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3. Ephrins and Eph Signaling

Ephrins are membrane-bound ligands of the tyrosine kinase Eph receptors. The tissue expression patterns of Eph receptors and ephrin ligands are complementary, similar to that of semaphorin and their plexin receptors, so that each of the two types of proteins is expressed along the boundaries of apposing domains.60 Ephrins and their receptors participate in determining the body plan of the developing embryo by providing a repulsive signal during embryogenesis which prevents intermingling of structurally and functionally distinct domains. Ephrins are classified into two classes, depending on their interaction with the plasma membrane: class A ephrins (A1–A5) are GPI-anchored, while class B (B1–B3) are single-pass transmembrane proteins. The structure of the Eph receptors and ephrin ligands is described in detail in Chapter 2. Eph receptors generally, though not exclusively, bind only to one class of ephrins, and are classified accordingly (A1–A8, B1–B6). The interactions between Eph receptors and ephrin ligands within each class are promiscuous, with the exception of the EphB4 receptor in the vascular system, which binds with high affinity only to ephrin-B2. Since both receptors and ligands are attached to the plasma membrane, ephrin signaling requires cell-to-cell contact.

Similar to semaphorin and its receptors, Eph and ephrins were initially studied as neuronal guidance proteins (reviewed in Refs. 61 and 62). The role of the Eph-ephrin system in vascular morphogenesis became apparent when disruption of the mouse ephrin-B2 gene was found to result in defective arterial and venous angiogenesis and in embryonic lethality, though vasculogenesis of the major blood vessels was not affected.63 The expression pattern of ephrin-B2 was complementary to that of one of its ligands, EphB4, such that they were expressed preferentially in arteries or in veins, respectively. It was subsequently observed that EphB4 is expressed exclusively in endothelial cells, and that an EphB4 loss-of-function mutation duplicates the ephrin-B2-null phenotype.64 As discussed in more detail in Chapter 2, the reciprocal expression of ephrin-B2 and EphB4 functions as a mechanism for defining the identity of arteries and veins and for maintaining the boundary between them, as well as between blood vessels and their

surrounding tissue.63,65,66 This is similar to the function of the Eph receptor-ephrin ligand system in the nervous system, where it marks the boundary between the developing nerves and their surrounding tissue, thus constituting a guidance mechanism.61

The roles of receptor versus ligand are interchangeable in Ephephrin signaling, since ephrin also transduces intracellular signals upon engagement by Eph (reviewed in Ref. 67) (Fig. 2). Thus Eph receptors initiate forward signaling, while ephrins give rise to reverse signaling. This bidirectional signaling is similar to the interaction between plexA1 and the transmembrane semaphorin sema6D.68 Despite the absence of a cytoplasmic domain, GPI-anchored class A ephrins also transduce intracellular signals upon interaction with EphA receptors.69−72 The

Fig. 2. Forward signaling via the Eph receptor and reverse signaling via the ephrin ligand.

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mechanism by which this signaling occurs has not been fully elucidated (see below).

3.1. Forward signaling

Ephrin-stimulated forward signaling via Eph receptor requires clustering of the ephrins on the surface of the apposing cell.73,74 Based on structural studies, it was proposed that the Eph signaling unit is a tetramer.75 Eph tetramers undergo autophosphorylation on multiple tyrosines,76 both in the juxtamembrane region, the kinase domain and the carboxy-terminus region. The phosphorylation of the juxtamembrane region releases its steric inhibition of the kinase domain,77,78 and facilitates interaction with phosphotyrosines-binding proteins. Most of these interactions are mediated by Src homology 2 (SH2) domains of the Eph binding partners, and among others, include Src family kinases (SFK), the p85 regulatory unit of phosphatidylinositol 3-kinase, p120RasGAP, and several adaptor proteins (reviewed in Ref. 76). One of the proteins interacting with the cytoplasmic domain of EphA and EphB receptors via an SH2 domain is the non-receptor tyrosine kinase Abl.79 Recent results indicate that EphB4 stimulation by the ephrin-B2 ligand activates Abl, which then phosphorylates the adaptor protein Crk on Tyr221 and consequently uncouples it from the Crk-associated substrate p130(CAS).80 In turn, dissociation of Crk from P130 (CAS) inhibits cell migration.81

The carboxy-termini of all Eph receptors conform to the PDZbinding motif consensus sequence VXV,82 and interact with the PDZ domains of several adaptor proteins (reviewed in Ref. 83). Some of these PDZ ligands, PICK1, syntenin and GRIP1,82 are adaptor proteins that contain between 1 (PICK1) to 7 (GRIP1) PDZ domains. PICK1 clusters and becomes phosphorylated upon binding to EphB2, though the functional significance of this phosphorylation is not known. Eph receptors of both class A and class B bind and phosphorylate AF-6,84 a large adaptor protein which binds Ras. By analogy to Bcr, another AF-6 tyrosine kinase,85 AF-6 phosphorylation by Eph receptors may facilitate Ras binding to AF-6, thus segregating it away from its effector Raf-1 and consequently downregulating the Raf/MEK/ERK pathway.82,84

Similar to plexins, the main effectors of the Eph receptors are cytoskeleton and cell adhesion proteins. The functions of these proteins are regulated by the Eph receptors via several signaling pathways. The kinase domain of the EphA receptors binds ephexin, a RhoA, Rac1 and Cdc42-activating GEF,86 and upon stimulation by the ephrin-A ligand activates ephexin via tyrosine phosphorylation by Src.87,88 This phosphorylation increases the specificity of EphA-activated ephexin towards RhoA, resulting in relative inhibition of Cdc42 and Rac1. The coupling of RhoA activation and inhibition of Cdc42 and Rac1 facilitates cell collapse, similar to the effects of semaphorin. Additional RhoGEFs mediate Eph-induced rearrangement of the cytoskeleton.89−92 Of these, the vascular smooth muscle-expressed ephexin homologue Vsm-RhoGEF, is specific to the vascular system and binds EphA receptors.90 Similar to ephexin, Vsm-RhoGEF is activated by tyrosine phosphorylation upon EphA stimulation, subsequently activating RhoA and inducing stress fiber assembly. While most of the evidence suggests that Eph regulation of Rho-GTPases involves a tyrosine phosphorylation cascade, it appears that this is not always the case, as Cdc42 and Rac1 were inhibited even in cells expressing kinase-dead EphB3.93 The manner in which the inhibition occurred was not determined, however, and a mechanism involving the activation of endogenous ephrin-B by the kinase-dead EphB3 could not be ruled out. Signaling via the ephrin ligand appears to play a role in the vascular system — overexpression of kinase-dead EphB4 was sufficient to alter the normal developmental program of the vascular system in the mouse,94 suggesting that ephrin-B2 can function as a signaling receptor. Instead of the typical angiogenic patterning where a vascular network forms upon the emergence and interconnection of new vessels, the pre-existing blood vessels increased in diameter without sprouting new capillaries. This growth pattern was observed in developing and postnatal angiogenesis, as well as in tumors and normal growth. Given that the EphB4 receptor exerts a repulsive signal via ephrin-B2, it is possible that when the normal angiogenic program was suppressed by the expression of kinase-dead EphB4, endothelial cell proliferation, which is also activated by ephrin-B2,95 was diverted into circumferential enlargement of the initial vascular network.