SHEP1 function in cell migration is impaired by a single amino acid mutation that disrupts association with the scaffolding protein cas but not with Ras GTPases

SHEP1 is a signaling protein that contains a guanine nucleotide exchange factor-like domain, which binds Ras family GTPases and also forms a stable complex with the scaffolding protein Crk-associated substrate (Cas). SHEP1 and Cas have several common functions, such as increasing c-Jun N-terminal kinase activity, promoting T cell activation, and regulating the actin cytoskeleton. However, it is unclear whether a physical association between SHEP1 and Cas is required for these activities. We reported previously that SHEP1 is tyrosine-phosphorylated downstream of the EphB2 receptor; in this study, we further demonstrate that activated EphB2 inhibits SHEP1 association with Cas. To investigate whether phosphorylation negatively regulates the SHEP1-Cas complex, we have identified by mass spectrometry several SHEP1 tyrosine phosphorylation sites downstream of EphB2; of particular interest among them is tyrosine 635 in the Cas association/exchange factor domain. Mutation of this tyrosine to glutamic acid, but not to phenylalanine, disrupts Cas binding to SHEP1 without inhibiting Ras GTPase binding. The glutamic acid mutation also makes SHEP1 unable to promote Cas-Crk association, membrane ruffling, and cell migration toward epidermal growth factor (EGF), implying that these activities of SHEP1 depend upon a physical interaction with Cas. Association with Cas also seems to be necessary for EGF-induced SHEP1 tyrosine phosphorylation, which is mediated by a Src family kinase. It is noteworthy that EGF stimulation does not cause dissociation of SHEP1 from Cas. These data show that SHEP1 regulates membrane ruffling and cell migration and that binding to Cas is probably critical for these functions. Furthermore, the SHEP1-Cas complex may have different roles downstream of EphB2 and the EGF receptor.     

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.     

Splice variants and expression patterns of SHEP1, BCAR3 and NSP1, a gene family involved in integrin and receptor tyrosine kinase signaling

SHEP1, BCAR3 and NSP1 are the three members of a family of cytoplasmic proteins involved in cell adhesion/migration and antiestrogen resistance. All three proteins contain an SH2 domain and an exchange factor-like domain that binds both Ras GTPases and the scaffolding protein Cas. SHEP1, BCAR3 and NSP1 mRNAs are widely expressed in tissues, and SHEP1 and BCAR3 have multiple splice variants that differ in their 5' untranslated regions and in some cases the beginning of their coding regions. Interestingly, our data suggest that SHEP1 is highly expressed in blood vessels in mouse breast cancer models. In contrast, BCAR3 and NSP1 are more highly expressed than SHEP1 in breast cancer cells. These expression patterns suggest differential roles for the three genes during breast cancer progression in either the vasculature or the tumor cells.     

The kinase-null EphB6 receptor undergoes transphosphorylation in a complex with EphB1.

Uniquely for the Eph family of receptor tyrosine kinases, the EphB6 receptor is catalytically inactive due to the alteration of several critical residues in its kinase domain. This has cast doubt upon its ability to participate in cytoplasmic signaling events. We show here that despite its lack of kinase activity, EphB6 undergoes inducible tyrosine phosphorylation upon stimulation with the Eph-B receptor subfamily ligand ephrin-B1. We also demonstrate, for the first time, evidence of cross-talk between Eph receptors. Overexpression of a catalytically active member of the Eph-B subfamily, EphB1, resulted in increased EphB6 phosphorylation. EphB1-induced EphB6 phosphorylation was ligand-dependent and required the functional catalytic activity of EphB1. EphB1 not only transphosphorylated EphB6, but together they also formed a stable hetero-complex. In addition, we identify the proto-oncogene c-Cbl as an EphB6-binding protein. Although EphB6-Cbl association appeared to be constitutive, Cbl required a functional phosphotyrosine binding domain in order to bind the receptor, whereas its RING finger motif ubiquitin-transfer domain was not necessary. Our findings demonstrate that EphB6 is an actively signaling receptor that undergoes transphosphorylation upon ligand binding and that can initiate specific cytoplasmic signaling events.     

Manipulation of EphB2 regulatory motifs and SH2 binding sites switches MAPK signaling and biological activity

Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.     

Eph receptor-ephrin bidirectional signals that target Ras and Rho proteins

The ability of cells to respond to their surrounding environment and relay signals to the cell interior is essential for numerous processes during the development and maintenance of tissues. Eph receptors and their membrane-bound ligands, the ephrins, are unique in the receptor tyrosine kinase family in that their signaling is bidirectional, through both the receptor and the ligand. Eph receptors and ephrins are essential for a variety of biological processes, and play a particularly important role in regulating cell shape and cell movement. Recent data have linked Eph receptor-ephrin signaling complexes to the Ras and Rho families of small molecular weight GTPases and also to heterotrimeric G proteins. Understanding the signaling networks involved is an important step to understand the molecular basis for normal and defective cell-cell communication through Eph receptors and ephrins.     

Cupredoxin-cancer interrelationship: azurin binding with EphB2, interference in EphB2 tyrosine phosphorylation, and inhibition of cancer growth

Azurin is a member of a family of metalloproteins called cupredoxins. Although previously thought to be involved in electron transfer, azurin has recently been shown to preferentially enter cancer cells than normal cells and induce apoptosis in such cells. Azurin also demonstrates structural similarity to a ligand known as ephrinB2, which binds its cognate receptor tyrosine kinase EphB2 to initiate cell signaling. Eph/ephrin signaling is known to be involved in cancer progression. We now demonstrate that azurin binds to the EphB2-Fc receptor with high affinity. We have localized a C-terminal domain of azurin (Azu 96-113) that exhibits structural similarity to ephrinB2 at the G-H loop region known to be involved in receptor binding. A synthetic peptide (Azu 96-113) as well as a GST fusion derivative GST-Azu 88-113 interferes with the growth of various human cancer cells. In a prostate cancer cell line DU145 lacking functional EphB2, azurin or its GST-fusion derivatives had little cytotoxic effect. However, in DU145 cells expressing functional EphB2, azurin and GST-Azu 88-113 demonstrated significant cytotoxicity, whereas ephrinB2 promoted cell growth. Azurin inhibited the ephrinB2-mediated autophosphorlyation of the EphB2 tyrosine residue, thus interfering in upstream cell signaling and contributing to cancer cell growth inhibition.     

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase.

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.     

The Eph Tyrosine Kinase Receptors EphB2 and EphA2 Are Novel Proteolytic Substrates of Tissue Factor/Coagulation Factor VIIa

Tissue factor (TF) binds the serine protease factor VIIa (FVIIa) to form a proteolytically active complex that can trigger coagulation or activate cell signaling. Here we addressed the involvement of tyrosine kinase receptors (RTKs) in TF/FVIIa signaling by antibody array analysis and subsequently found that EphB2 and EphA2 of the Eph RTK family were cleaved in their ectodomains by TF/FVIIa. We used N-terminal Edman sequencing and LC-MS/MS analysis to characterize the cleaved Eph isoforms and identified a key arginine residue at the cleavage site, in agreement with the tryptic serine protease activity of FVIIa. Protease-activated receptor 2 (PAR2) signaling and downstream coagulation activity was non-essential in this context, in further support of a direct cleavage by TF/FVIIa. EphB2 was cleaved by FVIIa concentrations in the subnanomolar range in a number of TF expressing cell types, indicating that the active cellular pool of TF was involved. FVIIa caused potentiation of cell repulsion by the EphB2 ligand ephrin-B1, demonstrating a novel proteolytical event to control Eph-mediated cell segregation. These results define Eph RTKs as novel proteolytical targets of TF/FVIIa and provide new insights into how TF/FVIIa regulates cellular functions independently of PAR2.     

In vivo tyrosine phosphorylation sites of activated ephrin-B1 and ephB2 from neural tissue

EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 previously identified as phosphorylation sites in 293 cells (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38, 14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex.     

Dancing with the dead: Eph receptors and their kinase-null partners

Eph receptor tyrosine kinases and their ligands, ephrins, are membrane proteins coordinating a wide range of biological functions both in developing embryos and in adult multicellular organisms. Numerous studies have implicated Eph receptors in the induction of opposing responses, including cell adhesion or repulsion, support or inhibition of cell proliferation and cell migration, and progression or suppression of multiple malignancies. Similar to other receptor tyrosine kinases, Eph receptors rely on their ability to catalyze tyrosine phosphorylation for signal transduction. Interestingly, however, Eph receptors also actively utilize three kinase-deficient receptor tyrosine kinases, EphB6, EphA10, and Ryk, in their signaling network. The accumulating evidence suggests that the unusual flexibility of the Eph family, allowing it to initiate antagonistic responses, might be partially explained by the influence of the kinase-dead participants and that the exact outcome of an Eph-mediated action is likely to be defined by the balance between the signaling of catalytically potent and catalytically null receptors. We discuss in this minireview the emerging functions of the kinase-dead EphB6, EphA10, and Ryk receptors both in normal biological responses and in malignancy, and analyze currently available information related to the molecular mechanisms of their action in the context of the Eph family.     

Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity.

During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors were recently shown to associate with NMDA receptors at synaptic sites and were suggested to play a role in synaptogenesis. Here we show that Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2(-/-) hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) was impaired, and two forms of synaptic depression were completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescued the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions.     

Biphasic functions of the kinase-defective Ephb6 receptor in cell adhesion and migration

EphB6 is a unique member in the Eph family of receptor tyrosine kinases in that its kinase domain contains several alterations in conserved amino acids and is catalytically inactive. Although EphB6 is expressed both in a variety of embryonic and adult tissues, biological functions of this receptor are largely unknown. In the present study, we examined the function of EphB6 in cell adhesion and migration. We demonstrated that EphB6 exerted biphasic effects in response to different concentrations of the ephrin-B2 ligand; EphB6 promoted cell adhesion and migration when stimulated with low concentrations of ephrin-B2, whereas it induced repulsion and inhibited migration upon stimulation with high concentrations of ephrin-B2. A truncated EphB6 receptor lacking the cytoplasmic domain showed monophasic-positive effects on cell adhesion and migration, indicating that the cytoplasmic domain is essential for the negative effects. EphB6 is constitutively associated with the Src family kinase Fyn. High concentrations of ephrin-B2 induced tyrosine phosphorylation of EphB6 through an Src family kinase activity. These results indicate that EphB6 can both positively and negatively regulate cell adhesion and migration, and suggest that tyrosine phosphorylation of the receptor by an Src family kinase acts as the molecular switch for the functional transition.     

Sizes of interface residues account for cross‐class binding affinity patterns in Eph receptor–ephrin families

Eph receptors comprise the largest known family of receptor tyrosine kinases in mammals. They bind members of a second family, the ephrins. As both Eph receptors and ephrins are membrane bound, interactions permit unusual bidirectional cell–cell signaling. Eph receptors and ephrins each form two classes, A and B, based on sequences, structures, and patterns of affinity: Class A Eph receptors bind class A ephrins, and class B Eph receptors bind class B ephrins. The only known exceptions are the receptor EphA4, which can bind ephrinB2 and ephrinB3 in addition to the ephrin‐As (Bowden et al., Structure 2009;17:1386–1397); and EphB2, which can bind ephrin‐A5 in addition to the ephrin‐Bs (Himanen et al., Nat Neurosci 2004;7:501–509). A crystal structure is available of the interacting domains of the EphA4‐ephrin B2 complex (wwPDB entry 2WO2) (Bowden et al., Structure 2009;17:1386–1397). In this complex, the ligand‐binding domain of EphA4 adopts an EphB‐like conformation. To understand why other cross‐class EphA receptor–ephrinB complexes do not form, we modeled hypothetical complexes between (1) EphA4–ephrinB1, (2) EphA4–ephrinB3, and (3) EphA2–ephrinB2. We identify particular residues in the interface region, the size variations of which cause steric clashes that prevent formation of the unobserved complexes. The sizes of the sidechains of residues at these positions correlate with the pattern of binding affinity. Proteins 2014; 82:349–353. © 2013 Wiley Periodicals, Inc.     

Binding of Rac1, Rnd1, and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 effector domain

Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, beta-strands 3 and 4 and a short alpha-helical segment of the plexin domain. The 2.0 A resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel beta-sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.     

Signal transfer by Eph receptors

The Eph receptors are a unique family of receptor tyrosine kinases that enforce cellular position in tissues through mainly repulsive signals generated upon cell-cell contact. Together, Eph receptors and their membrane-anchored ligands. the ephrins, are key molecules for establishing tissue organization through signaling pathways that control axonal projection, cell migration, and the maintenance of cellular boundaries. Through their SH2 (Src Homology 2) and PDZ (postsynaptic density protein, disks large, zona occludens) domains, several signaling molecules have been demonstrated to interact with the activated cytoplasmic domain of Eph receptors by using the yeast two-hybrid system and in vitro biochemical assays. Most proteins found to interact with Eph receptors are well-known regulators of cytoskeletal organization and cell adhesion, and also cell proliferation. Promoting growth, however, does not appear to be a primary role of Eph receptors. Explaining which signaling interactions identified for the Eph receptors have physiological significance, how Eph receptor signaling cascades are propagated, and characterizing the intrinsic signaling properties of the ephrins are all exciting questions currently being investigated.     

EphB1 recruits c-Src and p52Shc to activate MAPK/ERK and promote chemotaxis

Eph receptors and their ligands (ephrins) play an important role in axonal guidance, topographic mapping, and angiogenesis. The signaling pathways mediating these activities are starting to emerge and are highly cell- and receptor-type specific. Here we demonstrate that activated EphB1 recruits the adaptor proteins Grb2 and p52Shc and promotes p52Shc and c-Src tyrosine phosphorylation as well as MAPK/extracellular signal-regulated kinase (ERK) activation. EphB1-mediated increase of cell migration was abrogated by the MEK inhibitor PD98059 and Src inhibitor PP2. In contrast, cell adhesion, which we previously showed to be c-jun NH2-terminal kinase (JNK) dependent, was unaffected by ERK1/2 and Src inhibition. Expression of dominant-negative c-Src significantly reduced EphB1-dependent ERK1/2 activation and chemotaxis. Site-directed mutagenesis experiments demonstrate that tyrosines 600 and 778 of EphB1 are required for its interaction with c-Src and p52Shc. Furthermore, phosphorylation of p52Shc by c-Src is essential for its recruitment to EphB1 signaling complexes through its phosphotyrosine binding domain. Together these findings highlight a new aspect of EphB1 signaling, whereby the concerted action of c-Src and p52Shc activates MAPK/ERK and regulates events involved in cell motility.     

Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina

Optic nerve formation requires precise retinal ganglion cell (RGC) axon pathfinding within the retina to the optic disc, the molecular basis of which is not well understood. At CNS targets, interactions between Eph receptor tyrosine kinases on RGC axons and ephrin ligands on target cells have been implicated in formation of topographic maps. However, studies in chick and mouse have shown that both Eph receptors and ephrins are also expressed within the retina itself, raising the possibility that this receptor-ligand family mediates aspects of retinal development. Here, we more fully document the presence of specific EphB receptors and B-ephrins in embryonic mouse retina and provide evidence that EphB receptors are involved in RGC axon pathfinding to the optic disc. We find that as RGC axons begin this pathfinding process, EphB receptors are uniformly expressed along the dorsal-ventral retinal axis. This is in contrast to the previously reported high ventral-low dorsal gradient of EphB receptors later in development when RGC axons map to CNS targets. We show that mice lacking both EphB2 and EphB3 receptor tyrosine kinases, but not each alone, exhibit increased frequency of RGC axon guidance errors to the optic disc. In these animals, major aspects of retinal development and cellular organization appear normal, as do the expression of other RGC guidance cues netrin, DCC, and L1. Unexpectedly, errors occur in dorsal but not ventral retina despite early uniform or later high ventral expression of EphB2 and EphB3. Furthermore, embryos lacking EphB3 and the kinase domain of EphB2 do not show increased errors, consistent with a guidance role for the EphB2 extracellular domain. Thus, while Eph kinase function is involved in RGC axon mapping in the brain, RGC axon pathfinding within the retina is partially mediated by EphB receptors acting in a kinase-independent manner.     

Retinal axon growth cones respond to EphB extracellular domains as inhibitory axon guidance cues

Axon pathfinding relies on cellular signaling mediated by growth cone receptor proteins responding to ligands, or guidance cues, in the environment. Eph proteins are a family of receptor tyrosine kinases that govern axon pathway development, including retinal axon projections to CNS targets. Recent examination of EphB mutant mice, however, has shown that axon pathfinding within the retina to the optic disc is dependent on EphB receptors, but independent of their kinase activity. Here we show a function for EphB1, B2 and B3 receptor extracellular domains (ECDs) in inhibiting mouse retinal axons when presented either as substratum-bound proteins or as soluble proteins directly applied to growth cones via micropipettes. In substratum choice assays, retinal axons tended to avoid EphB-ECDs, while time-lapse microscopy showed that exposure to soluble EphB-ECD led to growth cone collapse or other inhibitory responses. These results demonstrate that, in addition to the conventional role of Eph proteins signaling as receptors, EphB receptor ECDs can also function in the opposite role as guidance cues to alter axon behavior. Furthermore, the data support a model in which dorsal retinal ganglion cell axons heading to the optic disc encounter a gradient of inhibitory EphB proteins which helps maintain tight axon fasciculation and prevents aberrant axon growth into ventral retina. In conclusion, development of neuronal connectivity may involve the combined activity of Eph proteins serving as guidance receptors and as axon guidance cues.     

Ephrin-B reverse signaling is mediated by a novel PDZ-RGS protein and selectively inhibits G protein-coupled chemoattraction

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. We describe a cytoplasmic protein, PDZ-RGS3, which 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, or BDNF, act as 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.