Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity.

The EPH-related transmembrane tyrosine kinases constitute the largest known family of receptor-like tyrosine kinases, with many members displaying specific patterns of expression in the developing and adult nervous system. A family of cell surface-bound ligands exhibiting distinct, but overlapping, specificities for these EPH-related kinases was identified. These ligands were unable to act as conventional soluble factors. However, they did function when presented in membrane-bound form, suggesting that they require direct cell-to-cell contact to activate their receptors. Membrane attachment may serve to facilitate ligand dimerization or aggregation, because antibody-mediated clustering activated previously inactive soluble forms of these ligands.

Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.

A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.

PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands.

Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses. PDZ proteins apparently play critical roles in such protein localizations. Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates. We report that Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins. In addition, we find that Eph receptors and ligands colocalize with PDZ proteins at synapses. Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse.

Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis.

We report that the many Eph-related receptor tyrosine kinases, and their numerous membrane-bound ligands, can each be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan.

Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone.

The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1-3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.

Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration.

BACKGROUND: In the trunk of avian embryos, neural crest migration through the somites is segmental, with neural crest cells entering the rostral half of each somitic sclerotome but avoiding the caudal half. Little is known about the molecular nature of the cues-intrinsic to the somites-that are responsible for this segmental migration of neural crest cells. RESULTS: We demonstrate that Eph-related receptor tyrosine kinases and their ligands are essential for the segmental migration of avian trunk neural crest cells through the somites. EphB3 localizes to the rostral half-sclerotome, including the neural crest, and the ligand ephrin-B1 has a complementary pattern of expression in the caudal half-sclerotome. To test the functional significance of this striking asymmetry, soluble ligand ephrin-B1 was added to interfere with receptor function in either whole trunk explants or neural crest cells cultured on alternating stripes of ephrin-B1 versus fibronection. Neural crest cells in vitro avoided migrating on lanes of immobilized ephrin-B1; the addition of soluble ephrin-B1 blocked this inhibition. Similarly, in whole trunk explants, the metameric pattern of neural crest migration was disrupted by addition of soluble ephrin-B1, allowing entry of neural crest cells into caudal portions of the sclerotome. CONCLUSIONS: Both in vivo and in vitro, the addition of soluble ephrin-B1 results in a loss of the metameric migratory pattern and a disorganization of neural crest cell movement. These results demonstrate that Eph-family receptor tyrosine kinases and their transmembrane ligands are involved in interactions between neural crest and sclerotomal cells, mediating an inhibitory activity necessary to constrain neural precursors to specific territories in the developing nervous system.

Elk-L3, a novel transmembrane ligand for the Eph family of receptor tyrosine kinases, expressed in embryonic floor plate, roof plate and hindbrain segments.

The Eph family of receptor tyrosine kinases has 13 distinct members and seven ligands for these receptors have been described to date. These receptors and their ligands have been implicated in regulating neuronal axon guidance and in patterning of the developing nervous system and may also serve a patterning and compartmentalization role outside of the nervous system as well. The ligands are all membrane-attached, and this attachment appears to be crucial for their normal function; five of the known ligands are linked to the membrane via a glycosyl phosphotidylinositol (GPI) linkage, while two of the ligands are transmembrane proteins. Despite the large number of Eph family receptors and ligands, they can be divided into just two major subclasses based on their binding specificities. All the GPI-anchored ligands bind and activate one subclass of the Eph receptors (that represented by Eck) while the two transmembrane ligands bind and activate the other major subclass of receptors (represented by Elk). Here we report the identification and characterization of the third, and most divergent, member of the transmembrane group of Eph ligands, which we term Elk-L3 (Elk-related receptor ligand number 3). Elk-L3 is notable for its remarkably restricted and prominent expression in the floor plate and roof plate of the developing neural tube and its rhombomere-specific expression in the developing hindbrain. The Elk-L3 gene has been localized to mouse chromosome 11 and human chromosome 17.

EphA2 promotes infiltrative invasion of glioma stem cells in vivo through cross-talk with Akt and regulates stem cell properties.

Diffuse infiltrative invasion is a major cause for the dismal prognosis of glioblastoma multiforme (GBM), but the underlying mechanisms remain incompletely understood. Using human glioma stem cells (GSCs) that recapitulate the invasive propensity of primary GBM, we find that EphA2 critically regulates GBM invasion in vivo. EphA2 was expressed in all seven GSC lines examined, and overexpression of EphA2 enhanced intracranial invasion. The effects required Akt-mediated phosphorylation of EphA2 on serine 897. In vitro the Akt-EphA2 signaling axis is maintained in the absence of ephrin-A ligands and is disrupted upon ligand stimulation. To test whether ephrin-As in tumor microenvironment can regulate GSC invasion, the newly established Efna1;Efna3;Efna4 triple knockout mice (TKO) were used in an ex vivo brain slice invasion assay. We observed significantly increased GSC invasion through the brain slices of TKO mice relative to wild-type (WT) littermates. Mechanistically EphA2 knockdown suppressed stem cell properties of GSCs, causing diminished self-renewal, reduced stem marker expression and decreased tumorigenicity. In a subset of GSCs, the reduced stem cell properties were associated with lower Sox2 expression. Overexpression of EphA2 promoted stem cell properties in a kinase-independent manner and increased Sox2 expression. Disruption of Akt-EphA2 cross-talk attenuated stem cell marker expression and neurosphere formation while having minimal effects on tumorigenesis. Taken together, the results show that EphA2 endows invasiveness of GSCs in vivo in cooperation with Akt and regulates glioma stem cell properties.

Distribution of Eph-related molecules in the developing and mature cochlea.

Receptors and ligands of the Eph family have recently been shown to influence the development of a variety of tissues. In the present study, the temporal and spatial distribution of Eph receptors and ligands were investigated in the embryonic and postnatal cochlea using Northern blot and immunohistochemical analysis. The results of Northern blot experiments revealed that a large number of Eph family members were present in embryonic cochlear and vestibular ganglia. Immunohistochemical studies revealed that ligands and receptors of the GPI subclass were distributed in complementary patterns within the differentiating spiral limbus, inner sulcus and outer sulcus. The distribution of these molecules became more restricted beginning in the first postnatal week. In contrast, members of the transmembrane subclass of Eph ligands were largely associated with cochlear neurons and their target hair cells. Expression of these ligands appeared to increase during the second postnatal week, corresponding to the period of peripheral nerve fiber reorganization in the cochlea. Together, these studies suggest that multiple Eph family members play unique roles in formation of the cochlea.

Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting.

Genetic deletion studies have shown that haploinsufficiency of Delta-like ligand (Dll) 4, a transmembrane ligand for the Notch family of receptors, results in major vascular defects and embryonic lethality. To better define the role of Dll4 during vascular growth and differentiation, we selected the postnatal retina as a model because its vasculature develops shortly after birth in a highly stereotypic manner, during which time it is accessible to experimental manipulation. We report that Dll4 expression is dynamically regulated by VEGF in the retinal vasculature, where it is most prominently expressed at the leading front of actively growing vessels. Deletion of a single Dll4 allele or pharmacologic inhibition of Dll4/Notch signaling by intraocular administration of either soluble Dll4-Fc or a blocking antibody against Dll4 all produced the same set of characteristic abnormalities in the developing retinal vasculature, most notably enhanced angiogenic sprouting and increased endothelial cell proliferation, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. In a model of ischemic retinopathy, Dll4 blockade also enhanced angiogenic sprouting and regrowth of lost retinal vessels while suppressing ectopic pathological neovascularization. Our data demonstrate that Dll4 is induced by VEGF as a negative feedback regulator and acts to prevent overexuberant angiogenic sprouting, promoting the timely formation of a well differentiated vascular network.

Ephrins and their receptors: a repulsive topic?

This review gives a historical perspective to the field of ephrins and their receptors and offers speculation regarding the possible functions of this family in axonal guidance.

Distinct and overlapping expression patterns of ligands for Eph-related receptor tyrosine kinases during mouse embryogenesis.

Recent studies have implicated Eph-related receptor tyrosine kinases and their membrane-bound ligands in restricting or stimulating the movement of cells and axons. Members of these large families of receptors and ligands fall into two major binding specificity classes, in which the GPI-anchored subgroup of ligands can each bind to all members of a subgroup of receptors, whereas the transmembrane ligands interact with a distinct subgroup of receptors. Analysis of expression patterns is therefore important in order to understand which receptor-ligand interactions occur in vivo. We have cloned mouse orthologues of five members of the ligand family and analysed in detail their developmental expression, in comparison with each other, and with the receptor specificity class they can interact with. We find that B61, AL-1/RAGS, LERK4, and ELF-1, members of the GPI-anchored subgroup of ligands, have both distinct and overlapping aspects to their expression in early mesoderm, somites, and branchial arches; in complex, dynamic patterns in the limb; and in spatial domains and specific neurons in the CNS. Similarly, Elk-L is expressed in hindbrain segments, the roof plate, and floor plate, which overlaps with that of other transmembrane ligands, but has distinct expression in somites. The expression domains of ligands are complementary to those of the corresponding receptors in a number of tissues, including the midbrain, hindbrain, and differentiating limbs, consistent with potential roles in restricting cell movement. In addition, we find that there are some overlaps in expression of receptors and ligands, for example in somites and the early limb. Taken together with previous studies showing that Eph-related receptors also have distinct but overlapping expression patterns, these data indicate that each ligand may have stage- and tissue-specific interactions with an individual member or multiple members of the receptor family.

Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras.

Activation of receptor tyrosine kinases such as those for epidermal growth factor (EGF), platelet-derived growth factor, or nerve growth factor converts the inactive, GDP-bound form of Ras to the active, GTP-bound form, and a dominant negative mutant of Ras interferes with signalling from such receptors. The mechanisms by which receptor tyrosine kinases and Ras are coupled, however, are not well understood. Many cytoplasmic proteins regulated by such receptors contain Src-homology (SH) 2 and 3 domains, and the SH2- and SH3-containing protein Grb2, like its homologue from Caenorhabditis elegans, Sem-5, appears to play an important role in the control of Ras by receptor tyrosine kinases. Here we show that overexpression of Grb2 potentiates the EGF-induced activation of Ras and mitogen-activated protein kinase by enhancing the rate of guanine nucleotide exchange on Ras. Cellular Grb2 appears to form a complex with a guanine-nucleotide-exchange factor for Ras, which binds to the ligand-activated EGF receptor, allowing the tyrosine kinase to modulate Ras activity.

Axon guidance in the mouse optic chiasm: retinal neurite inhibition by ephrin "A"-expressing hypothalamic cells in vitro.

In the mammalian visual system, retinal axons undergo temporal and spatial rearrangements as they project bilaterally to targets on the brain. Retinal axons cross the neuraxis to form the optic chiasm on the hypothalamus in a position defined by overlapping domains of regulatory gene expression. However, the downstream molecules that direct these processes remain largely unknown. Here we use a novel in vitro paradigm to study possible roles of the Eph family of receptor tyrosine kinases in chiasm formation. In vivo, Eph receptors and their ligands distribute in complex patterns in the retina and hypothalamus. In vitro, retinal axons are inhibited by reaggregates of isolated hypothalamic, but not dorsal diencephalic or cerebellar cells. Furthermore, temporal retinal neurites are more inhibited than nasal neurites by hypothalamic cells. Addition of soluble EphA5-Fc to block Eph "A" subclass interactions decreases both the inhibition and the differential response of retinal neurites by hypothalamic reaggregates. These data show that isolated hypothalamic cells elicit specific, position-dependent inhibitory responses from retinal neurites in culture. Moreover, these responses are mediated, in part, by Eph interactions. Together with the in vivo distributions, these data suggest possible roles for Eph family members in directing retinal axon growth and/or reorganization during optic chiasm formation.

Vascular-specific growth factors and blood vessel formation.

A recent explosion in newly discovered vascular growth factors has coincided with exploitation of powerful new genetic approaches for studying vascular development. An emerging rule is that all of these factors must be used in perfect harmony to form functional vessels. These new findings also demand re-evaluation of therapeutic efforts aimed at regulating blood vessel growth in ischaemia, cancer and other pathological settings.

Stromal cells expressing ephrin-B2 promote the growth and sprouting of ephrin-B2(+) endothelial cells.

Ephrin-B2 is a transmembrane ligand that is specifically expressed on arterial endothelial cells (ECs) and surrounding cells and interacts with multiple EphB class receptors. Conversely, EphB4, a specific receptor for ephrin-B2, is expressed on venous ECs, and both ephrin-B2 and EphB4 play essential roles in vascular development. The bidirectional signals between EphB4 and ephrin-B2 are thought to be specific for the interaction between arteries and veins and to regulate cell mixing and the making of particular boundaries. However, the molecular mechanism during vasculogenesis and angiogenesis remains unclear. Manipulative functional studies were performed on these proteins in an endothelial cell system. Using in vitro stromal cells (OP9 cells) and a paraaortic splanchnopleura (P-Sp) coculture system, these studies found that the stromal cells expressing ephrin-B2 promoted vascular network formation and ephrin-B2(+) EC proliferation and that they also induced the recruitment and proliferation of alpha-smooth muscle actin (alpha-SMA)-positive cells. Stromal cells expressing EphB4 inhibited vascular network formation, ephrin-B2(+) EC proliferation, and alpha-SMA(+) cell recruitment and proliferation. Thus, these data suggest that ephrin-B2 and EphB4 mediate reciprocal interactions between arterial and venous ECs and surrounding cells to form each characteristic vessel. (Blood. 2001;98:1028-1037)

Bidirectional signalling through the EPH-family receptor Nuk and its transmembrane ligands.

Receptor tyrosine kinases of the EPH class have been implicated in the control of axon guidance and fasciculation, in regulating cell migration, and in defining compartments in the developing embryo. Efficient activation of EPH receptors generally requires that their ligands be anchored to the cell surface, either through a transmembrane (TM) region or a glycosyl phosphatidylinositol (GPI) group. These observations have suggested that EPH receptors can transduce signals initiated by direct cell-cell interaction. Genetic analysis of Nuk, a murine EPH receptor that binds TM ligands, has raised the possibility that these ligands might themselves have a signalling function. Consistent with this, the three known TM ligands have a highly conserved cytoplasmic region, with multiple potential sites for tyrosine phosphorylation. Here we show that challenging cells that express the TM ligands Elk-L or Htk-L with the clustered ectodomain of Nuk induces phosphorylation of the ligands on tyrosine, a process that can be mimicked both in vitro and in vivo by an activated Src tyrosine kinase. Co-culture of cells expressing a TM ligand with cells expressing Nuk leads to tyrosine phosphorylation of both the ligand and Nuk. These results suggest that the TM ligands are associated with a tyrosine kinase, and are inducibly phosphorylated upon binding Nuk, in a fashion reminiscent of cytokine receptors. Furthermore, we show that TM ligands, as well as Nuk, are phosphorylated on tyrosine in mouse embryos, indicating that this is a physiological process. EPH receptors and their TM ligands therefore mediate bidirectional cell signalling.

Eph family receptors and their ligands distribute in opposing gradients in the developing mouse retina.

The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal-low nasal and high ventral-low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.

Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion.

Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development.

Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells.

Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, we have expressed the EphB2 receptor (formerly Nuk/Cek5/Sek3) in neuronal NG108-15 cells, and have observed the tyrosine phosphorylation of multiple cellular proteins upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induced the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn formed a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also bound through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexed with activated EphB2 in vivo. We have localized an in vitro RasGAP-binding site to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2, and demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signalling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the + 3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signalling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture.