UniPR129 is a competitive small molecule Eph-ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations.

BACKGROUND AND PURPOSE: The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph-ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance. EXPERIMENTAL APPROACH: UniPR129 (the L-homo-Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin-A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2-ephrin-A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti-angiogenic effect was tested in vitro using cultures of HUVECs. KEY RESULTS: UniPR129 disrupted EphA2-ephrin-A1 interaction with Ki = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects. CONCLUSIONS AND IMPLICATIONS: The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph-ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound.

EphA4 constitutes a population-specific guidance cue for motor neurons.

Motor neurons in the ventral neural tube project axons specifically to their target muscles in the periphery. Although many of the transcription factors that specify motor neuron cell fates have been characterized, less is understood about the mechanisms that guide motor axons to their correct targets. We show that ectopic expression of EphA4 receptor tyrosine kinase alters the trajectories of a specific population of motor axons in the avian hindlimb. Most motor neurons in the medial portion of the lateral motor column (LMC) extend their axons aberrantly in the dorsal nerve trunk at the level of the crural plexus, in the presence of ectopic EphA4. This misrouting of motor axons is not accompanied by alterations in motor neuron identity, settling patterns in the neural tube, or the fasciculation of spinal nerves. However, ectopic EphA4 axons do make errors in pathway selection during sorting in the plexus at the base of the hindlimb. These results suggest that EphA4 in motor neurons acts as a population-specific guidance cue to control the dorsal trajectory of their axons in the hindlimb.

EphA4/ephrin-A5 interactions in muscle precursor cell migration in the avian forelimb.

Limb muscles derive from muscle precursor cells that lie initially in the lateral portion of the somitic dermomyotome and subsequently migrate to their target limb regions, where muscle-specific gene transcription is initiated. Although several molecules that control the generation and delamination of muscle precursor cells have been identified, little is known about the mechanisms that guide muscle precursor cell migration in the limb. We have examined the distribution of members of the Eph family during muscle precursor cell development. The EphA4 receptor tyrosine kinase and its ligand, ephrin-A5, are expressed by muscle precursor cells and forelimb mesoderm in unique spatiotemporal patterns during the period when muscle precursors delaminate from the dermomyotome and migrate into the limb. To test the function of EphA4/ephrin-A5 interactions in muscle precursor migration, we used targeted in ovo electroporation to express ephrin-A5 ectopically specifically in the presumptive limb mesoderm. In the presence of ectopic ephrin-A5, Pax7-positive muscle precursor cells are significantly reduced in number in the proximal limb, compared with controls, and congregate abnormally near the lateral dermomyotome. In stripe assays, isolated muscle precursor cells avoid substrate-bound ephrin-A5 and this avoidance is abolished by addition of soluble ephrin-A5. These data suggest that ephrin-A5 normally restricts migrating, EphA4-positive muscle precursor cells to their appropriate territories in the forelimb, disallowing entry into abnormal embryonic regions.

EphB/syndecan-2 signaling in dendritic spine morphogenesis.

We previously reported that the cell surface proteoglycan syndecan-2 can induce dendritic spine formation in hippocampal neurons. We demonstrate here that the EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and that this phosphorylation event is crucial for syndecan-2 clustering and spine formation. Syndecan-2 is tyrosine phosphorylated and forms a complex with EphB2 in mouse brain. Dominant-negative inhibition of endogenous EphB receptor activities blocks clustering of endogenous syndecan-2 and normal spine formation in cultured hippocampal neurons. This is the first evidence that Eph receptors play a physiological role in dendritic spine morphogenesis. Our observations suggest that spine morphogenesis is triggered by the activation of Eph receptors, which causes tyrosine phosphorylation of target molecules, such as syndecan-2, in presumptive spines.

Multiple signaling interactions of Abl and Arg kinases with the EphB2 receptor.

The Eph family of receptor tyrosine kinases and the Abl family of non-receptor tyrosine kinases have both been implicated in tissue morphogenesis. They regulate the organization of the actin cytoskeleton in the developing nervous system and participate in signaling pathways involved in axon growth. Both Eph receptors and Abl are localized in the neuronal growth cone, suggesting that they play a role in axon pathfinding. Two-hybrid screens identified regions of Abl and Arg that bind to the EphB2 and EphA4 receptors, suggesting a novel signaling connection involving the two kinase families. The association of full-length Abl and Arg with EphB2 was confirmed by co-immunoprecipitation and found to involve several distinct protein interactions. The SH2 domains of Abl and Arg bind to tyrosine-phosphorylated motifs in the juxtamembrane region of EphB2. A second, phosphorylation-independent interaction with EphB2 involves non-conserved sequences in the C-terminal tails of Abl and Arg. A third interaction between Abl and EphB2 is probably mediated by an intermediary protein because it requires tyrosine phosphorylation of EphB2, but not the binding sites for the Abl SH2 domain. The connection between EphB2 and Abl/Arg appears to be reciprocal. Activated EphB2 causes tyrosine phosphorylation of Abl and Arg, and vice versa. Interestingly, treatment of COS cells and B35 neuronal-like cells with ephrin-B1 to activate endogenous EphB2 decreased the kinase activity of endogenous Abl. These data are consistent with the opposite effects that Eph receptors and Abl have on neurite ougrowth and suggest that Eph receptors and Abl family kinases have shared signaling activities.

In vivo tyrosine phosphorylation sites of activated ephrin-B1 and ephB2 from neural tissue.

EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 previously identified as phosphorylation sites in 293 cells (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38, 14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex.

Ephrin-A6, a new ligand for EphA receptors in the developing visual system.

In the embryonic visual system, EphA receptors are expressed on both temporal and nasal retinal ganglion cell axons. Only the temporal axons, however, are sensitive to the low concentrations of ephrin-A ligands found in the anterior optic tectum. The poor responsiveness of nasal axons to ephrin-A ligands, which allows them to traverse the anterior tectum and reach their targets in the posterior tectum, has been attributed to constitutive activation of the EphA4 receptor expressed in these axons. EphA4 is highly expressed throughout the retina, but is preferentially phosphorylated on tyrosine (activated) in nasal retina. In a screen for EphA4 ligands expressed in chicken embryonic retina, we have identified a novel ephrin, ephrin-A6. Like ephrin-A5, ephrin-A6 has high affinity for EphA4 and activates this receptor in cultured retinal cells. In the embryonic day 8 (E8) chicken visual system, ephrin-A6 is predominantly expressed in the nasal retina and ephrin-A5 in the posterior tectum. Thus, ephrin-A6 has the properties of a ligand that activates the EphA4 receptor in nasal retinal cells. Ephrin-A6 binds with high affinity to several other EphA receptors as well and causes growth cone collapse in retinal explants, demonstrating that it can elicit biological responses in retinal neurons. Ephrin-A6 expression is high at E6 and E8, when retinal axons grow to their tectal targets, and gradually declines at later developmental stages. The asymmetric distribution of ephrin-A6 in retinal cells, and the time course of its expression, suggest that this new ephrin plays a role in the establishment of visual system topography.

Eph receptors and ephrin ligands: embryogenesis to tumorigenesis.

Protein tyrosine kinase genes are the largest family of oncogenes. This is not surprising since tyrosine kinases are important components of signal transduction pathways that control cell shape, proliferation, differentiation, and migration. At 14 distinct members, the Eph kinases constitute the largest family of receptor tyrosine kinases. Although they have been most intensively studied for their roles in embryonic development, increasing evidence also implicates Eph family proteins in cancer. This review will address the recent progress in understanding the function of Eph receptors in normal development and how disregulation of these functions could promote tumorigenesis.

Developmental regulation of EphA4 expression in the chick auditory brainstem.

The avian auditory brainstem nuclei nucleus magnocellularis (NM) and nucleus laminaris (NL) display highly precise patterns of neuronal connectivity. NM projects tonotopically to the dorsal dendrites of ipsilateral NL neurons and to the ventral dendrites of contralateral NL neurons. The precision of this binaural segregation is evident at the earliest developmental stage at which connections can be observed. We have begun to examine the possibility that Eph receptor tyrosine kinase signaling is involved in establishing these spatially segregated connections. The expression of the EphA4 tyrosine kinase was examined at several developmental stages. EphA4 is expressed in rhombomere 5, which contains progenitors for both NM and NL. In this rhombomere, the labeling becomes striped during the time that precursor cells migrate to the auditory anlage. At the precise time when NM-NL projections are forming, EphA4 expression in NL is asymmetric, with markedly higher expression in the dorsal NL neuropil than in the ventral neuropil, suggesting a possible role in guiding growing axons to the appropriate region. At later embryonic ages EphA4 expression is symmetric around NL, and is absent in NM. As auditory function matures, EphA4 expression decreases so that by 4 days after hatch no EphA4 antibody labeling is evident in the auditory brainstem nuclei.

The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2.

A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein. NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm.

Eph receptors and ephrins in the developing chick cerebellum: relationship to sagittal patterning and granule cell migration.

Spatiotemporal expression patterns of six members of the Eph gene family (EphA4, EphA3, EphB2, ephrin-B1, ephrin-A2, and ephrin-A5) were characterized immunocytochemically at various stages of chick cerebellar development. EphA4 expression is observed in the cerebellar anlage as early as embryonic day 5 (E5) and continues in the posthatch cerebellum. During the early period of cerebellar development (E3-E8), complementarity is observed between EphA4 and ephrin-A5 expression within the cerebellar-isthmal region. By E8, differential expression of EphA4 in parasagittal Purkinje cell bands is evident, and the expression remains banded in the posthatch cerebellum. Banded expression of the ephrin-A5 ligand complements EphA4 expression during the middle period (E9-E15). During this period, ephrin-A2 and EphA3 are coexpressed in a banded pattern and with variable correlation to EphA4. Variability in the banding expression is observed for EphA4, EphA3, ephrin-A5, and ephrin-A2 across different lobes, and graded complementarity in the expression pattern of EphA3 and ephrin-A5 is observed in the external granular layer between the posterior and anterior lobes. Analysis of Purkinje cell birth date in correlation with Eph-ephrin expression during the middle period reveals that early-born cells express EphA4, whereas late-born cells express ephrin-A5. Finally, EphA4 expression domains are respected by migrating granule cell ribbons, which express both ephrin-B1 and EphB2. These expression patterns suggest multiple roles for the Eph-ephrin system in cerebellar development, including demarcation/enforcement of boundaries of the cerebellar anlage, formation/maintenance of Purkinje cell compartments, and restriction of the early phase of granule cell migration to ribbons.

Expression of EphA4, ephrin-A2 and ephrin-A5 during axon outgrowth to the hindlimb indicates potential roles in pathfinding.

During neural development, spinal motor axons extend in a precise manner from the ventral portion of the developing spinal cord to innervate muscle targets in the limb. Although classical studies in avians have characterized the cellular interactions that influence motor axon pathfinding to the limb, less is known about the molecular mechanisms that mediate this developmental event. Here, we examine the spatiotemporal distributions of the EphA4 receptor tyrosine kinase (RTK) and its cognate ligands, ephrin-A2 and ephrin-A5, on motor neurons, their axons and their pathways to the avian hindlimb to determine whether these molecules may influence axonal projections. The expression patterns of EphA4, ephrin-A2 and ephrin-A5 mRNAs and proteins are highly complex and appear to exhibit some overlap during motor axon outgrowth and pathfinding to the hindlimb, reminiscent of the co-expression of Eph RTKs and ephrins in the retinotectal system. EphA4, similar to the carbohydrate moiety polysialic acid, strikingly marks the main dorsal, but not ventral, nerve trunk after axon sorting at the limb plexus region. Our results suggest that EphA4 RTK and its ligands may influence axon fasciculation and the sorting of axons at the limb plexus, contributing to the correct dorsoventral organization of nerve branches in the hindlimb.

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half-somite.

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.

Replacing two conserved tyrosines of the EphB2 receptor with glutamic acid prevents binding of SH2 domains without abrogating kinase activity and biological responses.

Eph receptor tyrosine kinases play key roles in pattern formation during embryonic development, but little is known about the mechanisms by which they elicit specific biological responses in cells. Here, we investigate the role of tyrosines 605 and 611 in the juxtamembrane region of EphB2, because they are conserved Eph receptor autophosphorylation sites and demonstrated binding sites for the SH2 domains of multiple signaling proteins. Mutation of tyrosines 605 and 611 to phenylalanine impaired EphB2 kinase activity, complicating analysis of their function as SH2 domain binding sites and their contribution to EphB2-mediated signaling. In contrast, mutation to the negatively charged glutamic acid disrupted SH2 domain binding without reducing EphB2 kinase activity. By using a panel of EphB2 mutants, we found that kinase activity is required for the changes in cell-matrix and cell - cell adhesion, cytoskeletal organization, and activation of mitogen-activated protein (MAP) kinases elicited by EphB2 in transiently transfected cells. Instead, the two juxtamembrane SH2 domain binding sites were dispensable for these effects. These results suggest that phosphorylation of tyrosines 605 and 611 is critical for EphB2-mediated cellular responses because it regulates EphB2 kinase activity.

The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization.

Eph receptor tyrosine kinases and their ephrin ligands have been implicated in embryonic vascular development and in in vivo models of angiogenesis. Eph proteins may also regulate tumor neovascularization, but this role has not been previously investigated. To screen for Eph proteins expressed in tumor blood vessels, we used tumor xenografts grown in nude mice from MDA-MB-435 human breast cancer cells or KS1767 human Kaposi's sarcoma cells. By immunohistochemistry, the ephrin-A1 ligand and one of its receptors, EphA2, were detected throughout tumor vasculature. Double-labeling with anti-CD34 antibodies demonstrated that both ephrin-A1 and EphA2 were expressed in xenograft endothelial cells and also tumor cells. Furthermore, EphA2 was tyrosine-phosphorylated in the xenograft tumors, indicating that it was activated, presumably by interacting with ephrin-A1. Ephrin-A1 and EphA2 were also detected in both the vasculature and tumor cells of surgically removed human cancers. In an in vitro angiogenesis model, a dominant negative form of EphA2 inhibited capillary tube-like formation by human umbilical vein endothelial cells (HUVECs), demonstrating a requirement for EphA receptor signaling. These data suggest that ephrin-A1 and EphA2 play a role in human cancers, at least in part by influencing tumor neovascularization. Eph proteins may represent promising new targets for antiangiogenic cancer treatments.

A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.

The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases.

Multiple in vivo tyrosine phosphorylation sites in EphB receptors.

Autophosphorylation regulates the function of receptor tyrosine kinases. To dissect the mechanism by which Eph receptors transmit signals, we have developed an approach using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to map systematically their in vivo tyrosine phosphorylation sites. With this approach, phosphorylated peptides from receptors digested with various endoproteinases were selectively isolated on immobilized anti-phosphotyrosine antibodies and analyzed directly by MALDI mass spectrometry. Multiple in vivo tyrosine phosphorylation sites were identified in the juxtamembrane region, kinase domain, and carboxy-terminal tail of EphB2 and EphB5, and found to be remarkably conserved between these EphB receptors. A number of these sites were also identified as in vitro autophosphorylation sites of EphB5 by phosphopeptide mapping using two-dimensional chromatography. Only two in vitro tyrosine phosphorylation sites had previously been directly identified for Eph receptors. Our data further indicate that in vivo EphB2 and EphB5 are also extensively phosphorylated on serine and threonine residues. Because phosphorylation at each site can affect receptor signaling properties, the multiple phosphorylation sites identified here for the EphB receptors suggest a complex regulation of their functions, presumably achieved by autophosphorylation as well as phosphorylation by other kinases. In addition, we show that MALDI mass spectrometry can be used to determine the binding sites for Src homology 2 (SH2) domains by identifying the EphB2 phosphopeptides that bind to the SH2 domain of the Src kinase.

An Eph receptor regulates integrin activity through R-Ras.

The ability of integrins to mediate cell attachment to extracellular matrices and to blood proteins is regulated from inside the cell. Increased ligand-binding activity of integrins is critical for platelet aggregation upon blood clotting and for leukocyte extravasation to inflamed tissues. Decreased adhesion is thought to promote tumor cell invasion. R-Ras, a small intracellular GTPase, regulates the binding of integrins to their ligands outside the cell. Here we show that the Eph receptor tyrosine kinase, EphB2, can control integrin activity through R-Ras. Cells in which EphB2 is activated become poorly adherent to substrates coated with integrin ligands, and a tyrosine residue in the R-Ras effector domain is phosphorylated. The R-Ras phosphorylation and loss of cell adhesion are causally related, because forced expression of an R-Ras variant resistant to phosphorylation at the critical site made cells unresponsive to the anti-adhesive effect of EphB2. This is an unusual regulatory pathway among the small GTPases. Reduced adhesiveness induced through the Eph/R-Ras pathway may explain the repulsive effect of the Eph receptors in axonal pathfinding and may facilitate tumor cell invasion and angiogenesis.

Eph receptors and ephrins demarcate cerebellar lobules before and during their formation.

The formation of the ten cerebellar lobules is an unsolved problem in brain development. We report a screen for the four subfamilies of Eph receptors and their ligands (ephrins) in developing mouse cerebellum, using soluble receptor-immunoglobulin and ligand-immunoglobulin fusion proteins, and antibodies against EphA and ephrin-B proteins. Our results identify Eph receptors and ephrins as the first molecules known to demarcate individual lobules during development. Staining for ephrin-A ligands is in lobule VIII as it forms, across the whole width of the cerebellum. Staining for three EphA receptors approximately coincides with presumptive lobules VI and/or VII before and just after birth, whereas a fourth EphA receptor (EphA4, which binds ligands of both subfamilies) has more widespread expression. Staining for EphB receptors is in lobules VII, VIII, and IX. Staining for ephrin-B ligands is much weaker, becomes detectable only after birth, and does not appear to be lobule-specific. Staining for all subfamilies spreads to at least some adjacent lobules as maturation proceeds. The lobule-specific patterns appear before the lobules form, and initially extend across the width of the cerebellum, in spite of the lesser conservation of the lateral extensions of the lobules. These expression patterns define previously unknown developmental units and suggest that Eph family proteins may contribute to cerebellar morphogenesis.

Signal transfer by Eph receptors.

The Eph receptors are a unique family of receptor tyrosine kinases that enforce cellular position in tissues through mainly repulsive signals generated upon cell-cell contact. Together, Eph receptors and their membrane-anchored ligands. the ephrins, are key molecules for establishing tissue organization through signaling pathways that control axonal projection, cell migration, and the maintenance of cellular boundaries. Through their SH2 (Src Homology 2) and PDZ (postsynaptic density protein, disks large, zona occludens) domains, several signaling molecules have been demonstrated to interact with the activated cytoplasmic domain of Eph receptors by using the yeast two-hybrid system and in vitro biochemical assays. Most proteins found to interact with Eph receptors are well-known regulators of cytoskeletal organization and cell adhesion, and also cell proliferation. Promoting growth, however, does not appear to be a primary role of Eph receptors. Explaining which signaling interactions identified for the Eph receptors have physiological significance, how Eph receptor signaling cascades are propagated, and characterizing the intrinsic signaling properties of the ephrins are all exciting questions currently being investigated.