Ret is a receptor protein tyrosine kinase that has been implicated in the development of the enteric nervous, endocrine, and renal systems. Mutations associated with multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B) have been shown to activate the intrinsic kinase and transforming ability of ret. Using the cytoplasmic domain of Ret as bait in a yeast two-hybrid screen of a mouse embryonic library, it was discovered that the src homology 2 (SH2) domain containing protein Grb10 binds Ret. Grb10 belongs to an emerging family of SH2 containing adapter proteins, the prototypical member being Grb7. The SH2 domain of Grb10 has been shown to specifically interact with Ret. Additionally, using an EGFR/Ret chimera, it has been shown that Grb10 binds Ret in an activation-dependent manner in vivo. This is the first description of a receptor protein tyrosine kinase that utilizes Grb10 as a signaling intermediate (Pandey, 1995b).
Pagliaccio (Pag) is a receptor tyrosine kinase of the Eph family that is expressed in Xenopus embryos in a diverse set of localized tissues. Pag is the Xenopus homolog of Hek-8 (human), Sek-1 (mouse), cek8 (chicken), and RTK-1 (zebrafish). The function of this protein has been investigated by injecting RNA encoding an epidermal growth factor receptor-Pag chimera into early Xenopus embryos. Activation of the chimeric receptor results in a kinase-dependent loss of cell-cell adhesion. This dissociation can be reversed by co-injection of RNA encoding C-cadherin, suggesting that one or more cadherins could be the functional targets for Pag activity (Winning, 1996).
The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. In vivo tyrosine phosphorylation sites of the Eph receptors (EphA3, EphA4, and EphB2) in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Furthermore, tyrosine-phosphorylated EphB2 is detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation was detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrates that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src. In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma is not abolished upon mutation of tyrosine 611 in EphB2. Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine-to-phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, show tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2. The mutational analysis also indicates that tyrosines 605 and 611 are important for EphB2 kinase activity. It is proposed that Src kinase is a downstream effector that mediates the neuron's response to Eph receptor activation (Zisch, 1998).
Eph family receptor tyrosine kinases (including EphA3, EphB4) direct neuronal pathfinding within migratory fields of cells expressing gradients of membrane-bound ligands. Other Eph family RTKs (EphB1 and EphA2) direct vascular network assembly, affecting endothelial migration, capillary morphogenesis, and angiogenesis. To explore how ephrins could provide positional labels for cell targeting, a test was performed to see whether endogenous endothelial and P19 cell receptors [EphB1 (ELK) and EphB2 (Nuk)] discriminate between different oligomeric forms of an ephrin-B1/Fc fusion ligand. Receptor tyrosine phosphorylation is stimulated by both dimeric and clustered multimeric ephrin-B1, yet only ephrin-B1 multimers (tetramers) promote endothelial capillary-like assembly, cell attachment, and the recruitment of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor complexes. Cell-cell contact among cells expressing both EphB1 and ephrin-B1 is required for EphB1 activation and recruitment of LMW-PTP to EphB1 complexes. The EphB1-binding site for LMW-PTP was mapped and shown to be required for tetrameric ephrin-B1 to recruit LMW-PTP and to promote attachment. Thus, distinct EphB1-signaling complexes are assembled and different cellular attachment responses are determined by a receptor switch mechanism responsive to distinct ephrin-B1 oligomers (Stein, 1998a).
The large subfamily of receptor tyrosine kinases (RTKs) for which EPH is the prototype are likely to have 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, whilst 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).
Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, the EphB2 receptor (formerly Nuk/Cek5/Sek3) was expressed in neuronal NG108-15 cells, and the tyrosine phosphorylation of multiple cellular proteins was observed upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induces the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn forms a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also binds through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexes with activated EphB2 in vivo. An in vitro RasGAP-binding site has been localized to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2; it has been demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signaling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the +3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signaling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture (Holland, 1997).
Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. Two-hybrid interaction cloning was used to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. The EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, were mapped to the juxtamembrane region. Within this subdomain, Tyr594 is required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruits Nck to native receptor complexes and activates c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocks Nck recruitment to EphB1, attenuated downstream JNK activation, and blocks cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK (Stein, 1998b).
Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, the yeast two-hybrid system was applied. The carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis reveal that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells, endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. These observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs (Hock, 1998).
Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses. PDZ proteins apparently play critical roles in such protein localizations. Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates. Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins. Eph receptors and ligands colocalize with PDZ proteins at synapses. Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse (Torres, 1998).
The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized cell-cell junctions. To identify potential receptor-binding targets of AF-6, the PDZ domain of AF-6 was screened against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors. Phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain (Buchert, 1999).
Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encode polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies have demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bind ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells (Lin, 1999).
An examination was carried out of the functional roles of two major autophosphorylation sites, Tyr-615 and Tyr-838, in the EphA8 receptor. Two-dimensional phosphopeptide mapping analysis demonstrates that Tyr-615 and Tyr-838 constitute major autophosphorylation sites in EphA8. Tyr-615 is phosphorylated to the highest stoichiometry, suggesting that phosphorylation at this site may have a physiologically important role. Upon conservative mutation of Tyr-838 located in the tyrosine kinase domain, the catalytic activity of EphA8 is strikingly reduced, both in vitro and in vivo, whereas a mutation at Tyr-615 in the juxtamembrane domain does not impair the tyrosine kinase activity. In vitro binding experiments reveal that phosphorylation at Tyr-615 in EphA8 mediates the preferential binding to Fyn-SH2 domain rather than Src and Ras GTPase-activating protein (Ras GAP)-SH2 domains. Additionally, a high level of EphA8 is detected in Fyn immunoprecipitates in intact cells, indicating that EphA8 and Fyn can physically associate in vivo. In contrast, the association of full-length Fyn to EphA8 containing mutation at either Tyr-615 or Tyr-838 is greatly reduced. These data indicate that phosphorylation of Tyr-615 is critical for determining the association with Fyn, whereas the integrity of Tyr-838 phosphorylation is required for efficient phosphorylation at Tyr-615 as well as other major sites. Finally, it was observed that cell attachment responses are attenuated by overexpression of wild type EphA8 receptor but to a much lesser extent by EphA8 mutants lacking phosphorylation at either Tyr-615 or Tyr-838. Furthermore, transient expression of kinase-inactive Fyn in EphA8-overexpressing cells blocks cell attachment responses attenuated by the EphA8 signaling. It is proposed that Fyn kinase is one of the major downstream targets for the EphA8 signaling pathway leading to a modification of cell adhesion, and that autophosphorylation at Tyr-838 is critical for positively regulating the EphA8 signaling event (Choi, 1999a).
The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. Ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5-treated retinal ganglion cell cultures, Rho is activated and Rac is downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduces the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway. Currently not much is known as to how ligand-induced activation of EphA receptor tyrosine kinases regulates the Rho and the Cdc42/Rac pathways. EphA receptors might interact via autophosphorylated juxtamembrane tyrosine residues, with RasGAP (Ras GTPase-activating protein), which is constitutively associated with RhoGAP (see Drosophila RhoGAP). RhoGAP is a negative regulator of Rho, and the strong activation of Rho by fc-ephrin-A5 in these experiments would require inactivation of RhoGAP activity. It remains to be shown if additional elements (p62 dok) of the RasGAP-RhoGAP complex are responsible for such an inhibition (Wahl, 2000).
EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB. The kinase activity of EphB may be important for subsequent steps in synapse formation, because perturbation of EphB tyrosine kinase activity affects the number of synaptic specializations that form in cultured neurons. These findings indicate that EphrinB activation of EphB promotes an association of EphB with NMDA receptors that may be critical for synapse development or function (Dalva, 2000).
As more examples of molecules that regulate aspects of synapse formation or maturation are described, it is becoming clear that there may be a variety of factors that regulate the process of synapse development. EphB receptors are localized at the postsynaptic membrane and associate with molecules such as PICK1 and AF6 that are established structural components of the synapse. In addition, EphB receptors interact with a host of intracellular effector molecules including Src, Nck, and Grb2, although the role for these molecules in synapse development is not yet clear. The binding of ephrinB to EphB2 may lead to the direct recruitment of NMDA receptor subunit NR1 and its associated subunits (NR2A-B) to the EphB complex. The binding of ephrinB to EphB also results in the recruitment of other proteins, including CaMKII and Grb10, to the EphB/NMDA receptor complex. These results indicate that EphB receptors are linked to structural and signaling molecules at the synapse that may enable an EphB receptor-driven signal to contribute to the development or function of synapses (Dalva, 2000).
The observation that EphB and NMDA receptors interact raises the intriguing possibility that cross-talk exists between these receptors to elicit changes in the functional properties of these proteins. One possibility is that the association of EphB and NMDA receptors could lead to changes in NMDA receptor function, perhaps via the phosphorylation of NMDA receptor subunits. The EphB RTK may phosphorylate the NMDA receptor either directly or via an associated kinase. Members of the Src family of tyrosine kinases, Src and Fyn, are good candidates to mediate EphB-dependent tyrosine phosphorylation of the NMDA receptor, since both of these Src family members bind Eph receptors and have been shown to regulate NMDA receptor function. CaMKII is another good candidate, because CaMKII is recruited to the EphB/NMDA receptor complex, and CaMKII has been shown to phosphorylate the NMDA receptor. Phosphorylation of the NMDA receptor may alter its channel conductance and NMDA receptor phosphorylation may underlie aspects of LTP. Thus, the assembly of the EphB and NMDA receptor complex may lead to changes in the channel properties of the NMDA receptor that could play an important role in synapse development or plasticity. This conclusion is supported by the observations that ephrin stimulation of EphBs enhances the formation of both pre- and post-synaptic specializations, and that blocking the ability of EphBs to signal via their receptor tyrosine kinase activity suppresses the number of postsynaptic specializations (Dalva, 2000).
Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. The cloning and characterization of ephexin is reported. Ephexin is a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility (Shamah, 2001).
The genomes of C. elegans and Drosophila (see CG3799) each contain a single gene with a high degree of homology to ephexin. In humans, in addition to an ephexin ortholog, there are at least three other genes that are highly homologous to ephexin: the previously characterized Dbl family member TIM and two uncharacterized cDNAs -- Neuroblastoma and KIAA0915. Using the ClustalX program, phylogenetic analysis of the DH-PH domains of the ephexin-related proteins was performed and they were compared to the DH-PH domains of Dbl and Vav1, two other Dbl family GEFs. This analysis revealed that the DH-PH domains of ephexin-related GEFs are more closely related to each other than to the DH-PH domains of Dbl and Vav1, and identified ephexin-related GEFs as a subfamily within the larger Dbl family of GEFs. Alignment of ephexin subfamily proteins revealed a high degree of conservation within the DH, PH, and SH3 domains, but little or no homology in the N-terminal regions, including the 14 amino acid hydrophobic sequence present in ephexin orthologs (Shamah, 2001).
For growth cones to respond to extracellular guidance cues in a directional manner, guidance receptors must transduce highly localized signals to the actin cytoskeleton specifically in the region of the growth cone where receptor activation occurs. The finding that ephexin interacts directly with EphA4 receptors independent of receptor kinase activity suggests a mechanism for localized actin regulation by EphA receptors. Specifically, EphA activation would result in the modulation of ephexin activity only at the site of ligand presentation, while ephexin molecules in other regions of the growth cone remain unaffected. Furthermore, because ephexin is directly associated with EphA receptors, its downstream effects on Rho GTPases may remain confined to the vicinity of the activated receptors. Thus, ephexin may be modulated by EphA receptors to elicit local changes in Rho GTPase activity and to induce spatially restricted retraction of the growth cone at points of contact with ephrin-A (Shamah, 2001).
The mechanism through which EphA receptors modulate ephexin activity is currently not clear. One intriguing possibility is that substrate specificity within the catalytic domain of ephexin can be differentially regulated to yield inhibitory effects toward some GTPases and potentiating effects toward others. Alternatively, EphA4 inhibition of ephexin signaling to Rac1 and Cdc42 might be an indirect effect of elevated RhoA activity, or vice versa. In addition, the access of ephexin to different Rho GTPases might be regulated by EphA receptors as a result of changes in Rho GTPase subcellular localization, and this could contribute to the observed changes in Rho GTPase activities (Shamah, 2001).
The EphA receptor-induced change in ephexin activity might result from a posttranslational modification of ephexin. One possibility is that EphA receptor activation leads to changes in the phosphorylation state of ephexin due to the activation of specific kinases or phosphatases. Ephexin might also be modified as a result of EphA-induced regulation of the PI3-kinase signaling pathway, since EphA4 receptors bind to multiple PI3-kinase regulatory subunits and several Eph receptors are known to activate PI3-kinase. Also, PI3-kinase lipid products have been shown to regulate the activity of Vav and alphaPIX, a Pak-interacting GEF (Shamah, 2001).
Alternatively, since the association of EphA4 receptors occurs in the catalytic DH-PH domains of ephexin, EphA modulation of ephexin might occur through reversible steric or allosteric hindrance of GEF activity. Further studies utilizing specific pharmacologic and dominant-negative reagents as well as more detailed structure-function analyses should help to elucidate the mechanism of regulation of ephexin by EphA receptors (Shamah, 2001).
In summary, the experiments described here identify ephexin as an important molecular link between EphA receptors and the Rho family of GTPases, and suggest a model for how EphA receptors may locally regulate the actin cytoskeleton during axon guidance. Additional studies with specific inhibitors of ephexin and the genetic disruption of ephexin will be required to determine the role of ephexin in axon guidance and cell migration events during development (Shamah, 2001).
Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, Xenopus Dishevelled (Xdsh) is found to form a complex with Eph receptors and ephrin-B ligands and mediate the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affects the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induces the activation of RhoA and Rho kinase in the EphB2-overexpressing cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. It is proposed that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity (Tanaka, 2003).
The function of the multi-PDZ domain scaffold protein GRIP1 (glutamate receptor interacting protein 1) in neurons is unclear. To explore the function of GRIP1 in hippocampal neurons, RNA interference (RNAi) was used to knock down the expression of GRIP1. Knockdown of GRIP1 by small interfering RNA (siRNA) in cultured hippocampal neurons causes a loss of dendrites, associated with mislocalization of the GRIP-interacting proteins GIuR2 (AMPA receptor subunit), EphB2 (receptor tyrosine kinase) and KIF5 (also known as kinesin 1; microtubule motor). The loss of dendrites by GRIP1-siRNA was rescued by overexpression of the extracellular domain of EphB2, and was phenocopied by overexpression of the intracellular domain of EphB2 and extracellular application of ephrinB-Fc fusion proteins. Neurons from EphB1-EphB2-EphB3 triple knockout mice showed abnormal dendrite morphogenesis. Disruption of the KIF5-GRIP1 interaction inhibited EphB2 trafficking and strongly impaired dendritic growth. These results indicate an important role for GRIP1 in dendrite morphogenesis by serving as an adaptor protein for kinesin-dependent transport of EphB receptors to dendrites (Hoogenraad, 2005).
Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. This study shows that the Rac-specific GTPase-activating protein α2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. α-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. These results reveal an α-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo (Wegmeyer, 2007).
Receptor tyrosine kinases of the EPH class have been implicated in the control of axon guidance and fasciculation, in regulating cell migration, and in defining compartments in the developing embryo. Efficient activation of EPH receptors generally requires that their ligands be anchored to the cell surface, either through a transmembrane (TM) region or a glycosyl phosphatidylinositol (GPI) group. These observations have suggested that EPH receptors can transduce signals initiated by direct cell-cell interaction. Genetic analysis of Nuk, a murine EPH receptor that binds TM ligands, has raised the possibility that these ligands might themselves have a signaling function. Consistent with this, the three known TM ligands have a highly conserved cytoplasmic region, with multiple potential sites for tyrosine phosphorylation. Challenging cells that express the TM ligands Elk-L or Htk-L with the clustered ectodomain of Nuk induces phosphorylation of the ligands on tyrosine, a process that can be mimicked both in vitro and in vivo by an activated Src tyrosine kinase. Co-culture of cells expressing a TM ligand with cells expressing Nuk leads to tyrosine phosphorylation of both the ligand and Nuk. These results suggest that the TM ligands are associated with a tyrosine kinase, and are inducibly phosphorylated upon binding Nuk, in a fashion reminiscent of cytokine receptors. Furthermore, TM ligands, as well as Nuk, are phosphorylated on tyrosine in mouse embryos, indicating that this is a physiological process. EPH receptors and their TM ligands therefore mediate bidirectional cell signaling (Holland, 1996).
Axonal pathfinding in the nervous system is mediated in part by cell-to-cell signaling events involving members of the Eph receptor tyrosine kinase (RTK) family and their membrane-bound ligands. Genetic evidence suggests that transmembrane ligands may transduce signals in the developing embryo. The cytoplasmic domain of the transmembrane ligand Lerk2 becomes phosphorylated on tyrosine residues after contact with the Nuk/Cek5 receptor ectodomain; this suggests that Lerk2 has receptorlike intrinsic signaling potential. Moreover, Lerk2 is an in vivo substrate for the platelet-derived growth factor receptor, which suggests crosstalk between Lerk2 signaling and signaling cascades activated by tyrosine kinases. It is proposed that transmembrane ligands of Eph receptors act not only as conventional RTK ligands but also as receptorlike signaling molecules (Bruckner, 1997).
Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with 'reverse' signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. These findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands (Bruckner, 1999).
Eph proteins are receptors with tyrosine-kinase activity which, with their ephrin ligands, mediate contact-dependent cell interactions that are implicated in the repulsion mechanisms that guide migrating cells and neuronal growth cones to specific destinations. Ephrin-B proteins have conserved cytoplasmic tyrosine residues that are phosphorylated upon interaction with an EphB receptor, and may transduce signals that regulate a cellular response. Because Eph receptors and ephrins have complementary expression in many tissues during embryogenesis, bidirectional activation of Eph receptors and ephrin-B proteins could occur at interfaces of their expression domains, for example at segment boundaries in the vertebrate hindbrain. Previous work has implicated Eph receptors and ephrin-B proteins in the restriction of cell intermingling between hindbrain segments. An analysis was carried out to see whether complementary expression of Eph receptors and ephrins restricts cell intermingling, and whether this requires bidirectional or unidirectional signaling. Bidirectional but not unidirectional signaling restricts the intermingling of adjacent cell populations, whereas unidirectional activation is sufficient to restrict cell communication through gap junctions. These results reveal that Eph receptors and ephrins regulate two aspects of cell behaviour that can stabilize a distinct identity of adjacent cell populations (Mellitzer, 1999).
Transmembrane B ephrins and their Eph receptors signal bidirectionally. However, neither the cell biological effects nor signal transduction mechanisms of the reverse signal are well understood. A cytoplasmic protein, PDZ-RGS3, is described that binds B ephrins through a PDZ domain, and has a regulator of heterotrimeric G protein signaling (RGS) domain. PDZ-RGS3 can mediate signaling from the ephrin-B cytoplasmic tail. SDF-1, a chemokine with a G protein-coupled receptor, and BDNF, both act as a chemoattractants for cerebellar granule cells, with SDF-1 action being selectively inhibited by soluble EphB receptor. This study reveals a pathway that links reverse signaling to cellular guidance, uncovers a novel mode of control for G proteins, and demonstrates a mechanism for selective regulation of responsiveness to neuronal guidance cues (Lu, 2001).
To investigate reverse signaling at a molecular level, a screen was performed for proteins that bind the B ephrin cytoplasmic domain, leading to identification of PDZ-RGS3 in a yeast two-hybrid assay. In situ hybridization shows a close overlap of expression patterns for PDZ-RGS3 with any one of the three known B ephrins in several parts of the nervous system. Taken together, these results indicate that PDZ-RGS3 is a genuine biological interaction partner of B ephrins (Lu, 2001).
PDZ domains are known to bind to a short conserved motif at the C terminus of many membrane proteins. A sequence fitting this motif is found at the C terminus of all known B ephrins, and the PDZ domain of PDZ-RGS3 binds the ephrin-B C terminus. Tyrosine residues are found in the binding motif (YYKV-carboxy terminus) suggesting potential control of binding by phosphorylation, although no evidence of this was seen, and the interaction did not appear to be regulated by EphB receptor binding. The presence of an RGS domain suggests PDZ-RGS3 might interact with downstream effector pathways. Accordingly, a Xenopus embryo cell dissociation assay PDZ-RGS3 mediates effects of the B ephrin cytoplasmic tail, in a manner dependent on both its PDZ and RGS domains. The involvement of the RGS domain in signaling, as well as the cerebellar expression of ephrins, led the authors to test cerebellar granule cells for an effect of reverse signaling on the action of the chemokine SDF-1, which acts through a GPCR. Soluble EphB2-Fc selectively regulates the guidance response to SDF-1, and this regulation is blocked by a truncated version of PDZ-RGS3 lacking the RGS domain (Lu, 2001).
Regarding the mechanism for signal transduction across the cell membrane, as with other PDZ proteins that bind B ephrins, the association with PDZ-RGS3 is seen constitutively, and does not appear to be modulated by treating cells with soluble EphB2-Fc. This suggests regulated association between B ephrin and PDZ-RGS3 is not a likely mechanism of signal transduction. An alternative could be regulation of clustering or subcellular localization. It is known that EphB2-Fc can cluster B ephrins and associated PDZ proteins into membrane rafts. Heterotrimeric G proteins have also been localized to rafts. Therefore, one model could be that Eph receptor binding clusters B ephrins into rafts, or other subcellular structures, and this could bring associated PDZ-RGS3 into proximity with the appropriate G proteins, resulting in inhibition of their activity. It is worth noting that not only the PDZ binding motif, but at least 33 amino acids of the B ephrin cytoplasmic tail are strongly conserved, and it is likely that additional protein interactions play a role in signaling, either through independent pathways or in collaboration with PDZ-RGS3 (Lu, 2001).
At the level of cell biological effects, these results show that reverse signaling induced by Eph receptor can regulate cellular guidance. Specifically, soluble EphB2-Fc selectively inhibits SDF-1 chemoattraction of cultured cerebellar granule neurons. Although reverse signaling through B ephrins has been investigated more extensively, soluble EphA receptors can affect adhesion in cell lines, and it will be interesting to see if this may reflect similar developmental functions or signaling pathways. The observations on the regulation of cerebellar granule cell guidance by EphB2-Fc, SDF-1, and BDNF led to a model for control of cell migration in cerebellar development. In principle, these observations could also fit with other developmental functions proposed for B ephrin reverse signaling in blood vessel formation, rhombomere compartmentation, and axon pathway selection -- all involving regulation of migration or morphogenesis (Lu, 2001).
The inward migration of cerebellar granule cells from the EGL is one of the best-characterized models of neuronal migration. The genetic demonstration that SDF-1 and its receptor CXCR4 are required for normal granule cell migration provided the first evidence of chemokines as regulators of neural development. Specifically, the phenotype of premature granule cell migration, taken together with the embryonic expression of SDF-1 in the pia mater overlying the cerebellum, suggest a model where SDF-1 would prevent premature inward migration of cerebellar granule cells by chemoattracting them toward the pia. The results support this model: SDF-1 expression occurs in the pia at postnatal stages that span the onset of granule cell migration, and SDF-1 acts as a chemoattractant for cultured cerebellar granule cells. Reverse signaling induced by soluble EphB2-Fc can inhibit the effect of SDF-1 on cerebellar granule cells. This provides functional evidence for an effect of ephrin signaling on cerebellar granule cells. A developmental role for the interaction of these signaling pathways is supported by the correlated expression of ephrin-B2, SDF-1, and their receptors during cerebellar development (Lu, 2001).
The number of cells in an organ is regulated by mitogens and trophic factors that impinge on intrinsic determinants of proliferation and apoptosis. This study reports on the identification of an additional mechanism to control cell number in the brain: EphA7 induces ephrin-A2 reverse signaling, which negatively regulates neural progenitor cell proliferation. Cells in the neural stem cell niche in the adult brain proliferate more and have a shorter cell cycle in mice lacking ephrin-A2. The increased progenitor proliferation is accompanied by a higher number of cells in the olfactory bulb. Disrupting the interaction between ephrin-A2 and EphA7 in the adult brain of wild-type mice disinhibits proliferation and results in increased neurogenesis. The identification of ephrin-A2 and EphA7 as negative regulators of progenitor cell proliferation reveals a novel mechanism to control cell numbers in the brain (Holmberg, 2005).
EphA2 is regulated by E-cadherin. In nonneoplastic epithelia, EphA2 is tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties require the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 is decreased, and EphA2 is redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restores a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreases cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2 (Zantek, 1999).
In humans, fetal cytotrophoblasts leave the placenta and enter the uterine wall, where they preferentially remodel arterioles. The fundamental mechanisms that govern these processes are largely unknown. Invasive cytotrophoblasts express several chemokines, as well as the receptors with which they interact. These ligand-receptor interactions stimulate cytotrophoblast migration to approximately the same level as a growth factor cocktail that includes serum. Additionally, cytotrophoblast commitment to uterine invasion is accompanied by rapid downregulation of EPHB4, a transmembrane receptor associated with venous identity, and upregulation of ephrin B1. Within the uterine wall, the cells also upregulated expression of ephrin B2, an EPH transmembrane ligand that is associated with arterial identity. In vitro cytotrophoblasts avoid EPHB4-coated substrates; upon co-culture with 3T3 cells expressing this molecule, their migration is significantly inhibited. As to the mechanisms involved, cytotrophoblast interactions with EPHB4 downregulate chemokine-induced but not growth factor-stimulated migration. It is proposed that EPHB4/ephrin B1 interactions generate repulsive signals that direct cytotrophoblast invasion toward the uterus, where chemokines stimulate cytotrophoblast migration through the decidua. the mucous membrane lining the uterus. When cytotrophoblasts encounter EPHB4 expressed by venous endothelium, ephrin B-generates repulsive signals and a reduction in chemokine-mediated responses limit cytotrophoblast interaction with veins. When cytotrophoblasts encounter ephrin B2 ligands expressed in uterine arterioles, migration is permitted. The net effect is preferential cytotrophoblast remodeling of arterioles, a hallmark of human placentation (Red-Horse, 2005).
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