Eph Receptors Proteins


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 Eph Receptors Proteins Background

Ephrins (Eph) family RTKs and their membrane-anchored protein ligands, the ephrins, also play important roles in establishing the relative positions of cells and tissues in developing embryos. The Eph family represents the largest subgroup of RTKs, with 14 members having been discovered so far in vertebrates. Eph receptors can be divided into two classes based on sequence similarity and ligand binding affinity. A-type Eph receptors (EphAl-8,10) bind promiscuously to glycosylphosphatidylinositol (GPI)-linked ephrin-A ligands (ephrin-Al-6), whereas B-type Eph receptors (EphBl-4, 6) bind promiscuously to transmembrane ephrin-B ligands (ephrin-Bl-3). The only known exceptions to this rule are EphA4, which can bind ephrin-B2 and 3 in addition to the ephrin-As, and EphB2, which can bind ephrin-A5 in addition to the ephrin-Bs. In going from amino to carboxy-terminus, Eph receptors are comprised of an extracellular domain with a globular ligand binding domain (LBD), a cysteine-rich epidermal growth factor (EGF)-like region, and two FN type III repeats, a transmembrane domain (TMD), and an intracellular domain with a juxtamembrane (JM) region, a protein tyrosine kinase domain (TKD), a sterile alpha motif (SAM), and a PDZ domain binding motif.

The interaction between Eph and ephrin results in the phosphorylation of two tyrosine residues in the receptor’s JM region. These phosphotyrosines are required for full activation of the receptor’s tyrosine kinase activity and also act as docking sites for Src homology 2 (SH2) domain-containing proteins, which in turn act to transmit the signal initiated by ephrin binding to intracellular effectors. Unlike the ligands for other RTKs, ephrins are membrane-bound and fail to elicit a response from their receptors when presented in a soluble form. On the other hand, soluble ephrins that have been artificially clustered are functional, indicating that Ephs and ephrins mediate cell contact-dependent interactions and that membrane anchorage facilitates the dimerization/multimerization of receptor-ligand complexes. This leads to activation of receptor catalytic activity and cross-phosphorylation of the JM tyrosine residues.

Eph and ephrin function is exemplified by the roles that these proteins play in visual topographic map formation and hindbrain segmentation. Retinal ganglion cell (RGC) axons make connections to the optic tectum in birds, fish, and amphibians or the superior colliculus (SC) in mammals in the same order in which they project from the retina, thus preserving nearest neighbour relationships and enabling spatially intact visual images to be transmitted to the brain. EphA3 shows increasing expression along the nasal to temporal axis of the retina and is also expressed by RGC axons, whereas ephrin-A2 and 5 are expressed in a low anterior to high posterior gradient across the tectum. The fact that temporal RGC axons project to the anterior tectum and nasal RGC axons project to the posterior tectum suggests that axons expressing high levels of EphA3 are excluded from tectal regions expressing high levels of ephrin-A2/5 by a receptor-mediated repulsive guidance mechanism. In support of this, temporal, but not nasal RGC axons strongly avoid growing out onto ephrin-A2/5-containing substrates in vitro. In vivo, patches of ectopic ephrin-A2 expression in the anterior tectum are similarly avoided by just temporal RGC axons. Ectopic expression of EphA3 in a subset of RGCs causes their axonal connections in the tectum to shift anteriorly. On the other hand, gene knockout of ephrin-A5 in mouse or ectopic expression of cytoplasmic domain-truncated EphA3 in the chicken retina results in temporal RGC axons projecting aberrantly to the posterior SC/tectum.

In the hindbrain, EphA4 and EphB2 and 3 are expressed by cells in r3 and 5, whereas ephrin-Bl-3 have a complementary distribution in r2 ,4, and 6. Lineage studies have demonstrated that cells can intermingle within a given rhombomere, but movement beyond that rhombomere’s boundaries is restricted. Intriguingly, there are large intracellular spaces between the boundaries of adjacent rhombomeres, perhaps indicating that a repulsive interaction between Eph-expressing cells in odd-numbered rhombomeres and ephrin-expressing cells in even-numbered rhombomeres contributes to hindbrain segmentation. In Xenopus and zebrafish embryos expressing cytoplasmic domain-truncated EphA4, cells with an r3/5 identity appear in the adjacent even-numbered rhombomeres. When cytoplasmic domain-truncated ephrin-B2 is expressed in a mosaic fashion in the zebrafish embryo, the expressing cells become randomly distributed in r2/4/6, but sort to the boundaries of r3/5. These findings indicate that Eph signaling in the hindbrain functions to restrict receptor-expressing cells to the odd-numbered rhombomeres by effecting their repulsion from ligand-expressing cells. Conversely, mosaic expression of truncated EphA4 or EphB2 results in the expressing cells becoming randomly distributed within r3/5 and sorting to the boundaries of r2/4/6. This surprising finding indicates that the ephrin-B2 ligands expressed by even-numbered rhombomere cells can themselves effect the repulsion of Eph-expressing cells, presumably as a result of a ‘reverse’ signaling mechanism. As such, hindbrain segmentation appears to depend on bidirectional signaling from ephrin-Eph complexes formed between cells at the interfaces of even and odd-numbered rhombomeres. Further support for the existence of bidirectional signaling comes from the finding that intermingling between adjacent cell populations in culture is severely restricted when one group expresses EphA4 or EphB2 and the other expresses ephrin-B2, but not when one group expresses wild-type EphA4/EphB2 and the other expresses truncated ephrin-B2 or vice versa.