EphB-EphrinB Signaling
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EphB-EphrinB Signaling
The Eph family forms the largest group of RTKs (Receptor Tyrosine Kinases) comprising 14 members in mammals that play critical roles in diverse biological processes during development as well as in the mature animal. They are activated by membrane-bound ligands called Ephrins, which are classified into two subclasses based on their mode of membrane anchorage. The Ephrin-A ligands are GPI (Glycosylphosphatidylinositol)-linked and prefer to bind to EphA Receptors. The Ephrin-B ligands (Ephrin-B1-B3), which possess a transmembrane moiety and a short cytoplasmic domain, bind to EphB Receptors (EphB1-B6). The interactions between Ephrins and Eph Receptors are generally promiscuous within each subclass (Ref.1). Upon stimulation by Ephrin ligands, Eph Receptors activate signaling cascades in various biological systems. Interactions between Ephs and Ephrins are implicated in repulsive axon guidance, cell migration, topographic mapping, and angiogenesis. Besides their functions in development, Ephs and Ephrins are implicated in the regulation of NMDA (N-Methyl D-Aspartate)-dependent synaptic function in mature synapses. The expression of EphB2 is regulated in an activity-dependent manner by stimuli known to induce synaptic plasticity.

The Eph Receptor extracellular domain is composed of the ligand-binding Glob (Globular domain), a Cys (Cystein)-rich region and two Fn (Fibronectin) Type-III repeats. The cytoplasmic part of Eph Receptors is divided into four functional units; the juxtamembrane domain that contains two conserved residues, a classical protein tyrosine kinase domain (Eph Kinase), a SAM (Sterile Alpha Motif) and PBM (PDZ-domain Binding Motif). Upon the formation of cell-cell contact, signaling through the Eph Receptors results in modulation of integrin activity and reorganization of the actin cytoskeleton. As a result, Ephs generate adhesive or repulsive signals, and in the neural system guide the movement of axonal growth cones, cell migration, and synapse formation (Ref.2). On ligand engagement each member of the receptor dimmer autophosphorylate several tyrosine residues that is located in the intracellular part of the partner receptor. Autophosphorylation of juxtamembrane tyrosine residues is required for full activation of the protein tyrosine kinase domain of the receptor. Once the receptor is activated, adaptor molecules SH2, SH3 and GRB4 (Growth Factor Receptor Bound protein-4) associate with it to transmit signals into the cell. The signaling pathways following Ephrin-B activation is classified into the phosphotyrosine-dependent pathway and PDZ-dependent pathway. In the former, Ephrin-B is tyrosine-phosphorylated upon interaction with EphB Receptors by the SFK (Src Family Kinases). On the other hand, LMW-PTP (Low Molecular Weight Protein Tyrosine Phosphatase) is recruited to the activated Ephrin-B1 in a delayed kinetics via interaction between the PBM of PTP and the carboxyl tail of Ephrin-B1, after which Ephrin-B1 is dephosphorylated and the phosphotyrosine-dependent reverse signaling is switched off. The tyrosine phosphorylation of Ephrin-B1 results in the binding of the adaptor protein GRB4 via interaction with its SH2 domain. The latter in turn recruits various signaling molecules such as FAK (Focal Adhesion Kinase), Axin, Abi1 (Abl interacting protein-1), CAP (Cbl Associated Protein) and Cbl to the Ephrin-B signaling complexes via its multiple SH3 domains, eventually leading to disassembly of F-Actin and rounding cell morphology (Ref.3). Unlike other RTKs, EphB2 induces a pronounced downregulation of GTP-bound H-Ras and consequently of the MAPK (Mitogen Activated Protein Kinase)/ERK (Extracellular Signal-Regulated Kinase) pathway. EphB2 inhibits cell adhesion through phosphorylation of RRas (member of the Ras family of small GTPases), which suppresses the ability of RRas to support integrin activity. EphB Receptors interact with Intersectin and Kalirin group of exchange factors for Rho family GTPases. Intersectin activates CDC42 and Kalirin, an exchange factor for Rac, co-clusters with activated EphB2 and localize activated Rac to sites of EphB-Ephrin interaction without changing the overall level of Rac activation. These interactions regulate the EphB Receptor-mediated morphogenesis and maturation of dendritic spines in cultured hippocampal and cortical neurons (Ref.4).

Ephrin-Bs can also initiate reverse signaling through PDZ-domain-mediated associations. The GTPase-activating protein PDZ-RGS3, which catalyzes the hydrolysis of GTP to GDP in the G-Alpha subunits of heterotrimeric G-Proteins, binds to the PDZ-binding motif of Ephrin-B molecules. Signaling through PDZ-RGS3 mediates de-adhesion of embryo cells expressing Ephrin-B1. It also inhibits SDF1 (Stromal Cell Derived Factor-1)-mediated cerebellar granule cell chemotaxis through the CXCR4 (Chemokine (C-X-C motif) Receptor-4) GPCR (G-Protein-Coupled Chemokine Receptor). During cerebellar development, Ephrin-B activation by EphB Receptors may attenuate granule cell attraction to SDF1, which is expressed at the pial surface, and allow cells to migrate from the external to the internal granule cell layer (Ref.5). This signaling mechanism has broad implications for cell migratory behavior in other systems as well. Additionally, Ephrin-B1 reverse signal\ling alters the subcellular localization and/or tyrosine-phosphorylation levels of other proteins involved in cytoskeletal regulation, including Paxillin, FAK, PAK1 and Rho GTPases including Rac and CDC42. HNRNPK (Heterogeneous Ribonucleoprotein-K) specifically interacts with the SH3 domain of Vav and translates Ephrin signals through Vav activation into regulatory events on the level of gene expression.

Eph Receptors and their ligands have been implicated in developmental patterning events, including assembly of the vasculature, retinotectal axonal targeting, and developmental segmentation of embryonic tissues into boundary zones defined by reciprocal spatial gradients of ligands and their receptors. Eph proteins also play a critical role in the cellular organization and function of non-neural tissues. In the cardiovascular system, Ephrin-B2 and EphB4 are preferentially expressed on arterial and venous endothelium, respectively (Ref.6). EphB2, EphB3 and Ephrin-B1 have also been implicated in embryonic vascularization and their localization suggests that signaling occurs not only between the arterial and venous compartments but also between endothelial cells and the surrounding mesenchyme. Intriguingly, EphB proteins may also contribute to the organization of the vascular network by mediating neuro-arterial interactions. Eph proteins probably regulate angiogenic processes associated with tumor growth. EphB4 and Ephrin-B2 also play a role in erythropoiesis by influencing hematopoietic cell lineages that share a common ancestry with endothelial cells (Ref.7). Furthermore, Eph proteins play a role in platelet clustering and hence may be important for blood clotting at sites of vascular injury.