EphA3, Erythropoietin-Producing Hepatocellular Carcinoma Cell Receptor A3
A fragment of EphA3 was first cloned in 1991 by Sajjadi et al., in an expression-cloning screen of a chicken embryonic cDNA library using antibodies against phosphotyrosine (Sajjadi et al. 1991). Using this cDNA fragment as a probe, full-length cDNA from both chicken and mouse embryos were identified (Sajjadi et al. 1991). Human EphA3 was independently isolated as a cell surface antigen of a pre-B-cell leukemia cell line (Boyd et al. 1992; Wicks et al. 1992). EphA3 is a member of a large family of tyrosine kinase receptors, which plays many critical roles in both physiological and pathological conditions (Pasquale 2008).
The full-length human EphA3 protein consists of 983 amino acids, organized into the typical extracellular, transmembrane, and intracellular domains as that of other Eph family tyrosine kinase receptors (Wicks et al. 1992). A splice variant, which contains only the extracellular domain, was also identified in the mouse (Sajjadi et al. 1991). The relative abundance and difference in function between the full-length and truncated receptors have not been analyzed at present. Since the truncated form lacks intracellular signaling capacity but is capable of binding to Eph ligands (see the Ligands section for details), it may serve to down modulate EphA3 receptor function. Due to promiscuity of ligand–receptor interactions in the Eph family, it is possible that this truncated form may regulate functions of many other Eph receptors. Similar truncated form has been identified for TrkB, the receptor for the neurotrophic factor brain-derived neurotrophic factor, and has been shown to inhibit full-length TrkB receptor function in developing mouse brain (Carim-Todd et al. 2009).
EphA3 is expressed both in the nervous system and in nonneural tissues. In the developing mouse brain, EphA3 expression is detectable as early as E12, but is undetectable after P10 (Kilpatrick et al. 1996; Kudo et al. 2005; Lai and Lemke 1991). Both mRNA and protein were detected in the developing cerebral cortex, ganglionic eminence, and presumptive caudate-putamen at E12. Expression was also found in the dorsal thalamus and in spinal motor neurons (Iwamasa et al. 1999; Kilpatrick et al. 1996; Kudo et al. 2005; Mackarehtschian et al. 1999). High levels of protein expression were found in the thalamocortical axons. What is intriguing is that there is an extensive overlap with the adhesion molecule L1 expression, suggesting that EphA3 and L1 interact or at least regulate similar targets. Indeed it has been shown that EphA3 is physically associated with L1, and requires L1 protein for ephrin-A5-induced growth cone collapse (Demyanenko et al. 2011). It will be extremely interesting to examine how EphA3 and L1 interact to regulate axon guidance.
The most striking expression patterns of EphA3 were found in the developing chick and wallaby retina with a nasal (low) to temporal (high) gradient (Cheng et al. 1995; Stubbs et al. 2000). This gradient plays a critical role in the specification of retinotectal topographic axon projection map as discussed in the Biological Functions section. How this graded expression is regulated is not entirely clear at present. There is evidence suggesting that two homeobox transcription factors, SOHo1 and GH6, expressed in opposing gradients as that of EphA3 in the retina, inhibit EphA3 transcription, defining the expression in the temporal region (Schulte and Cepko 2000). Indeed, ecotopic expression of either transcription factor resulted in inhibition of EphA3 expression and a partial disruption of retinal axon guidance to the tectum. Two members of the winged-helix (WH) transcription factors, chick brain factor (CBF) 1 and 2, have also been shown to influence EphA3 expression in the chick retina, with CBF1 inhibiting and CBF2 inducing EphA3 expression (Takahashi et al. 2003; Takahashi et al. 2009). Extracellular signals have also been shown to regulate EphA3 expression. For example, the T-cell co-stimulatory signal CD28 and the type 1 insulin-like growth factor can enhance both EphA3 protein levels (Smith et al. 2004b). In contrast, interleukin-1ß reduces the expression (Li et al. 1998). However, the regulation of EphA3 expression is incompletely understood, and it remains unclear how different signals coordinate the expression important for its biological functions.
Ligands and Mechanisms of Interaction with EphA3
Two ephrins, ephrin-A1 (initially named B61) and ephrin-A2 (Elf-1), were identified as ligands of EphA3 in 1994 using EphA3 extracellular domain fusion proteins as affinity probes in expression screening experiments (Beckmann et al. 1994; Cheng and Flanagan 1994). Two new ligands, ephrin-A3 and ephrin-A4, were identified by Kozlosky and colleagues using similar methods in the following year (Kozlosky et al. 1995). An additional ligand, ephrin-A5, was identified as a retinal axon guidance molecule (RAGS) (Drescher et al. 1995), and as an expressed sequence tag with homology to ephrin-B1 (Cerretti et al. 1995). Ephrin-A5 was also independently isolated by Lackmann and colleagues from the conditioned medium of human placenta (Lackmann et al. 1996). This ligand was found to be identical to AL-1, a ligand purified with affinity chromatography using the extracellular domain of the related receptor EphA5 from the conditioned medium of a human breast cancer cell line BT20 (Winslow et al. 1995). It is now known that the ligand–receptor interactions of the Eph family are highly promiscuous, and thus EphA3 receptor can interact with all the A-subclass ephrins. A careful study by Lackmann and colleagues in 1997 showed that while different bivalent ephrin-A-Fc fusion proteins interact with EphA3 with similar high affinity, the monomeric form of ephrin-A5 can form a much more stable complex with EphA3 than that of ephrin-A3 (Lackmann et al. 1997), suggesting that ephrin-A5 is the preferred high affinity ligand. Whether ephrins interact with Eph receptors in dimeric/oligomeric forms or monomeric form in vivo is not clear at present. Although nearly all functional assays reported have used multimeric forms of ephrins, monomeric ephrins may indeed have important functions in development or adult, since both ephrin-A1 and ephrin-A5 have been isolated initially from conditioned media as released monomeric form (Holzman et al. 1990; Lackmann et al. 1996; Winslow et al. 1995), it would not be surprising if novel functions were discovered for released monomer ephrins in the future.
The interaction of EphA3 with the ligand ephrin-A5 has been extensively analyzed (Lackmann et al. 1998; Smith et al. 2004a). The ligand-binding domain of EphA3 is located to the N-terminal region to the first IgG domain, encoded by exon III (Lackmann et al. 1998). This is consistent with findings in other Eph receptors (Himanen and Nikolov 2003; Labrador et al. 1997). Ligand–receptor interaction involves a high affinity and a low-affinity binding site. The former is responsible for ligand–receptor dimerization, and the latter facilitates ligand–receptor tetramer formation (Himanen et al. 2001; Himanen and Nikolov 2003; Smith et al. 2004a). In addition, a ligand independent receptor dimerization domain has also been identified using both protein domain deletion and functional mutagenesis approaches in the cysteine-rich hinge region of the receptor (Lackmann et al. 1998; Smith et al. 2004a). Similar analysis also identified three domains in ephrin-A5 that play key roles in interaction with EphA3 receptor (Day et al. 2005). Two of the sites interact with the high- and low-affinity interaction domains identified in EphA3 receptor, and a third novel interface, which was proposed to bind to the cysteine-rich receptor domain of Eph receptors in adjacent ligand–receptor tetramers, allowing polymerization of large number of the ligand–receptor complexes and effective functional activation.
What is interesting about ephrin–Eph interaction is that simple ligand–receptor binding is not sufficient to fully activate the receptors (Pabbisetty et al. 2007; Stein et al. 1998; Vearing and Lackmann 2005). It has been shown that addition of monomeric ephrin-A5 failed to induce EphA3 kinase activation as assayed by autophosphorylation on tyrosine (Vearing et al. 2005). In contrast, dimeric ephrin-A5-Fc was found to be sufficient to induce EphA3 autophosphorylation. This difference is in part explained by the observation that monomeric ligands have a much lower binding affinity than the dimeric ligands (Pabbisetty et al. 2007). However, ephrin-A5 dimer-induced partial EphA3 activation is not enough to induce a cellular response as demonstrated by actin cytoskeleton contraction, which required additional receptor clustering (Vearing et al. 2005). Similar observations have been reported for ephrin-B1 (Stein et al. 1998). Correlated with the ability to affect cellular functions, only the fully activated Eph receptors are capable of recruiting downstream signaling molecules, although much remains uncharacterized (Stein et al. 1998; Vearing and Lackmann 2005). However, it is not known whether highly clustered ephrins are necessary to induce the full range of biological functions of all Eph receptors. For example, it has been reported that no differences were observed in EphA2 receptor autophosphorylation or endothelial cell migration by dimer or further cross-linked ephrin-A1-Fc (Brantley-Sieders et al. 2004). This issue is complicated by the possibility that different Eph receptors may respond differently to ephrin dimers. In addition, few studies have addressed this issue carefully since ephrin-Fc preparations may contain a mixture of dimers and larger aggregates. Adding to the complexity of ephrin–Eph interactions, it has been shown that interaction of ephrin-A5 and EphA3 in cis silences EphA3 receptor activation, possibly modulating concentration of actively signaling EphA3 receptor molecules (Carvalho et al. 2006).
Since both the EphA3 receptor and its ephrin ligands are membrane anchored, the question of how the ligand–receptor complex can be internalized by endocytosis becomes an interesting question. Elegant mutational and crystallographic studies by Nikolov and colleagues (Janes et al. 2005) showed that although the protease ADAM10 (A disintegrin and metalloprotease 10) is constitutively associated with EphA3, association of ephrin-A5 to EphA3 receptor creates a new binding site for ADAM10, and allows the cleavage of ephrin-A5 releasing the EphA3/ephrin-A5 complex for internalization. In addition, only ephrin-A5 ligand expressed in trans to ADAM10 can be cleaved after EphA3 binding. Such a mechanism ensures only ligands interacting with the receptor are cleaved.
Activation and Signal Transduction
Similar to other receptor tyrosine kinases, EphA3 is activated by ligand binding. Upon ligand binding, the two tyrosine residues located at the juxtamembrane segment of EphA3 become phosphorylated. Crystallographic analysis showed that the unphosphorylated juxtamembrane domain tyrosine residues interact with the active site to prevent adoption of a catalytically active confirmation of the catalytic site (Davis et al. 2008). Mutagenesis studies showed that mutation of Y596F but not Y602F leads to the loss of EphA3 kinase activity as well as the ability to inhibit cell migration and induce cellular process and neuronal growth cone retraction (Davis et al. 2008; Hu et al. 2009; Shi et al. 2010). In contrast, restoration of the negative charge of phospho-Y596 with an Y596E mutation restores EphA3 kinase activity, suggesting that phosphorylation of Y596 is necessary to release inhibition of EphA3 kinase activity. The activation mechanism of EphA3 kinase is likely to be complex and involve many other amino acid residues in both the juxtamembrane domain and the kinase domain. Significantly, both Y742 and S768 also contribute to the regulation of EphA3 kinase activity (Davis et al. 2008).
Although phospho-Y602 is not required for EphA3 kinase activity, it is necessary for its biological functions as measured with either morphological changes (membrane blebbing and cellular process retraction) or cell migration (Hu et al. 2009; Lawrenson et al. 2002; Shi et al. 2010). In fact, Y779 phosphorylation is also important for these biological activities. Both Y602F and Y779F mutations cause a partial loss of biological activity, while the double mutant Y602/779F resulted in a complete loss of biological activity while maintaining its kinase activity (Shi et al. 2010). These observations are consistent with the notion that phosphorylated Y602 and Y779 serve as docking sites for downstream signaling molecules that mediate EphA3 biological effects, and that different tyrosine residues together with their respective signaling pathways collaborate to achieve full biological activity for the receptor.
Downstream of the receptor, activation of EphA3 in T cells by ephrin-A1 results in tyrosine phosphorylation of c-cbl proto-oncogene (Sharfe et al. 2003). The process is dependent on the Src family kinases, indicating that Src mediates effects of EphA3 activation on c-cbl phosphorylation. c-cbl may serve as a regulator of receptor ubiquitination and degradation, thus downregulating EphA3 protein levels upon activation by ephrins (Sharfe et al. 2003).
It has also been shown that the adaptor protein Nck binds to the phosphotyrosine residue Y602 through its Src Homology domain 2 (SH2), and blocking Nck signaling also reduces EphA3-mediated inhibition of cell migration and process retraction (Hu et al. 2009). Nck is known to interact with multiple signals that regulate cytoskeleton dynamics, including p21-activated kinase ( PAKI), and the Wiskott–Aldrich syndrome family proteins that control actin reorganization (Li et al. 2001).
EphA3 is widely expressed in both neural and nonneural tissues during development and plays multiple roles during embryogenesis.
Substrate detachment and adhesion. Binding of EphA3 by clustered ephrin-A5 has been shown to induce detachment and rounding of HEK293 cells and melanoma cells, but promote adhesion of pre-B and T-cell leukemic cells (Lawrenson et al. 2002; Wimmer-Kleikamp et al. 2008). The opposing effects in these two different types of cells are due to the presence of a protein tyrosine phosphatase activity in the leukemic cells, which prevents autophosphorylation of EphA3. The emerging model is that when EphA3 is fully activated and autophosphorylated on the tyrosine residues, it triggers the activation of Rho-GTPase and leads to cell detachment and repulsion (Lawrenson et al. 2002). In contrast, when receptor activity is down modulated by phosphatase activity, interactions between EphA3 and its ligands lead to cell adhesion. Although the identity of the phosphatase that dephosphorylates EphA3 in pre-B leukemia cells has yet to be identified, evidence indicates that PTP1B may play a similar role in EphA3 dephosphorylation in HEK293 cells and in glioblastoma cells by interacting with the activated receptor at the plasma membrane as well as in endosomal vesicles (Nievergall et al. 2010).
Axon guidance. The earliest hint for a critical function of EphA3 and its ligands in embryonic development came when ephrin-A5 was found to be the repulsive axon guidance signal (RAGS) responsible for generating the topographic retinal axon termination map in the optical tectum in the brain (Cheng et al. 1995; Drescher et al. 1995). The landmark study by Drescher and colleagues, together with the studies by Cheng and colleagues, which demonstrated the expression of EphA3 and ephrin-A2, another ligand, are expressed in opposing gradients in the projecting retina and the target tectum, unraveled an age-old mystery of how axon terminals are topographically mapped onto target brain tissues. Further studies demonstrated that ephrin-A2 and ephrin-A5 together serve as repulsive axon guidance signals for the formation of the retinotectal project map (Ciossek et al. 1998; Feldheim et al. 2000), and that EphA3 as well as other EphA receptors mediate the effects of these ephrins (Brown et al. 2000; Feldheim et al. 2004). Further studies now show that ephrin–Eph interactions play a general role in the specification of topographic maps in several other axon pathways including hippocamposeptal projections (Yue et al. 2002), thalamocortical projections (Dufour et al. 2003; Uziel et al. 2002, 2006), the dopaminergic pathways (Cooper et al. 2008; Passante et al. 2008; Sieber et al. 2004; Yue et al. 1999) and possibly throughout the nervous system. In addition to guiding axons, EphA3 has been shown to play a role in the segregation of axial motor and sensory axon tracks during development through interaction with ephrin-A–EphA transaxonal interaction (Gallarda et al. 2008).
Heart development. EphA3 plays critical roles in development of extraneuronal tissues as well during embryogenesis. One clearly defined function is heart development (Stephen et al. 2007). Inactivation of EphA3 resulted in perinatal heart failure and death in about 75% of newborn mice. Postmortem examinations showed that the EphA3-null mice had enlarged atria and presence of blood in the lung and liver due to capillary disruption caused by exceedingly high cardiac filling pressure. The EphA3-deficient heart showed defects in the atrioventricular valves and in the atrioventricular septum, which separates the right atrium from the left ventricle. Analysis of EphA3 expression in early embryos revealed that the receptor is transcribed in the developing atrioventricular and outflow tract endocardial cushions at E10.5, and in the mesenchymal cap of the developing septum primum at E12.5. The endocardial cushions later generate the atrioventricular valves and septa. A ligand, ephrin-A1, was found to be expressed in the neighboring cells. In the EphA3-null embryos, the endocardial cushions appear to be smaller, possibly due to cell migration defects from an altered cytoskeletal structure of these cells. Consistent with ephrin-A1 expression in the developing heart, deletion of the gene also results in heart defects (Frieden et al. 2010). However, ephrin-A1 KO hearts showed thickened aortic and mitral valves and elevated mesenchymal marker expression, a different phenotype than that of EphA3-null mice, suggesting distinct functions. These observations, together with results from studies in the nervous system, indicate that EphA3 receptor and the ephrin-A ligands, may participate in the development and function of many different tissues and organs during embryogenesis and in adult.
Cancer. In addition to functions in normal animals, EphA3 mutations have been associated with tumor formation. EphA3 has been identified as a melanoma tumor antigen, and is overexpressed in several other tumors including lung cancer, kidney tumors, and brain tumors (Chiari et al. 2000). EphA3 mutations have been identified in primary lung and colon cancer in tumor genome sequencing projects (Wood et al. 2006; Ding et al. 2008). Mutational profiling of kinases in cancer also identified an EphA3 mutation in the cys-rich extracellular domain involved in ligand binding and receptor tetramerization in human pancreatic cancer (Corbo et al. 2010). EphA3 mutations have also been reported in glioblastoma, melanoma, and liver tumor samples (Bae et al. 2009; Balakrishnan et al. 2007). Mutations were identified in both the extracellular and kinase domains, with no hotspot preferences. Although the exact nature of these mutations are currently unknown, it has also been shown that EphA3 is among genes mutated or lost in the loss of heterozygocity in head and neck squamous cell carcinoma suggesting that this receptor serves as a tumor suppressor (Lee et al. 2010).
EphA3, a tyrosine kinase receptor of the Eph family, plays critical roles in the regulation of both physiological and pathological processes. The receptor is required for axon guidance to proper targets in the nervous system, and for heart development. EphA3 mutations have been detected in many different types of tumors. Since EphA3 expression is found in many different tissues during embryonic development, it is within reason that new functions will be attributed to this receptor in the future. However, understanding of EphA3 function and how it conveys the signals in the cells are at their infant stage. For example, the role EphA3 plays in carcinogenesis remains undefined, since both overexpression and mutations have been found in tumors. In addition, downstream signaling pathways for EphA3 are poorly understood at present. It is not clear whether EphA3 uses similar signaling strategies as that of other Eph receptors or uses unique pathways. Furthermore, roles of the truncated receptor in development and disease remain to be elucidated.
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