The activation of ezrin-radixin-moesin proteins is regulated by netrin-1 through Src kinase and RhoA/Rho kinase activities and mediates netrin-1-induced axon outgrowth

The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42. The molecular mechanisms that link netrin-1/DCC to the actin machinery remain unclear. In this study we seek to demonstrate that the actin-binding proteins ezrin-radixin-moesin (ERM) are effectors of netrin-1/DCC signaling in embryonic cortical neurons. We show that ezrin associates with DCC in a netrin-1-dependent manner. We demonstrate that netrin-1/DCC induces ERM phosphorylation and activation and that the phosphorylation of DCC is required in that context. Moreover, Src kinases and RhoA/Rho kinase activities mediate netrin-1-induced ERM phosphorylation in neurons. We also observed that phosphorylated ERM proteins accumulate in growth cone filopodia, where they colocalize with DCC upon netrin-1 stimulation. Finally, we show that loss of ezrin expression in cortical neurons significantly decreases axon outgrowth induced by netrin-1. Together, our findings demonstrate that netrin-1 induces the formation of an activated ERM/DCC complex in growth cone filopodia, which is required for netrin-1-dependent cortical axon outgrowth.     

Trio mediates netrin-1-induced Rac1 activation in axon outgrowth and guidance

The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio(-/-) cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio(-/-) spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.     

Tyrosine Phosphorylation of the Rho Guanine Nucleotide Exchange Factor Trio Regulates Netrin-1/DCC-Mediated Cortical Axon Outgrowth

The chemotropic guidance cue netrin-1 mediates attraction of migrating axons during central nervous system development through the receptor Deleted in Colorectal Cancer (DCC). Downstream of netrin-1, activated Rho GTPases Rac1 and Cdc42 induce cytoskeletal rearrangements within the growth cone. The Rho guanine nucleotide exchange factor (GEF) Trio is essential for Rac1 activation downstream of netrin-1/DCC, but the molecular mechanisms governing Trio activity remain elusive. Here, we demonstrate that Trio is phosphorylated by Src family kinases in the embryonic rat cortex in response to netrin-1. In vitro, Trio was predominantly phosphorylated at Tyr²⁶²² by the Src kinase Fyn. Though the phospho-null mutant Trio^Y2622F retained GEF activity toward Rac1, its expression impaired netrin-1-induced Rac1 activation and DCC-mediated neurite outgrowth in N1E-115 neuroblastoma cells. Trio^Y2622F impaired netrin-1-induced axonal extension in cultured cortical neurons and was unable to colocalize with DCC in growth cones, in contrast to wild-type Trio. Furthermore, depletion of Trio in cortical neurons reduced the level of cell surface DCC in growth cones, which could be restored by expression of wild-type Trio but not Trio^Y2622F. Together, these findings demonstrate that Trio^Y2622 phosphorylation is essential for the regulation of the DCC/Trio signaling complex in cortical neurons during netrin-1-mediated axon outgrowth.     

Depolarization recruits DCC to the plasma membrane of embryonic cortical neurons and enhances axon extension in response to netrin-1

The netrin-1 receptor Deleted in Colorectal Cancer (DCC) is required for the formation of major axonal projections by embryonic cortical neurons, including the corpus callosum, hippocampal commissure, and cortico-thalamic tracts. The presentation of DCC by axonal growth cones is tightly regulated, but the mechanisms regulating DCC trafficking within neurons are not well understood. Here, we investigated the mechanisms regulating DCC recruitment to the plasma membrane of embryonic cortical neurons. In embryonic spinal commissural neurons, protein kinase A (PKA) activation recruits DCC to the plasma membrane and enhances axon chemoattraction to netrin-1. We demonstrate that PKA activation similarly recruits DCC and increases embryonic cortical neuron axon extension, which, like spinal commissural neurons, respond to netrin-1 as a chemoattractant. We then determined if depolarization might recruit DCC to the plasma membrane. Neither netrin-1 induced axon extension, nor levels of plasma membrane DCC, were altered by depolarizing embryonic spinal commissural neurons with elevated levels of KCl. In contrast, depolarizing embryonic cortical neurons increased the amount of plasma membrane DCC, including at the growth cone, and increased axon outgrowth evoked by netrin-1. Inhibition of PKA, phosphatidylinositol-3-kinase, protein kinase C, or exocytosis blocked the depolarization-induced recruitment of DCC and suppressed axon outgrowth. Inhibiting protein synthesis did not affect DCC recruitment, nor were the distributions of trkB or neural cell adhesion molecule (NCAM) influenced by depolarization, consistent with selective mobilization of DCC. These findings identify a role for membrane depolarization modulating the response of axons to netrin-1 by regulating DCC recruitment to the plasma membrane.     

Tyrosine phosphorylation of netrin receptors in netrin-1 signaling

Deleted in colorectal cancer (DCC) and neogenin are receptors of netrins, a family of guidance cues that promote axon outgrowth and guide growth cones in developing nervous system. The intracellular mechanisms of netrins, however, remain elusive. In this paper, we show that both DCC and neogenin become tyrosine phosphorylated in cortical neurons in response to netrin-1. Using a site-specific antiphosphor DCC antibody, we show that Y1420 phosphorylation is increased in netrin-1-stimulated neurons and that tyrosine-phosphorylated DCC is located in growth cones. In addition, we show that tyrosine-phosphorylated DCC selectively interacts with the Src family kinases Fyn and Lck, but not Src, c-Abl, Grb2, SHIP1, Shc, or tensin, suggesting a role of Fyn or Lck in netrin-1-DCC signaling. Of interest to note is that tyrosine-phosphorylated neogenin and uncoordinated 5 H2 (Unc5H2) not only bind to the Src homology 2 (SH2) domains of Fyn and SHP2, but also interact with the SH2 domain of SHIP1, suggesting a differential signaling between DCC and neogenin/Unc5H2. Furthermore, we demonstrate that inhibition of Src family kinase activity attenuated netrin-1-induced neurite outgrowth. Together, these results suggest a role of Src family kinases and tyrosine phosphorylation of netrin-1 receptors in regulating netrin-1 function.     

Amyloid Precursor Protein Regulates Netrin-1-mediated Commissural Axon Outgrowth

The multifunctional protein netrin-1 was initially discovered as the main attractive cue for commissural axon guidance by acting through its receptor DCC. Recently, we have shown that netrin-1 also interacts with the orphan transmembrane receptor amyloid precursor protein (APP). APP is cleaved by proteases, generating amyloid-β peptide, the main component of the amyloid plaques that are associated with Alzheimer disease. Our previous work demonstrated that via its interaction with APP, netrin-1 is a negative regulator of amyloid-β production in adult brain, but the biological relevance of APP/netrin-1 interaction under non-pathological conditions was unknown. We show here that during commissural axon navigation, APP, expressed at the growth cone, is part of the DCC receptor complex mediating netrin-1-dependent axon guidance. APP interacts with DCC in the presence of netrin-1 and enhances netrin-1-mediated DCC intracellular signaling, such as MAPK activation. Inactivation of APP in mice is associated with reduced commissural axon outgrowth. Thus, APP functionally acts as a co-receptor for DCC to mediate axon guidance.     

Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1

During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1–induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator. Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase–deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1–induced Rac1 activation. Hsc70 was required for netrin-1–mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance.     

Phosphatidylinositol transfer protein-alpha in netrin-1-induced PLC signalling and neurite outgrowth

Neurite extension is essential for wiring the nervous system during development. Although several factors are known to regulate neurite outgrowth, the underlying mechanisms remain unclear. Here, we provide evidence for a role of phosphatidylinositol transfer protein-alpha (PlTPalpha) in neurite extension in response to netrin-1, an extracellular guidance cue. PlTPalpha interacts with the netrin receptor DCC (deleted in colorectal cancer) and neogenin. Netrin-1 stimulates PlTPalpha binding to DCC and to phosphatidylinositol (5) phosphate [Pl(5)P], increases its lipid-transfer activity and elevates hydrolysis of phosphatidylinositol bisphosphate (PlP2). In addition, the stimulated PIP2 hydrolysis requires PlTPalpha. Furthermore, cortical explants of PlTPalpha mutant mice are defective in extending neurites in response to netrin-1. Commissural neurons from chicken embryos expressing a dominant-negative PlTPalpha mutant show reduced axon outgrowth. Morpholino-mediated knockdown of PlTPalpha expression in zebrafish embryos leads to dose-dependent defects in motor-neuron axons and reduced numbers of spinal-cord neurons. Taken together, these results identify a crucial role for PlTPalpha in netrin-1-induced neurite outgrowth, revealing a signalling mechanism for DCC/neogenin and PlTPalpha regulation.     

Glu-256 is a main structural determinant for oligomerisation of human arginase I

The role of RasGAP was investigated in the model system of Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) stimulated by fibroblast growth factor 1 (FGF1). The injection of the SH2-SH3-SH2 domains of RasGAP suppressed Ras activity, extracellular signal-regulated protein kinase 2 (ERK2) phosphorylation and Mos synthesis. The SH2 domain of Src, and PP2, an inhibitor of Src, also abolished Ras activity, ERK2 phosphorylation and Mos synthesis. In addition, Src activity was blocked by the SH2-SH3-SH2 domains of RasGAP. Immunoprecipitation of a chimera composed of the extracellular domain of the platelet-derived growth factor (PDGF) receptor and the intracellular domain of FGFR1 stimulated by PDGF-BB demonstrates the recruitment of phosphorylated RasGAP. This study shows that the transduction cascade induced by the FGFR1-FGF1 interaction in Xenopus oocytes involves RasGAP as a co-activator of Src to stimulate the Ras/mitogen-activated protein kinase cascade and Mos synthesis. It emphasises a new positive regulatory role for RasGAP in FGFR transduction.     

The adaptor protein Nck-1 couples the netrin-1 receptor DCC (deleted in colorectal cancer) to the activation of the small GTPase Rac1 through an atypical mechanism

Netrins are a family of secreted proteins that guide the migration of cells and axonal growth cones during development. DCC (deleted in colorectal cancer) is a receptor for netrin-1 implicated in mediating these responses. Here, we show that DCC interacts constitutively with the SH3/SH2 adaptor Nck in commissural neurons. This interaction is direct and requires the SH3 but not SH2 domains of Nck-1. Moreover, both DCC and Nck-1 associate with the actin cytoskeleton, and this association is mediated by DCC. A dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in N1E-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of mammalian Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance.     

Rac1 and Cdc42 but not RhoA or Rho kinase activities are required for neurite outgrowth induced by the Netrin-1 receptor DCC (deleted in colorectal cancer) in N1E-115 neuroblastoma cells

Netrins are chemotropic guidance cues that attract or repel growing axons during development. DCC (deleted in colorectal cancer), a transmembrane protein that is a receptor for netrin-1, is implicated in mediating both responses. However, the mechanism by which this is achieved remains unclear. Here we report that Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using N1E-115 neuroblastoma cells, we found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In contrast, down-regulation of RhoA and its effector Rho kinase stimulates the ability of DCC to induce neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1 but not Cdc42 or RhoA. We detected that stimulation of DCC receptors with netrin-1 resulted in a 4-fold increase in Rac1 activation. These results implicate the small GTPases Rac1, Cdc42, and RhoA as essential components that participate in signaling the response of axons to netrin-1 during neural development.     

Myosin X regulates netrin receptors and functions in axonal path-finding

Netrins regulate axon path-finding during development, but the underlying mechanisms are not well understood. Here, we provide evidence for the involvement of the unconventional myosin X (Myo X) in netrin-1 function. We find that Myo X interacts with the netrin receptor deleted in colorectal cancer (DCC) and neogenin, a DCC-related protein. Expression of Myo X redistributes DCC to the cell periphery or to the tips of neurites, whereas its silencing prevents DCC distribution in neurites. Moreover, expression of DCC, but not neogenin, stimulates Myo X-mediated formation and elongation of filopodia, suggesting that Myo X function may be differentially regulated by DCC and neogenin. The involvement of Myo X in netrin-1 function was further supported by the effects of inhibiting Myo X function in neurons. Cortical explants derived from mouse embryos expressing a motor-less Myo X exhibit reduced neurite outgrowth in response to netrin-1 and chick commissural neurons expressing the motor-less Myo X, or in which Myo X is silenced using microRNA (miRNA), show impaired axon projection in vivo. Taken together, these results identify a novel role for Myo X in regulating netrin-1 function.     

p200 RhoGAP promotes cell proliferation by mediating cross-talk between Ras and Rho signaling pathways

p200 RhoGAP, a member of the Rho GTPase-activating protein (RhoGAP) family, was previously implicated in the regulation of neurite outgrowth through its RhoGAP activity. Here we show that ectopic expression of p200 RhoGAP stimulates fibroblast cell proliferation and cell cycle progression, leading to transformation. The morphology of the foci induced by p200 RhoGAP is distinct from that formed by Rac or Rho activation but similar to that induced by oncogenic Ras, raising the possibility that p200 RhoGAP may engage Ras signaling. Expression of p200 RhoGAP results in a significant increase of Ras-GTP and the activation of two downstream signaling pathways of Ras, ERK1/2 and phosphatidylinositol 3-kinase. Inhibition of Ras or ERK1/2, but not phosphatidylinositol 3-kinase, effectively suppresses the foci formation induced by p200 RhoGAP, suggesting that the Ras-ERK pathway is required for p200 RhoGAP-mediated cell transformation. p200 RhoGAP co-localizes with p120 RasGAP in cells and forms a complex with p120 RasGAP, and this interaction is mediated by the C-terminal region and the Src homology 3 domain of p200 RhoGAP and p120 RasGAP, respectively. Mutations of p200 RhoGAP that disrupt interaction with p120 RasGAP abolish its Ras activation and cell transforming activities. Interestingly, the RhoGAP activity of the N-terminal RhoGAP domain in p200 RhoGAP is also required for its full transforming activity, and expression of a dominant negative RhoA mutant that blocks RhoA cycling between the GDP- and GTP-bound states suppresses p200 RhoGAP transformation. These results suggest that a Rho GTPase-activating protein may have a positive input to cell proliferation and provide evidence that p200 RhoGAP can mediate cross-talks between Ras- and Rho-regulated signaling pathways in cell growth regulation.     

AMPK interacts with DSCAM and plays an important role in Netrin-1 induced neurite outgrowth

Down syndrome cell adhesion molecule (DSCAM) acts as a netrin-1 receptor and mediates attractive response of axons to netrin-1 in neural development. However, the signaling mechanisms of netrin-DSCAM remain unclear. Here we report that AMP-activated protein kinase (AMPK) interacts with DSCAM through its γ subunit, but does not interact with DCC (deleted in colorectal cancer), another major receptor for netrin-1. Netrin-treatment of cultured cortical neurons leads to increased phosphorylation of AMPK. Both AMPK mutant with dominant-negative effect and AMPK inhibitor can significantly suppress netrin-1 induced neurite outgrowth. Together, these findings demonstrate that AMPK interacts with DSCAM and plays an important role in netrin-1 induced neurite outgrowth. Our study uncovers a previously unknown component, AMPK, in netrin-DSCAM signaling pathway.     

DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1

During nervous system development, spinal commissural axons project toward and across the ventral midline. They are guided in part by netrin-1, made by midline cells, which attracts the axons by activating the netrin receptor DCC. However, previous studies suggest that additional receptor components are required. Here, we report that the Down's syndrome Cell Adhesion Molecule (DSCAM), a candidate gene implicated in the mental retardation phenotype of Down's syndrome, is expressed on spinal commissural axons, binds netrin-1, and is necessary for commissural axons to grow toward and across the midline. DSCAM and DCC can each mediate a turning response of these neurons to netrin-1. Similarly, Xenopus spinal neurons exogenously expressing DSCAM can be attracted by netrin-1 independently of DCC. These results show that DSCAM is a receptor that can mediate turning responses to netrin-1 and support a key role for netrin/DSCAM signaling in commissural axon guidance in vertebrates.     

DCC-dependent phospholipase C signaling in netrin-1-induced neurite elongation

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cgamma (PLCgamma) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.     

Plexin‐B1 is a GTPase activating protein for M‐Ras,remodelling dendrite morphology

Plexins are receptors for axonal guidance molecules known as semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin‐B1, induces axonal growth cone collapse by functioning as an R‐Ras GTPase activating protein (GAP). Here, we report that Plexin‐B1 shows GAP activity for M‐Ras, another member of the Ras family of GTPases. In cortical neurons, the expression of M‐Ras was upregulated during dendritic development. Knockdown of endogenous M‐Ras—but not R‐Ras—reduced dendritic outgrowth and branching, whereas overexpression of constitutively active M‐Ras, M‐Ras(Q71L), enhanced dendritic outgrowth and branching. Sema4D suppressed M‐Ras activity and reduced dendritic outgrowth and branching, but this reduction was blocked by M‐Ras(Q71L). M‐Ras(Q71L) stimulated extracellular signal‐regulated kinase (ERK) activation, inducing dendrite growth, whereas Sema4D suppressed ERK activity and down‐regulation of ERK was required for a Sema4D‐induced reduction of dendrite growth. Thus, we conclude that Plexin‐B1 is a dual functional GAP for R‐Ras and M‐Ras, remodelling axon and dendrite morphology, respectively.     

Netrin-1 acts as a survival factor via its receptors UNC5H and DCC

The membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration by acting as receptors for netrin-1. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Here we show that the netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, we demonstrate that UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Finally, we present evidence that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H.     

The dependence receptors DCC and UNC5H as a link between neuronal guidance and survival

Netrins are secreted proteins that play a crucial role in neuronal migration and in axon guidance during the development of the nervous system. Netrin-1 has been shown to interact with the transmembrane receptors DCC and UNC5H and these receptors appeared of key importance in mediating the chemotropic activity of netrin-1. Before the discovery of DCC as a netrin-1 receptor, the gene encoding DCC was proposed as a putative tumor suppressor gene because one DCC allele was deleted in roughly 70% of colorectal cancers and its expression was often reduced or absent in colorectal cancer tissues. A putative explanation for such dual roles has recently emerged with the observation that DCC belongs to the growing family of dependence receptors. Such receptors share the property of inducing apoptosis in the absence of ligand, hence creating a cellular state of dependence on the ligand. The other netrin-1 receptors UNC5H were also subsequently proposed to be dependence receptors, suggesting that netrin-1 may not only be a chemotropic factor for neurons but also a survival factor. We describe here netrin-1 and its receptors, together with the molecular signaling pathways initiated upon netrin-1 binding or upon netrin-1 withdrawal leading respectively to axonal/neuronal guidance or cell death induction. We then conclude to the possible roles of DCC and UNC5H pro-apoptotic activities in both nervous system development and tumorigenesis.     

EphrinA/EphA signal facilitates insulin-like growth factor-I-induced myogenic differentiation through suppression of the Ras/extracellular signal-regulated kinase 1/2 cascade in myoblast cell lines

Insulin-like growth factor-I (IGF-I) activates not only the phosphatidylinositol 3-kinase (PI3K)-AKT cascade that is essential for myogenic differentiation but also the extracellular signal-regulated kinase (ERK) 1/2 cascade that inhibits myogenesis. We hypothesized that there must be a signal that inhibits ERK1/2 upon cell-cell contact required for skeletal myogenesis. Cell-cell contact-induced engagement of ephrin ligands and Eph receptors leads to downregulation of the Ras-ERK1/2 pathway through p120 Ras GTPase-activating protein (p120RasGAP). We therefore investigated the significance of the ephrin/Eph signal in IGF-I-induced myogenesis. EphrinA1-Fc suppressed IGF-I-induced activation of Ras and ERK1/2, but not that of AKT, in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT activated by IGF-I. IGF-I-dependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-I-mediated myogenesis. EphrinA1-Fc did not promote IGF-I-dependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-I-induced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-I-mediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that the ephrinA/EphA signal facilitates IGF-I-induced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines.