis to probe receptor function. Based on exon structure we have dissected the extracellular region of EphA3 receptor into evolutionarily conserved subdomains and used kinetic BIAcore analysis, mRNA injection into zebrafish embryos, and receptor transphosphorylation analysis to study their function. We show that ligand binding is restricted to the N-terminal region encoded by exon III, and we identify an independent, C-terminal receptor-dimerization domain. Recombinant proteins encoding either region in isolation can function as receptor antagonists in zebrafish. We propose a two-step mechanism of Eph receptor activation with distinct ligand binding and ligand-independent receptor-receptor oligomerization events.

Animals↗

PubMed2005

Upregulation of EphA3 receptor after spinal cord injury.

Spinal cord injury (SCI) releases a cascade of events that leads to the onset of an inhibitory milieu for axonal regeneration. Some of these changes result from the presence of repulsive factors that may restrict axonal outgrowth after trauma. The Eph receptor tyrosine kinase (RTK) family has emerged as a key repellent cue known to be involved in neurite outgrowth, synapse formation, and axonal pathfinding during development. Given the nonpermissive environment for axonal regeneration after SCI, we questioned whether re-expression of one of these molecules occurs during regenerative failure. We examined the expression profile of EphA3 at the mRNA and protein levels after SCI, using the NYU contusion model. There is a differential distribution of this molecule in the adult spinal cord and EphA3 showed an increase in expression after several injury models like optic nerve and brain injury. Standardized semi-quantitative RT-PCR analysis demonstrated a time-dependent change in EphA3 mRNA levels, without alterations in beta-actin levels. The basal level of EphA3 mRNA in the adult spinal cord is low and its expression was induced 2 days after trauma (the earliest time point analyzed) and this upregulation persisted for 28 days post-injury (the latest time point examined). These results were corroborated at the protein level by immunohistochemical analysis and the cell phenotype identified by double labeling studies. In control animals, EphA3 immunoreactivity was observed in motor neurons of the ventral horn but not in lesioned animals. In addition, GFAP-positive cells were visualized in the ventral region of injured white matter. These results suggest that upregulation of EphA3 in reactive astrocytes may contribute to the repulsive environment for neurite outgrowth and may be involved in the pathophysiology generated after SCI.

Animals↗

PubMed1998

Expression and tyrosine phosphorylation of Eph receptors suggest multiple mechanisms in patterning of the visual system.

The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells, In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization.

Amino Acid Sequence↗

PubMed2003

Ephrin-A1 induces c-Cbl phosphorylation and EphA receptor down-regulation in T cells.

Eph receptor tyrosine kinases are expressed by T lineage cells, and stimulation with their ligands, the ephrins, has recently been shown to modulate T cell behavior. We show that ephrin-A1 stimulation of Jurkat T cells induces tyrosine phosphorylation of EphA3 receptors and cytoplasmic proteins, including the c-cbl proto-oncogene. Cbl phosphorylation was also observed in peripheral blood T cells. In contrast, stimulation of Jurkat cells with the EphB receptor ligand ephrin-B1 does not cause Cbl phosphorylation. EphA activation also induced Cbl association with Crk-L and Crk-II adapters, but not the related Grb2 protein. Induction of Cbl phosphorylation upon EphA activation appeared to be dependent upon Src family kinase activity, as Cbl phosphorylation was selectively abrogated by the Src family inhibitor 4-amino-5(4-chlorophenyl-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine, while EphA phosphorylation was unimpaired. Ephrin-A1 stimulation of Jurkat cells was also found to cause down-regulation of endogenous EphA3 receptors from the cell surface and their degradation. In accordance with the role of Cbl as a negative regulator of receptor tyrosine kinases, overexpression of wild-type Cbl, but not its 70-Z mutant, was found to down-regulate EphA receptor expression. Receptor down-regulation could also be inhibited by blockage of Src family kinase activity. Our findings show that EphA receptors can actively signal in T cells, and that Cbl performs multiple roles in this signaling pathway, functioning to transduce signals from the receptors as well as regulating activated EphA receptor expression.

Cell Line↗

PubMed2004

Loss-of-function analysis of EphA receptors in retinotectal mapping.

EphA tyrosine kinases are thought to act as topographically specific receptors in the well-characterized projection map from the retina to the tectum. Here, we describe a loss-of-function analysis of EphA receptors in retinotectal mapping. Expressing patches of a cytoplasmically truncated EphA3 receptor in chick retina caused temporal axons to have reduced responsiveness to posterior tectal repellent activity in vitro and to shift more posteriorly within the map in vivo. A gene disruption of mouse EphA5, replacing the intracellular domain with beta-galactosidase, reduced in vitro responsiveness of temporal axons to posterior target membranes. It also caused map abnormalities in vivo, with temporal axons shifted posteriorly and nasal axons anteriorly, but with the entire target still filled by retinal axons. The anterior shift of nasal axons was not accompanied by increased responsiveness to tectal repellent activity, in contrast to the comparable anterior shift in ephrin-A knock-outs, helping to resolve a previous ambiguity in interpreting the ephrin gene knock-outs. The results show the functional requirement for endogenous EphA receptors in retinotectal mapping, show that the receptor intracellular domain is required for a forward signaling response to topographic cues, and provide new evidence for a role of axon competition in topographic mapping.

Animals↗

PubMed2002

Domain-specific olivocerebellar projection regulated by the EphA-ephrin-A interaction.

Neural maps in the vertebrate central nervous system often show discontinuously segregated, domain-to-domain patterns. However, the molecular mechanism that establishes such maps is not well understood. Here we show that in the chicken olivocerebellar system, EphA receptors and ephrin-As are expressed with distinct levels and combinations in mapping domains. When ephrin-A2 is retrovirally overexpressed in the cerebellum, the olivocerebellar map is disrupted, excluding axons with high receptor activity from ectopic expression domains. Conversely, overexpression of a truncated EphA3 receptor in the cerebellum reduces endogenous ligand activity to undetectable levels and causes aberrant mapping, with high receptor axons invading high ligand domains. In vitro, ephrin-A2 inhibits outgrowth of inferior olive axons in a region-specific manner. These results suggest that Eph receptors and ephrins constitute domain-specific positional information, and the spatially accurate receptor-ligand interaction is essential to guide inferior olive axons to their correct target domains.

Animals↗

PubMed2001

Differential effects of overexpression of two forms of ephrin-A5 on neonatal rat cardiomyocytes.

Eph receptors constitute the largest family of receptor tyrosine kinases. Multiple transcripts of ephrin-A5, the cognate ligand of the EphA3 receptor, were found in neonatal rat cardiomyocytes. Two cDNA clones encoding the full-length ephrin-A5 (ephrin-A5 alpha) and a 27-amino acid deletion form (ephrin-A5 beta) were isolated. To examine the role of ephrin-A5 in cardiomyocytes, the cDNAs were inserted into adenoviral vectors, termed Ad.ephrin-A5 alpha and Ad.ephrin-A5 beta, respectively, and overexpressed in cardiomyocytes. The effect of ephrin-A5 on cardiomyocyte gene expression was investigated using a cDNA expression array and Western blot analysis. The results showed that both ephrin-A5 alpha and ephrin-A5 beta downregulated cyclin D2, cyclin-dependent kinase-4 proteins, and their cognate receptor EphA3, which were associated with reduced bromodeoxyuridine incorporation in cardiomyocytes. Whereas ephrin-A5 alpha and ephrin-A5 beta also induced differential gene expression, only ephrin-A5 beta significantly upregulated the transcription of brain natriuretic peptide and downregulated ras-related protein RAB2, protein kinase C inhibitor protein-1, clusterin, and insulin-like growth factor-binding protein. The results suggest that the two forms of ephrin-A5 share similar function while differ in regulating different sets of genes in cardiomyocytes.

Adenoviridae↗

PubMed2003

Dissecting the EphA3/Ephrin-A5 interactions using a novel functional mutagenesis screen.

The EphA3 receptor tyrosine kinase preferentially binds ephrin-A5, a member of the corresponding subfamily of membrane-associated ligands. Their interaction regulates critical cell communication functions in normal development and may play a role in neoplasia. Here we describe a random mutagenesis approach, which we employed to study the molecular determinants of the EphA3/ephrin-A5 recognition. Selection and functional characterization of EphA3 point mutants with impaired ephrin-A5 binding from a yeast expression library defined three EphA3 surface areas that are essential for the EphA3/ephrin-A5 interaction. Two of these map to regions identified previously in the crystal structure of the homologous EphB2-ephrin-B2 complex as potential ligand/receptor interfaces. In addition, we identify a third EphA3/ephrin-A5 interface that falls outside the structurally characterized interaction domains. Functional analysis of EphA3 mutants reveals that all three Eph/ephrin contact areas are essential for the assembly of signaling-competent, oligomeric receptor-ligand complexes.

Binding Sites↗

PubMed1999

Marsupial retinocollicular system shows differential expression of messenger RNA encoding EphA receptors and their ligands during development.

The protracted development of the wallaby (Macropus eugenii) has allowed study of messenger RNAs encoding Eph receptors EphA3 and EphA7 and ligands ephrin-A2 and -A5 in the retina and superior colliculus at intervals throughout the development of the retinocollicular projection: from birth, before retinal innervation, to postnatal day 95, when the projection is mature. Reverse transcription-polymerase chain reaction showed messenger RNAs for both receptors and ligands were expressed at all ages. EphA7 was expressed more highly in the rostral superior colliculus. Ephrin-A2 and -A5 were expressed more highly in the caudal colliculus. EphA3 was expressed in a complementary manner, more highly in temporal than in nasal retina. There are higher levels of expression of the ligands when the projection is only coarsely topographically organised. This suggests a role for them and their receptor EphA3 in this stage, by repulsive interactions which restrict temporal axons to rostral superior colliculus. This is the first account in a marsupial mammal of the appearance of this molecular family, substantiating its ubiquitous role in topographically organised neuronal connections. Nevertheless, expression is not the same as in the mouse, suggesting differences in the details of topographic coding between species.

Animals↗

PubMed2006

Ephrin-A5 modulates the topographic mapping and connectivity of commissural axons in murine hippocampus.

Entorhinal and commissural/associational projections show a non-overlapping distribution in the hippocampus proper and the dentate gyrus. The expression of Ephrins and their Eph receptors in the developing hippocampus indicates that this family of axonal guidance molecules may modulate the formation of these connections. Here we focused on the role of the ephrin-A5 ligand in the development of the main hippocampal afferents. In situ hybridization showed that ephrin-A5 mRNA was detected mainly in the principal cells of the hippocampus proper and in the dentate gyrus throughout postnatal development. Immunocytochemical analyses revealed prominent expression of the EphA3 receptor, a putative receptor for ephrin-A5, in the main cells and the neuropil of the developing hippocampus. Tracing experiments in ephrin-A5(-/-) mice showed that commissural projections were transiently altered in the hippocampus proper at P5, but they were mistargeted throughout the postnatal development in the dentate gyrus. Immunocytochemistry with anti-calbindin antibodies revealed that the dentate mossy fiber projection was not altered in ephrin-A5(-/-) mice. Electron microscopy studies showed alterations in the density of synapses and spines in commissural/associational layers, but not in entorhinal layers, and in the mossy fibers in these animals. Taken together, these findings indicate that ephrin-A5 signaling is involved in the formation and maturation of synapses in the hippocampus.

Animals↗

PubMed2002

Differential splicing generates Tvl-1/RFXANK isoforms with different functions.

Earlier studies have shown that Tvl-1 gives rise to at least two differentially spliced mRNAs, one of which (Tvl-S) encodes a protein that lacks amino acids 91-112. DNA binding of RFX complexes assembled in the presence of Tvl-S is impaired. As a result, Tvl-S does not support the expression of Class II major histocompatibility complex (MHC) genes. Here, we show that the reason Tvl-S is inactive as a transcriptional regulator of Class II MHC genes is that the RFX complexes assembled in the presence of Tvl-S are unstable. Additionally, we show that interferon-gamma, which induces Class II MHC gene expression in 293 cells, promotes a shift in the splicing pattern of RFXANK/Tvl-1 toward the transcriptionally active Tvl-L isoform, suggesting that differential splicing of Tvl-1 is a signal-regulated process. Finally, we show that Tvl-1 regulates the expression of non-MHC genes. One such gene encodes the ephrin receptor EphA3. Since both Tvl-L and Tvl-S are identical in their ability to induce the expression of EphA3, we conclude that Tvl-1 regulates the expression of non-MHC genes by RFX-independent mechanisms.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Two homeobox genes define the domain of EphA3 expression in the developing chick retina.

Graded expression of the Eph receptor EphA3 in the retina and its two ligands, ephrin A2 and ephrin A5 in the optic tectum, the primary target of retinal axons, have been implicated in the formation of the retinotectal projection map. Two homeobox containing genes, SOHo1 and GH6, are expressed in a nasal-high, temporal-low pattern during early retinal development, and thus in opposing gradients to EphA3. Retroviral misexpression of SOHo1 or GH6 completely and specifically repressed EphA3 expression in the neural retina, but not in other parts of the central nervous system, such as the optic tectum. Under these conditions, some temporal ganglion cell axons overshot their expected termination zones in the rostral optic tectum, terminating aberrantly at more posterior locations. However, the majority of ganglion cell axons mapped to the appropriate rostrocaudal locations, although they formed somewhat more diffuse termination zones. These findings indicate that other mechanisms, in addition to differential EphA3 expression in the neural retina, are required for retinal ganglion axons to map to the appropriate rostrocaudal locations in the optic tectum. They further suggest that the control of topographic specificity along the retinal nasal-temporal axis is split into several independent pathways already at a very early time in development.

Animals↗

PubMed2004

Recruitment of Eph receptors into signaling clusters does not require ephrin contact.

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.

Cell Line↗

PubMed2001

Differential expression of Eph receptors and ephrins correlates with the formation of topographic projections in primary and secondary visual circuits of the embryonic chick forebrain.

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. We have analyzed the distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon during the period when visual connections are being established. We have found that both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules was observed. Finally, we also show that a similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway.

Animals↗

PubMed2003

Persistence of graded EphA/Ephrin-A expression in the adult frog visual system.

Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the adult or, if downregulated after metamorphosis, to be reexpressed after optic nerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic frogs, including mature adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nerve injury.

Affinity Labels↗

PubMed1998

Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region.

The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. Here we show that in vivo tyrosine phosphorylation sites of the Eph receptors EphA3, EphA4, and EphB2 in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Furthermore, tyrosine-phosphorylated EphB2 was detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation was detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrated that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src. In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma was not abolished upon mutation of tyrosine 611 in EphB2. Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine to phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, showed tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2. Our mutational analysis also indicated that tyrosines 605 and 611 are important for EphB2 kinase activity. We propose Src kinase as a downstream effector that mediates the neuron's response to Eph receptor activation.

Animals↗

PubMed2000

An ephrin-A-dependent signaling pathway controls integrin function and is linked to the tyrosine phosphorylation of a 120-kDa protein.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, have been implicated in the development of the retinotectal projection. Here, glycosylphosphatidylinositol-anchored A-ephrins are not only expressed in the tectum but also on retinal axons, raising the possibility that they function in this context as receptors. We now show that activation of ephrin-A2 or ephrin-A5 by one of their receptors, ephA3, results in a beta 1-integrin-dependent increased adhesion of ephrin-A-expressing cells to laminin. In the search for an ephrin-A-dependent signaling pathway controlling integrin activation, we identified a 120-kDa raft membrane protein that is tyrosine-phosphorylated specifically after ephrin-A activation. Tyrosine phosphorylation of this protein is not seen after stimulating ephrin-A2-expressing cells with basic fibroblast growth factor, epidermal growth factor, insulin growth factor, or fetal calf serum containing a large set of different growth factors. The role of p120 as a mediator of an ephrin-A-integrin coupling is supported by the finding that inhibiting tyrosine phosphorylation of p120 correlates with an abolishment of the beta 1-dependent cell adhesion.

Cell Adhesion↗