The large subfamily of receptor tyrosine kinases (RTKs) for which EPH is the prototype have likely roles in intercellular communication during normal mammalian development, but the biochemical signaling pathways utilized by this family are poorly characterized. Two in vitro autophosphorylation sites have been identified within the juxtamembrane domain of the Eph family member Sek, and a candidate binding protein for the activated Sek kinase. Specific antibodies define Sek as a 130 kDa glycoprotein with protein kinase activity expressed in keratinocytes, while a bacterially expressed gst-Sek kinase domain fusion protein autophosphorylates exclusively on tyrosine residues, confirming that Sek encodes an authentic protein tyrosine kinase. Two dimensional phosphopeptide mapping and site-directed mutagenesis define juxtamembrane residue Y602 as a major site of in vitro autophosphorylation in Sek, while Y596 is phosphorylated to a lower stoichiometry. Complimentary approaches of in vitro binding assays and BIAcore analysis reveal a high affinity association between the Y602 Sek autophosphorylation site and the cytoplasmic tyrosine kinase p59fyn, an interaction mediated through the SH2 domain of this intracellular signaling molecule. Moreover, these data identify the novel phosphotyrosyl motif pYEDP as mediating high affinity association with fyn-SH2, extending the previously defined consensus motif for this interaction. The extensive conservation of this fyn-binding motif within the juxtamembrane domain of Eph family RTKs suggests that signaling through fyn, or fyn-related, tyrosine kinases may be utilized by many members of this large subclass of transmembrane receptors (Ellis, 1996).
The Eph family of receptor protein-tyrosine kinases (RTKs) has recently been implicated in patterning and wiring events in the developing nervous system. Eph receptors are unique among other RTKs: they fall into two large subclasses that show distinct ligand specificities and they themselves might function as 'ligands', thereby activating bidirectional signaling. To gain insight into the mechanisms of ligand-receptor interaction, the ligand binding domain in Eph receptors has been mapped. By using a series of deletion and domain substitution mutants, an N-terminal globular domain of the Nuk/Cek5 receptor has been shown to be the ligand binding domain of the transmembrane ligand Lerk2. The Cek5 globular domain is sufficient to confer Lerk2-dependent transforming activity on the Cek9 orphan receptor. The same domain is used for binding of both transmembrane and glycosylphosphatidyl-anchored ligands. These studies have determined the first structural elements involved in ligand-receptor interaction and will allow more fine-tuned genetic experiments to elucidate the mechanism of action for these important guidance molecules (Labrador, 1997).
Reported here is the crystal structure at 2.9 A resolution of the amino-terminal ligand-binding domain of the EphB2 receptor (also known as Nuk). The domain folds into a compact jellyroll beta-sandwich composed of 11 antiparallel beta-strands. Using structure-based mutagenesis, an extended loop has been identified that is important for ligand binding and class specificity. This loop, which is conserved within but not between Eph RTK subclasses, packs against the concave beta-sandwich surface near positions at which missense mutations cause signaling defects, localizing the ligand-binding region on the surface of the receptor (Himanen, 1998).
The sterile alpha motif (SAM) domain is a protein interaction module that is present in diverse signal-transducing proteins. SAM domains are known to form homo- and hetero-oligomers. The crystal structure of the SAM domain from an Eph receptor tyrosine kinase, EphB2, reveals two large interfaces. In one interface, adjacent monomers exchange amino-terminal peptides that insert into a hydrophobic groove on each neighbor. A second interface is composed of the carboxyl-terminal helix and a nearby loop. A possible oligomer, constructed from a combination of these binding modes, may provide a platform for the formation of larger protein complexes (Thanos, 1999).
The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal lobes. To study EphA4 kinase signaling in these processes, mice were generated expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. These findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts (Kullander, 2001b).
The Eph receptor tyrosine kinase family is regulated by autophosphorylation within the juxtamembrane region and the kinase activation segment. The X-ray crystal structure has been solved to 1.9 Å resolution of an autoinhibited, unphosphorylated form of EphB2 comprised of the juxtamembrane region and the kinase domain. The structure, supported by mutagenesis data, reveals that the juxtamembrane segment adopts a helical conformation that distorts the small lobe of the kinase domain, and blocks the activation segment from attaining an activated conformation. Phosphorylation of conserved juxtamembrane tyrosines would relieve this autoinhibition by disturbing the association of the juxtamembrane segment with the kinase domain, while liberating phosphotyrosine sites for binding SH2 domains of target proteins. It is proposed that the autoinhibitory mechanism employed by EphB2 is a more general device through which receptor tyrosine kinases are controlled (Wybenga-Groot, 2001).
The Eph-related family of receptor tyrosine kinases consists of at least 13 members, several of which display distinctive expression patterns in the developing and adult nervous system. In order to study functional interactions between B61-related ligands and Eph-related receptors, chimeric receptors, containing an Eph-related ectodomain and the cytoplasmic domain of the TrkB neurotrophin receptor were constructed. Expression and activation of such chimeric receptors in NIH 3T3 cells induce transformation in focus formation assays. Membrane-bound LERK2 ligand signals through three different Eph-related receptors: Cek5, Cek10 and Elk. LERK2, however, fails to interact functionally with the Cek9 receptor. Quantitative analysis including binding assays indicates that Cek10 is the preferred LERK2 receptor. Preliminary mutagenesis of the LERK2 protein suggests a negative regulatory role for its cytoplasmic domain in LERK2 signaling (Brambilla, 1995).
The Eph family of receptor tyrosine kinases and their cell surface bound ligands have been implicated in a number of developmental processes, including axon pathfinding and fasciculation, as well as patterning in the central nervous system. To better understand the complex signaling events taking place, a comparative analysis has been undertaken of ligand-receptor interactions between a subset of ligands, those that are tethered to the cell surface via a transmembrane domain, and a subset of Eph receptors, the so-called Elk subclass. Based on binding characteristics, receptor autophosphorylation, and cellular transformation assays, it has been found that the transmembrane-type ligands Lerk2 and Elf2 have common and specific receptors within the Elk subclass of receptors. The common receptors Cek10 and Elk bind and signal in response to Lerk2 and Elf2, whereas the Myk1 receptor is specific for Elf2. Elf2, however, fails to signal through Cek5 in a cellular transformation assay, suggesting that Lerk2 may be the preferred Cek5 ligand in vivo. A recently identified third transmembrane-type ligand, Elf3, binds Cek10 specifically, but weakly, and only induces focus formation when activated by C-terminal truncation. This suggests that the physiological Elf3 receptor may have yet to be identified. Knowledge regarding functional ligand-receptor interactions as presented in this study will be important for the design and interpretation of in vivo experiments, e.g., loss-of-function studies in transgenic mice (Brambilla, 1996).
Many Eph-related receptor tyrosine kinases, and each of their numerous membrane-bound ligands, can be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan (Gale, 1996).
The Eph receptors are the largest known family of receptor protein tyrosine kinases; they play important roles, along with their ligands (ephrins), in the neural development, angiogenesis, and vascular network assembly. Ephrin-A2, -A3 and -A5 bind to, and activate the EphA8 receptor tyrosine kinase. An examination was performed to see if there are other additional ephrin ligands interacting with the EphA8 receptor tyrosine kinase expressed in NIH3T3 fibroblasts. For this purpose, chimeric ephrin-A1, -A4, -B1, -B2 or -B3 ligands were constructed consisting of the Fc portion of human IgG fused to ephrin ligand carboxyl-terminus. Both ephrin-A1 and ephrin-A4 chimeric ligands efficiently bind to the EphA8 receptor expressed in NIH3T3 fibroblasts, whereas the transmembrane ligands including ephrin-B1, -B2 and -B3 do not. Both the EphA8-TrkB chimeric receptor and the EphA8 receptor expressed in NIH3T3 fibroblasts are efficiently tyrosine-phosphorylated upon stimulating with epthin-A1 or -A4 but none of transmembrane ephrin-B proteins. These results strongly indicate that the EphA8 receptor functions exclusively as an glycosyl phosphatidylinositol (GPI)-linked ephrin ligand-dependent receptor protein tyrosine kinase (Choi, 1999b).
The Eph family is thought to exert its function through the complementary expression of receptors and ligands. EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection (Hornberger, 1999).
Eph receptor tyrosine kinases and their ligands (ephrins) are highly conserved protein families implicated in patterning events during development, particularly in the nervous system. In a number of functional studies, strict conservation of structure and function across distantly related vertebrate species has been confirmed. Soluble human EphA3 (HEK) exerts a dominant negative effect on somite formation and axial organization during zebrafish embryogenesis: this observation has been used to probe receptor function. Based on exon structure, the extracellular region of the EphA3 receptor has been dissected into evolutionarily conserved subdomains; kinetic BIAcore analysis, mRNA injection into zebrafish embryos, and receptor transphosphorylation analysis were all used to study the function of these domains. Ligand binding is restricted to the N-terminal region encoded by exon III, and an independent, C-terminal receptor-dimerization domain is identified. Recombinant proteins encoding either region in isolation can function as receptor antagonists in zebrafish. A two-step mechanism for Eph receptor activation with distinct ligand binding and ligand-independent receptor-receptor oligomerization events is proposed (Lackmann, 1998).
Fertilization in the female reproductive tract depends on intercellular signaling mechanisms that coordinate sperm presence with oocyte meiotic progression. To achieve this coordination in C. elegans, sperm release an extracellular signal, the major sperm protein (MSP), to induce oocyte meiotic maturation and ovulation. MSP binds to multiple receptors, including the VAB-1 Eph receptor protein-tyrosine kinase on oocyte and ovarian sheath cell surfaces. Canonical VAB-1 ligands called ephrins negatively regulate oocyte maturation and MPK-1 mitogen-activated protein kinase (MAPK) activation. MSP and VAB-1 regulate the signaling properties of two Ca2+ channels that are encoded by the NMR-1 N-methyl D-aspartate type glutamate receptor subunit and ITR-1 inositol 1,4,5-triphosphate receptor. Ephrin/VAB-1 signaling acts upstream of ITR-1 to inhibit meiotic resumption, while NMR-1 prevents signaling by the UNC-43 Ca2+/calmodulin-dependent protein kinase II (CaMKII). MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation, and UNC-43 acts redundantly in oocytes to promote oocyte maturation and MAPK activation. These results support a model in which VAB-1 switches from a negative regulator into a redundant positive regulator of oocyte maturation upon binding to MSP. NMR-1 mediates this switch by controlling UNC-43 CaMKII activation at the oocyte cortex (Corrigan, 2005).
Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. This study uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. The segregation of notochord and neural fates is an autonomous property of the mother cell, and the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. These cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. It has been demonstrated that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. A model is proposed whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification (Picco, 2007).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.