Homotypic receptor-receptor interactions regulating Eph signaling

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, and mediate many other cell-cell communication events. The Ephs and ephrins both localize to the plasma membrane and, upon cell-cell contact, form extensive signaling assemblies at the contact sites. Recent structural, biochemical and cell-biological studies revealed that these assemblies are generated not only via Eph-ephrin interactions, but also via homotypic interactions between neighboring receptor molecules. In addition, Eph-Eph interactions mediate receptor pre-clustering, which ensures fast and efficient activation once ligands come into contact range. Here we summarize the current knowledge about the homotypic Eph-Eph interactions and discuss how they could modulate the initiation of Eph/ephrin signaling.     

Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph-Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.     

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.     

Eph/ephrin signaling in epidermal differentiation and disease

Eph receptor tyrosine kinases mediate cell-cell communication by interacting with ephrin ligands residing on adjacent cell surfaces. In doing so, these juxtamembrane signaling complexes provide important contextual information about the cellular microenvironment that helps orchestrate tissue morphogenesis and maintain homeostasis. Eph/ephrin signaling has been implicated in various aspects of mammalian skin physiology, with several members of this large family of receptor tyrosine kinases and their ligands present in the epidermis, hair follicles, sebaceous glands, and underlying dermis. This review focuses on the emerging role of Eph receptors and ephrins in epidermal keratinocytes where they can modulate proliferation, migration, differentiation, and death. The activation of Eph receptors by ephrins at sites of cell-cell contact also appears to play a key role in the maturation of intercellular junctional complexes as keratinocytes move out of the basal layer and differentiate in the suprabasal layers of this stratified, squamous epithelium. Furthermore, alterations in the epidermal Eph/ephrin axis have been associated with cutaneous malignancy, wound healing defects and inflammatory skin conditions. These collective observations suggest that the Eph/ephrin cell-cell communication pathway may be amenable to therapeutic intervention for the purpose of restoring epidermal tissue homeostasis and integrity in dermatological disorders.     

Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation

The Eph receptors and their ephrin ligands play crucial roles in a large number of cell–cell interaction events, including those associated with axon pathfinding, neuronal cell migration and vasculogenesis. They are also involved in the patterning of most tissues and overall cell positioning in the development of the vertebrate body plan. The Eph/ephrin signaling system manifests several unique features that differentiate it from other receptor tyrosine kinases, including initiation of bi-directional signaling cascades and the existence of ligand and receptor subclasses displaying promiscuous intra-subclass interactions, but very rare inter-subclass interactions. In this review we briefly discuss these features and focus on recent studies of the unique and expansive high-affinity Eph/ephrin assemblies that form at the sites of cell–cell contact and are required for Eph signaling initiation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Tissue transglutaminase clusters soluble A-type ephrins into functionally active high molecular weight oligomers

The Eph receptors and their ligands, the ephrins, are thought to act at points of close cell-cell contact to elicit bi-directional signaling in receptor and ligand expressing cells. However, when cultured in vitro, some A-type ephrins are released from the cell surface and it is unclear if these soluble ephrins participate in Eph receptor activation. We show that soluble ephrin A5 is subject to oligomerization. Ephrins A1 and A5 are substrates for a cross-linking enzyme, tissue transglutaminase, which mediates the formation of oligomeric ephrin. Transglutaminase-cross-linked ephrin binds to A-type Eph receptors, stimulates Eph kinase activity, and promotes invasion and migration of HeLa cells. Transglutaminase-mediated oligomerization of soluble ephrin potentially represents a novel mechanism of forward signaling through Eph receptors and may extend the influence of A-type ephrins beyond cell contact mediated signaling.     

Eph receptors and ephrins

Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape, and mobility. Eph signaling is crucial for the development of many tissues and organs including the nervous and cardiovascular systems. Both Ephs and ephrins are membrane-bound and their interactions at sites of cell-cell contact initiate unique bi-directional signaling cascades where information is transduced in both the receptor- and the ligand-expressing cells. Recent studies summarized in this review reveal how the signaling process is triggered upon ligand-receptor binding via the formation of a 2:2 circular heterotetramer. This fixes the orientation of the participating molecules and facilitates phosphorylation of their cytoplasmic domains which then interact with downstream signaling factors. The elucidation of the structural details of Eph-ephrin recognition and binding should yield insight into the future development of novel therapeutic agents targeting cardiovascular function, nerve regeneration, and cancer.     

Eph receptor and ephrin function in breast,gut, and skin epithelia

Epithelial cells are tightly coupled together through specialized intercellular junctions, including adherens junctions, desmosomes, tight junctions, and gap junctions. A growing body of evidence suggests epithelial cells also directly exchange information at cell-cell contacts via the Eph family of receptor tyrosine kinases and their membrane-associated ephrin ligands. Ligand-dependent and -independent signaling via Eph receptors as well as reverse signaling through ephrins impact epithelial tissue homeostasis by organizing stem cell compartments and regulating cell proliferation, migration, adhesion, differentiation, and survival. This review focuses on breast, gut, and skin epithelia as representative examples for how Eph receptors and ephrins modulate diverse epithelial cell responses in a context-dependent manner. Abnormal Eph receptor and ephrin signaling is implicated in a variety of epithelial diseases raising the intriguing possibility that this cell-cell communication pathway can be therapeutically harnessed to normalize epithelial function in pathological settings like cancer or chronic inflammation.     

Eph receptor and ephrin function in breast,gut, and skin epithelia

Epithelial cells are tightly coupled together through specialized intercellular junctions, including adherens junctions, desmosomes, tight junctions, and gap junctions. A growing body of evidence suggests epithelial cells also directly exchange information at cell-cell contacts via the Eph family of receptor tyrosine kinases and their membrane-associated ephrin ligands. Ligand-dependent and -independent signaling via Eph receptors as well as reverse signaling through ephrins impact epithelial tissue homeostasis by organizing stem cell compartments and regulating cell proliferation, migration, adhesion, differentiation, and survival. This review focuses on breast, gut, and skin epithelia as representative examples for how Eph receptors and ephrins modulate diverse epithelial cell responses in a context-dependent manner. Abnormal Eph receptor and ephrin signaling is implicated in a variety of epithelial diseases raising the intriguing possibility that this cell-cell communication pathway can be therapeutically harnessed to normalize epithelial function in pathological settings like cancer or chronic inflammation.     

Ectodomain structures of Eph receptors

Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are important mediators of cell-cell communication that regulate axon guidance, long-term potentiation, and stem cell development, among others. By now, many Eph receptors and ephrins have also been found to play important roles in the progression of cancer. Since both the receptor and the ligand are membrane-bound, their interaction leads to the multimerization of both molecules to distinct clusters within their respective plasma membranes, resulting in the formation of discrete signaling centers. In addition, and unique to Eph receptors and ephrins, their interaction initiates bi-directional signaling cascades where information is transduced in the direction of both the receptor- and the ligand-bearing cells. The Ephs and the ephrins are divided into two subclasses, A and B, based on their affinities for each other and on sequence conservation. Crystal structures and other biophysical studies have indicated that isolated extracellular Eph and ephrin domains initially form high-affinity heterodimers around a hydrophobic loop of the ligand that is buried in a hydrophobic pocket on the surface of the receptor. The dimers can then further arrange by weaker interactions into higher-order Eph/ephrin clusters observed in vivo at the sites of cell-cell contact. Although the hetero-dimerization is a universal way to initiate signaling, other extracellular domains of Ephs are involved in the formation of higher-order clusters. The structures also show important differences defining the unique partner preferences of the two ligand and receptor subclasses, namely, how subclass specificity is determined both by individual interacting residues and by the precise architectural arrangement of ligands and receptors within the complexes.     

The ephrins and Eph receptors in angiogenesis.

Eph receptors are a unique family of receptor tyrosine kinases that play critical roles in embryonic patterning, neuronal targeting, vascular development and adult neovascularization. Engagement of Eph receptors by ephrin ligands mediates critical steps of angiogenesis, including juxtacrine cell-cell contacts, cell adhesion to extracellular matrix, and cell migration. Recent evidence from in vitro angiogenesis assays and analysis of mice deficient for one or more members of the Eph family establishes the role of Eph signaling in sprouting angiogenesis and blood vessel remodeling during vascular development. Furthermore, elevated expression of Eph receptors and ephrin ligands is associated with tumors and associated tumor vasculature, suggesting that Eph receptors and their ephrin ligands also play critical roles in tumor angiogenesis and tumor growth. This review will focus on the relevance of Eph receptor signaling in embryonic and adult neovascularization, and possible contributions to tumor growth and metastasis.     

The role of proteases in regulating Eph/ephrin signaling

Proteases regulate a myriad of cell functions, both in normal and disease states. In addition to protein turnover, they regulate a range of signaling processes, including those mediated by Eph receptors and their ephrin ligands. A variety of proteases is reported to directly cleave Ephs and/or ephrins under different conditions, to promote receptor and/or ligand shedding, and regulate receptor/ligand internalisation and signaling. They also cleave other adhesion proteins in response to Eph-ephrin interactions, to indirectly facilitate Eph-mediated functions. Proteases thus contribute to Eph/ephrin mediated changes in cell-cell and cell-matrix interactions, in cell morphology and in cell migration and invasion, in a manner which appears to be tightly regulated by, and co-ordinated with, Eph signaling. This review summarizes the current literature describing the function and regulation of protease activities during Eph/ephrin-mediated cell signaling.     

The role of proteases in regulating Eph/ephrin signaling

Proteases regulate a myriad of cell functions, both in normal and disease states. In addition to protein turnover, they regulate a range of signaling processes, including those mediated by Eph receptors and their ephrin ligands. A variety of proteases is reported to directly cleave Ephs and/or ephrins under different conditions, to promote receptor and/or ligand shedding, and regulate receptor/ligand internalisation and signaling. They also cleave other adhesion proteins in response to Eph-ephrin interactions, to indirectly facilitate Eph-mediated functions. Proteases thus contribute to Eph/ephrin mediated changes in cell-cell and cell-matrix interactions, in cell morphology and in cell migration and invasion, in a manner which appears to be tightly regulated by, and co-ordinated with, Eph signaling. This review summarizes the current literature describing the function and regulation of protease activities during Eph/ephrin-mediated cell signaling.     

Cell-cell signaling via Eph receptors and ephrins

Eph receptors are the largest subfamily of receptor tyrosine kinases regulating cell shape, movements, and attachment. The interactions of the Ephs with their ephrin ligands are restricted to the sites of cell-cell contact since both molecules are membrane attached. This review summarizes recent advances in our understanding of the molecular mechanisms underlining the diverse functions of the molecules during development and in the adult organism. The unique properties of this signaling system that are of highest interest and have been the focus of intense investigations are as follows: (i) the signal is simultaneously transduced in both ligand-expressing cells and receptor-expressing cells, (ii) signaling via the same molecules can generate opposing cellular reactions depending on the context, and (iii) the Ephs and the ephrins are divided into two subclasses with promiscuous intrasubclass interactions, but rarely observed intersubclass interactions.     

EphA receptor signaling--complexity and emerging themes

The impact of Eph and ephrin signaling on cell behavior is complex and highly context dependent. Forward signaling initiated by Eph receptor activation and reverse signaling initiated by ephrin activation often mediate opposite effects. The apparent ligand-independent functions of Eph receptors recognized recently add another layer of complexity. This review will attempt to sort out the information generated recently on signaling by the A subfamily of Eph receptors and ephrin ligands. We will focus on EphA/ephrin-A signaling in the context of several physiological and disease processes, where new progresses have been made lately and unifying themes are emerging amid previous confusions. For more comprehensive survey of literature on Eph/ephrin signaling pathways and networks, readers are referred to outstanding reviews both in this volume and in other recent publications.     

Ligand recognition by A‐class Eph receptors: crystal structures of the EphA2 ligand‐binding domain and the EphA2/ephrin‐A1 complex

Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B‐class receptors. Here, we present the crystal structures of an A‐class complex between EphA2 and ephrin‐A1 and of unbound EphA2. Although these structures are similar overall to their B‐class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A‐class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a ‘lock‐and‐key’‐type binding mechanism, in contrast to the ‘induced fit’ mechanism defining the B‐class molecules. This model is supported by structure‐based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell‐based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2‐specific homotypic cell adhesion interactions.     

Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.     

Association of Dishevelled with Eph tyrosine kinase receptor and ephrin mediates cell repulsion

Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, we discovered that Xenopus Dishevelled (Xdsh) forms a complex with Eph receptors and ephrin-B ligands and mediates the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affected the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induced the activation of RhoA and Rho kinase in the EphB2-overexpressed cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. We propose that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity.