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Eph receptors resemble plexins also in regulating integrin-dependent adhesion. This regulation is exerted via several pathways. When expressed in fibroblasts, activated EphB2 receptors phosphorylate a tyrosine in the effector domain of R-ras which blocks its binding to Raf-1 and, consequently, its ability to support integrin activation.96,97 Alternatively, stimulation of endogenous epithelial cell EphA2 by ephrin-A1 induces binding of the tyrosine phosphatase SHP2 to EphA2, dephosphorylation and dissociation of focal adhesion kinase (FAK) from EphA2, followed by inhibition of integrin-dependent cell adhesion.98 Conversely, Eph signaling can have an opposite effect on integrin-dependent attachment under certain conditions. Activation of EphB1 upon contact with a surface densely coated with ephrin-B1, augments integrin-mediated attachment of endothelial cells to the surface in a manner requiring the tyrosine kinase activity of EphB1.99 Integrin activation by EphB1 appears to be mediated by the low molecular weight protein tyrosine phosphatase (LMW-PTP), by the adaptor protein Nck, and by a Nck-interacting kinase (NIK), though the pathway between NIK and integrin is not known.100 A PI 3-K-dependent mechanism appears to mediate integrin activation in epithelial cells by EphA8,101 where binding of the p110γ PI 3-K catalytic subunit to the juxtamembrane region of the cytoplasmic domain of EphA8 may facilitate the access of this catalytic subunit to its lipid substrate. It appears that all the known mechanisms of integrin regulation depend on the tyrosine kinase activity of the Eph receptor (e.g. Refs. 93, 98 and 100).

3.2. Reverse signaling

Reverse signaling via transmembrane ephrins B ligands mirrors to a large extent features of EphB receptor forward signaling. Similar to EphB receptors, the cytoplasmic tail of ephrin-B ligands undergoes phosphorylation at three conserved tyrosines.102 As ephrins possess no catalytic activity, the tyrosines are not autophosphorylated but rather undergo phosphorylation by SFK following activation by EphB receptor ectodomains.103 EphB binding to ephrin-B induces SFK recruitment to the cytoplasmic domain of the latter, possibly triggered by ephrin-B clustering. Phosphorylation of one of the tyrosines (Tyr317 in

human ephrin-B1) generates an SH2-binding motif, to which the adaptor protein Grb4 binds and recruits several proteins via its three Src homology 3 (SH3) domains,104 most of which are involved in regulating the actin cytoskeleton and focal adhesions. Among the Grb4-binding proteins, the Cbl-associated protein (CAP) is known to mediate binding of additional cytoskeletal proteins via its own SH3 domains, e.g. vinculin, FAK and PAK1. Grb4 has 68% homology to Nck, the adaptor protein that binds EphB1. The same study104 has shown that ephrinB1 reverse signaling results in disassembly of stress fibers, loss of focal adhesions, and cell rounding in a manner dependent on Grb4 binding to the cytoplasmic domain of ephrin-B1.

Several studies found that ephrin-B regulation of cell adhesion involves Crk,105,106 an SH2/SH3 adaptor protein that couples integrin to cytoskeletal dynamics.107 As a further instance of analogy between ephrin ligands and their Eph receptors, it has been recently shown that while EphB4 induces phosphorylation of the adaptor protein Crk in response to stimulation by the ephrin-B2 ligand,80 activation of ephrinB2 by EphB4 leads to tyrosine phosphorylation of the Crk family proteins CrkII and CrkL, possibly by a SFK.108 In turn, this phosphorylation blocks the association of CrkII and CrkL with p130(CAS), resulting in reduction and alteration in the pattern of focal adhesions.

Though the expression of ephrin-B2 is highest in the arterial endothelium, it is also expressed on mural cells of both arteries and veins.108 Inactivation of ephrin-B2 in mouse vascular smooth muscle cells caused the disruption of microvessel integrity, likely as a result of incomplete coverage of the nascent microvessels by vascular smooth muscle cells (VSMC). At the level of cell function, the absence of ephrin-B2 expression resulted in irregular cell shape and random protrusion of lamellipodia, indicating loss of directionality during migration. The altered cell morphology was accompanied by a reduced number of focal adhesions and a lower activation level of FAK.

Like EphB receptors, Ephrin-B ligands have carboxy-terminus PDZbinding motifs of a similar consensus sequence (YKV), and bind several of the same adaptor proteins: PICK1, which clusters both Eph receptors and ephrin-B ligands, syntenin and GRIP1.84 Ephrin-B1 is targeted to sphingolipid/cholesterol-enriched and caveolae-containing

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subdomains of the plasma membrane (commonly referred to as rafts) thought to be sites of signaling protein clusters,109 and recruits to these regions two GRIP isoforms.110 This recruitment could conceivably initiate the formation of a larger signaling complex containing ephrin effectors via the seven PDZ domains of GRIP. PDZ-dependent interactions of ephrin with cytoplasmic proteins appears to be required for reverse signaling. Mouse knock-in of ephrin-B1 lacking the PDZ-binding motif resulted in embryonic lethality caused by impaired migration of neural crest cells,111 while a knock-in of a similarly truncated ephrin-B2 resulted in defective morphogenesis of the lymphatic system.112 In the latter case, it was also shown that the phosphorylatable tyrosines in the ephrin-B2 cytoplasmic domain were not essential for lymphatic vasculogenesis. In addition to the above adaptor proteins, ephrin-B2 interacts via its PDZ-binding motif with PDZ-regulator of heterotrimeric G protein signaling 3 (PDZ-RGS3).113 Expression of soluble EphB2, an ephrin-B2 receptor, inhibited cellular response to the chemoattractant SDF-1 during Xenopus development. Since SDF-1 signals through a G-coupled protein receptor, it is possible that ephrin-B2-activated PDZRGS3 contributed to the inhibition of SDF-1 signaling. It appears, therefore, that in addition to the other repulsive effects of ephrin, it counteracts chemoattractant signals.

GPI-anchored ephrin-A ligands are segregated into plasma membrane rafts.70 Targeting of ephrin-A to such subdomains facilitates the clustering required for the activation of their cognate Eph receptors,73,74 and brings the ephrin-A ligands in proximity to other signaling proteins that are compartmentalized into these subdomains.109 Activated ephrin-A5 recruits the SFK Fyn to the raft subdomains and induces its activation via a still unknown mechanism that may hypothetically involve ephrin-A interaction in cis with a putative transmembrane receptor. This results in an overall increase in the tyrosine phosphorylation level of other raft proteins. Unlike Eph receptors, however, ephrin-A signaling augments integrin-dependent cell adhesion69 and causes cell spreading rather than collapse.72 Ephrin-A and EphA receptors are laterally segregated into separate domains on the plasma membrane, so that each population can interact with its receptors or ligands, respectively, in trans. Forced collocation