Eph/Ephrin-Based Protein Complexes: The Importance of cis Interactions in Guiding Cellular Processes

Although intracellular signal transduction is generally represented as a linear process that transmits stimuli from the exterior of a cell to the interior via a transmembrane receptor, interactions with additional membrane-associated proteins are often critical to its success. These molecules play a pivotal role in mediating signaling via the formation of complexes in cis (within the same membrane) with primary effectors, particularly in the context of tumorigenesis. Such secondary effectors may act to promote successful signaling by mediating receptor-ligand binding, recruitment of molecular partners for the formation of multiprotein complexes, or differential signaling outcomes. One signaling family whose contact-mediated activity is frequently modulated by lateral interactions at the cell surface is Eph/ephrin (EphA and EphB receptor tyrosine kinases and their ligands ephrin-As and ephrin-Bs). Through heterotypic interactions in cis, these molecules can promote a diverse range of cellular activities, including some that are mutually exclusive (cell proliferation and cell differentiation, or adhesion and migration). Due to their broad expression in most tissues and their promiscuous binding within and across classes, the cellular response to Eph:ephrin interaction is highly variable between cell types and is dependent on the cellular context in which binding occurs. In this review, we will discuss interactions between molecules in cis at the cell membrane, with emphasis on their role in modulating Eph/ephrin signaling.

Blocking ephrinB2 with highly specific antibodies inhibits angiogenesis, lymphangiogenesis, and tumor growth

Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathologic conditions. Eph/ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptor-expressing cells. To investigate the functional consequences of blocking ephrinB2 activity, 2 highly specific human single-chain Fv (scFv) Ab fragments against ephrinB2 were generated and characterized. Both Ab fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor–independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2-blocking Abs caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific Ab-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases.

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.

EphrinB2 induces pelvic-urethra reflex potentiation via Src kinase-dependent tyrosine phosphorylation of NR2B

Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in pain-related neural plasticity at the spinal cord level have been identified. To test whether Src-family tyrosine kinase-dependent glutamatergic N-methyl-d-aspartate receptor NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate pelvic-urethra reflex potentiation, we recorded external urethra sphincter electromyogram reflex activity and analyzed protein expression in the lumbosacral (L6-S2) dorsal horn in response to intrathecal ephrinB2 injections. When compared with vehicle solution, exogenous ephrinB2 (5 μg/rat it)-induced reflex potentiation, in associated with phosphorylation of EphB1/2, Src-family kinase, NR2B Y1336 and Y1472 tyrosine residues. Both intrathecal EphB1 and EphB2 immunoglobulin fusion protein (both 10 μg/rat it) prevented ephrinB2-dependent reflex potentiation, as well as protein phosphorylation. Pretreatment with PP2 (50 μM, 10 μl it), an Src-family kinase antagonist, reversed the reflex potentiation, as well as Src kinase and NR2B phosphorylation. Together, these results suggest the ephrinB2-dependent EphBR activation, which subsequently provokes Src kinase-mediated N-methyl-d-aspartate receptor NR2B phosphorylation in the lumbosacral dorsal horn, is crucial for the induction of spinal reflex potentiation contributing to the development of visceral pain and/or hyperalgesia in the pelvic area.