EphA2 Signaling Following Endocytosis: Role of Tiam1

Eph receptors and their membrane‐bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell‐adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand‐mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1‐dependent Rac1 activation and ephrinA1‐induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling .     

EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

Eph signaling, which arises following stimulation by ephrins, is known to induce opposite cell behaviors such as promoting and inhibiting cell adhesion as well as promoting cell-cell adhesion and repulsion by altering the organization of the actin cytoskeleton and influencing the adhesion activities of integrins. However, crosstalk between Eph/ephrin with integrin signaling has not been fully elucidated in leukocytes, including monocytes and their related cells. Using a cell attachment stripe assay, we have shown that, following stimulation with ephrin-A1, kinase-independent EphA2 promoted cell spreading/elongation as well as adhesion to integrin ligand-coated surfaces in cultured U937 (monocyte) and J774.1 (monocyte/macrophage) cells as well as sublines of these cells expressing dominant negative EphA2 that lacks most of the intracellular region. Moreover, a pull-down assay showed that dominant negative EphA2 is recruited to the β2 integrin/ICAM1 and β2 integrin/VCAM1 molecular complexes in the subline cells following stimulation with ephrin-A1-Fc. Notably, this study is the first comprehensive analysis of the effects of EphA2 receptors on integrin-mediated cell adhesion in monocytic cells. Based on these findings we propose that EphA2 promotes cell adhesion by an unknown signaling pathway that largely depends on the extracellular region of EphA2 and the activation of outside-in integrin signaling.     

EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas)

Ephrins and Eph receptors are involved in axon guidance and cellular morphogenesis. An interaction between ephrin and Eph receptors elicits neuronal growth-cone collapse through cytoskeletal disassembly. When NIH3T3 cells were plated onto an ephrinA1-coated surface, the cells both adhered and spread. Adhesion and spreading proceeded concomitantly with changes in both the actin and microtubule cytoskeleton. EphA2, focal adhesion kinase (FAK) and p130(cas) were identified as the major ephrin-dependent phosphotyrosyl proteins during the ephrin-induced morphological changes. Mouse embryonic fibroblasts (MEFs) derived from FAK(-/-) and p130(cas-/-) mice had severe defects in ephrinA1-induced cell spreading, which were reversed after re-expression of FAK or p130(cas), respectively. Expression of a constitutively active EphA2 induced NIH3T3 cells to undergo identical, but ligand-independent, morphological changes. These data show that ephrinA1 can induce cell adhesion and actin cytoskeletal changes in fibroblasts in a FAK- and p130(cas)-dependent manner, through activation of the EphA2 receptor. The finding that ephrin Eph signalling can result in actin cytoskeletal assembly, rather than disassembly, has many implications for ephrin Eph responses in other cell types.     

EphrinA1 repulsive response is regulated by an EphA2 tyrosine phosphatase

Ephrin kinases and their ephrin ligands transduce repulsion of cells in axon guidance, migration, invasiveness, and tumor growth, exerting a negative signaling on cell proliferation and adhesion. A key role of their kinase activity has been confirmed by mutant kinase inactive receptors that shift the cellular response from repulsion to adhesion. Our present study aimed to investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in ephrinA1/EphA2 signaling. LMW-PTP, by means of dephosphorylation of EphA2 kinase, negatively regulates the ephrinA1-mediated repulsive response, cell proliferation, cell adhesion and spreading, and the formation of retraction fibers, thereby confirming the relevance of the net level of tyrosine phosphorylation of Eph receptors. LMW-PTP interferes with ephrin-mediated mitogen-activated protein kinase signaling likely through inhibition of p120RasGAP binding to the activated EphA2 kinase, thereby confirming the key role of mitogen-activated protein kinase inhibition by ephrinA1 repulsive signaling. We conclude that LMW-PTP acts as a terminator of EphA2 signaling causing an efficient negative feedback loop on the biological response mediated by ephrinA1 and pointing on tyrosine phosphorylation as the main event orchestrating the repulsive response.     

EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

Eph signaling, which arises following stimulation by ephrins, is known to induce opposite cell behaviors such as promoting and inhibiting cell adhesion as well as promoting cell-cell adhesion and repulsion by altering the organization of the actin cytoskeleton and influencing the adhesion activities of integrins. However, crosstalk between Eph/ephrin with integrin signaling has not been fully elucidated in leukocytes, including monocytes and their related cells. Using a cell attachment stripe assay, we have shown that, following stimulation with ephrin-A1, kinase-independent EphA2 promoted cell spreading/elongation as well as adhesion to integrin ligand-coated surfaces in cultured U937 (monocyte) and J774.1 (monocyte/macrophage) cells as well as sublines of these cells expressing dominant negative EphA2 that lacks most of the intracellular region. Moreover, a pull-down assay showed that dominant negative EphA2 is recruited to the β2 integrin/ICAM1 and β2 integrin/VCAM1 molecular complexes in the subline cells following stimulation with ephrin-A1-Fc. Notably, this study is the first comprehensive analysis of the effects of EphA2 receptors on integrin-mediated cell adhesion in monocytic cells. Based on these findings we propose that EphA2 promotes cell adhesion by an unknown signaling pathway that largely depends on the extracellular region of EphA2 and the activation of outside-in integrin signaling.     

EphA4 activity causes cell shape change and a loss of cell polarity in Xenopus laevis embryos.

The Eph family of receptor tyrosine kinases and their ephrin ligands are believed to limit cell-cell interactions during embryonic development via a repulsive mechanism. Little is known, however, about the intracellular effects of Eph signaling that lead to cellular repulsion. We have used scanning and transmission electron microscopy to examine the effects of EphA4 catalytic activity on cells in early embryos of Xenopus laevis. We show that ectopic EphA4 catalytic activity in superficial blastula cells leads to a more rounded cellular morphology, a loss of apical microvilli, and a loss of the apical/basolateral boundary, in addition to the previously reported loss of cell adhesion. These effects indicate that these epithelial cells have lost their apical/basolateral polarity. We also show that EphA4 catalytic activity causes a preferential loss of adherens junctions, compared to tight junctions. Furthermore, EphA4 catalytic activity was found to result in a change in filamentous actin levels in blastomeres. These results taken together suggest that the actin cytoskeleton might be a target of EphA4 signaling.     

EphA4 catalytic activity causes inhibition of RhoA GTPase in Xenopus laevis embryos

The Eph family of receptor tyrosine kinases is involved in limiting cell and tissue interactions via a repulsive mechanism. The mechanism of repulsion involves reorganizing the actin cytoskeleton, but little is known of the molecular components that connect the receptor to the actin cytoskeleton. Recent studies in retinal ganglion cells have demonstrated that EphA4 activates the small GTPase Rho. We have investigated the involvement of Rho in signaling downstream from EphA4. As a model system, we have used a chimeric receptor called EPP that we express and activate in early Xenopus embryos. Previous studies demonstrated that EPP activation leads to loss of cell-cell adhesion and change in cell shape, plus loss of aspects of cell polarity in epithelial cells, such as apical microvilli and the apical/basolateral boundary. In this study, we show that injecting inhibitors of Rho GTPases into early Xenopus embryos produces a phenotype very similar to that resulting from EPP activation. More importantly, expression of a constitutively active form of Xenopus RhoA (XRhoA) concurrent with activated EPP rescued embryos from the loss of cell-cell adhesion and change in cell shape associated with EPP. These data argue that, in contrast to the case in retinal ganglion cells, EphA4 in early Xenopus embryos acts to inhibit RhoA, suggesting that this receptor may regulate Rho differently (and therefore affect the cytoskeleton differently) in neuronal and non-neuronal cells. Furthermore, overexpression of ephexin, a novel guanine nucleotide exchange factor for Rho family GTPases, also blocks EPP-induced dissociation. This suggests that EphA4, which has been demonstrated to activate ephexin in cultured neuronal cells, may also target Rho GTPase via an ephexin-independent pathway.     

Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application

The Eph family of receptors, with 14 members in humans, makes up the largest group of receptor tyrosine kinases. These Eph receptors, along with their ligands, the 8 members of the ephrin family of ligands are involved in diverse developmental functions, including hindbrain development in vertebrates, tissue patterning, and angiogenesis. These Eph receptors and ephrin ligands have also been identified as important regulators in the development and progression of cancer. We have presented here a systematic and comprehensive investigation of the Eph/ephrin expression profiles of MCF-10A, MCF-7, and MDA-MB-231 cells representing normal breast, non-invasive breast tumor, and invasive tumor, respectively, based on their characteristic phenotypes in Matrigel matrix. The data have allowed us to correlate the gene expression profile with the cell phenotype that has potential application in tumor diagnostics. We demonstrate here that upregulation of EphA2, A7, A10, and ephrinA2 and B3 is likely involved in tumorigenesis and/or invasiveness, while downregulation of EphA1, A3, A4, A8, B3, B4, B6, and ephrinA1 and B1 may be particularly important in invasiveness. Based on these results we discuss the role of EphA2 and ephrinA1 combination in malignancy. The data have provided clues as to the importance of these molecules in the progression of breast cancer and specifically identified EphB6, a kinase-deficient receptor, which is downregulated in the most aggressive cell line, as reported for several other cancer types including neuroblastoma and melanoma suggesting its potential as a prognostic indicator in breast cancer as well.     

Inhibition of integrin-mediated cell adhesion but not directional cell migration requires catalytic activity of EphB3 receptor tyrosine kinase. Role of Rho family small GTPases

Genetic studies have shown that Eph receptor tyrosine kinases have both kinase-dependent and kinase-independent functions through incompletely understood mechanisms. We report here that ephrin-B1 stimulation of endogenous EphB kinases in LS174T colorectal epithelial cells inhibited integrin-mediated adhesion and HGF/SF-induced directional cell migration. Using 293 cells stably transfected with wild type (WT)- or kinase-deficient (KD-EphB3), we found that inhibition of integrin-mediated cell adhesion and induction of cell rounding was kinase-dependent. Unexpectedly, in two independent assays, both KD- and WT-EphB3 significantly inhibited directional cell migration. Upon ephrin-B1 stimulation, the activities of Rac1 and Cdc42 were reduced in both WT- and KD-EphB3-expressing cells that were induced to migrate. Pharmacological evidence demonstrates that a relative increase in RhoA signaling as a result of decreased Rac1/Cdc42 activities contributes to the inhibitory effects. Furthermore, EphB3-mediated inhibitory effect on cell adhesion but not migration was abolished by the integrin activating antibodies, suggesting that the inhibition of cell migration is not because of down-regulation of integrin function. These results uncover a differential requirement for EphB3 catalytic activity in the regulation of cell adhesion and migration, and suggest that while catalytic activity of EphB3 is required for inhibition of integrin-mediated cell adhesion, a distinct signaling pathway to Rho GTPases shared by WT- and KD-EphB3 receptor mediates inhibition of directional cell migration.     

The EphA3 receptor is expressed in a subset of rhabdomyosarcoma cell lines and suppresses cell adhesion and migration

Elevated expression of the Eph receptor tyrosine kinase EphA3 is associated with lymphocytic leukaemia, but little is known about its expression or function in solid tumours. Out of a panel of cancer cell lines, we found that EphA3 was expressed only on two rhabdomyosarcoma (RMS) cell lines of the embryonal histological subtype and on one of the alveolar RMS subtype, whereas it was not detected on two other cell lines of the alveolar subtype. Other EphA receptors (1-7) were, either not expressed in any, or expressed in all five RMS cell lines. Stimulation of EphA3-expressing TE671 and RD RMS cells with ephrinA5 resulted in loss of adhesion to fibronectin, decreased migration towards the stromal cell-derived growth factor-I (SDF-I), increased EphA3 phosphorylation, and increased Rho GTPase activity. In contrast, ectopic expression of EphA3 in the EphA3 negative CRL2061 cell line resulted in decreased cell adhesion. Finally, suppression of EphA3 expression by siRNA in RD cells results in increased SDF-I-mediated motility. These data indicate that EphA3 expression may define subsets of RMS tumours, and that EphA3 suppresses motility through regulation of Rho GTPases in RMS cells.     

EphrinA1 activates a Src/focal adhesion kinase-mediated motility response leading to rho-dependent actino/myosin contractility

Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell repulsive motility through heterotypic cell-cell interactions. Several Ephs, including EphA2, are greatly overexpressed in certain tumors, in correlation with poor prognosis and high vascularity in cancer tissues. The ability of several Eph receptors to regulate cell migration and invasion likely contribute to tumor progression and metastasis. We report here that in prostatic carcinoma cells ephrinA1 elicits a repulsive response that is executed through a Rho-dependent actino/myosin contractility activation, ultimately leading to retraction of the cell body. This appears to occur through assembly of an EphA2-associated complex involving the two kinases Src and focal adhesion kinase (FAK). EphrinA1-mediated repulsion leads to the selective phosphorylation of Tyr-576/577 of FAK, enhancing FAK kinase activity. The repulsive response elicited by ephrinA1 in prostatic carcinoma cells is mainly driven by a Rho-mediated phosphorylation of myosin light chain II, in which Src and FAK activation are required steps. Consequently, Src and FAK are upstream regulators of the overall response induced by ephrinA1/EphA2, instructing cells to retract the cell body and to move away, probably facilitating dissemination and tissue invasion of ephrin-sensitive carcinomas.     

An ephrin mimetic peptide that selectively targets the EphA2 receptor

Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologic versus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolar K(D) values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration.     

EphrinA1-EphA2 signal induces compaction and polarization of Madin-Darby canine kidney cells by inactivating Ezrin through negative regulation of RhoA

The epithelial cells exhibit either a columnar or a flat shape dependent on extracellular stimuli or the cell-cell adhesion. Membrane-anchored ephrinA stimulates EphA receptor tyrosine kinases as a ligand in a cell-cell contact-dependent manner. The mechanism through which ephrinA1/EphA2 signal regulates the cell morphology remains elusive. We demonstrate here that ephrinA1/EphA2 signal induces compaction and enhanced polarization (columnar change) of Madin-Darby canine kidney epithelial cells by regulating Ezrin, a linker that connects plasma membrane and actin cytoskeleton. Activation of EphA2 resulted in RhoA inactivation through p190RhoGAP-A and subsequent dephosphorylation of Ezrin on Thr-567 phosphorylated by Rho kinase. Consistently, the cells expressing an active mutant of Ezrin in which Thr-567 was replaced with Asp did not change their shape in response to ephrinA1. Furthermore, depletion of Ezrin led to compaction and enhanced polarization without ephrinA1 stimulation, suggesting the role for active Ezrin in keeping the flat cell shape. Ezrin localized to apical domain irrespective of ephrinA1 stimulation, whereas phosphorylated Ezrin on the apical domain was reduced by ephrinA1 stimulation. Collectively, ephrinA1/EphA2 signal negatively regulates Ezrin and promotes the alteration of cell shape, from flat to columnar shape.     

Redox-dependent downregulation of Rho by Rac

Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases.     

Expression Profile and Role of EphrinA1 Ligand After Spinal Cord Injury

Spinal cord injury (SCI) triggers the re-expression of inhibitory molecules present in early stages of development, contributing to prevention of axonal regeneration. Upregulation of EphA receptor tyrosine kinases after injury suggest their involvement in the nervous system’s response to damage. However, the expression profile of their ephrinA ligands after SCI is unclear. In this study, we determined the expression of ephrinA ligands after contusive SCI. Adult Sprague-Dawley female rats were injured using the MASCIS impactor device at the T10 vertebrae, and levels of ephrinA mRNA and protein determined at different time points. Identification of the cell phenotype expressing the ephrin ligand and colocalization with Eph receptors was performed with immunohistochemistry and confocal microscopy. Behavioral studies were made, after blocking ephrinA1 expression with antisense (AS) oligonucleotides, to assess hindlimb locomotor activity. Real-time PCR demonstrated basal mRNA levels of ephrin (A1, A2, A3, and A5) in the adult spinal cord. Interestingly, ephrinA1 was the only ligand whose mRNA levels were significantly altered after SCI. Although ephrinA1 mRNA levels increased after 2 weeks and remain elevated, we did not observe this pattern at the protein level as revealed by western blot analysis. Immunohistochemical studies showed ephrinA1 expression in reactive astrocytes, axons, and neurons and also their colocalization with EphA4 and A7 receptors. Behavioral studies revealed worsening of locomotor activity when ephrinA1 expression was reduced. This study suggests that ephrinA1 ligands play a role in the pathophysiology of SCI.     

An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase.

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.     

Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism

The Rho family GTPases Cdc42, Rac1 and RhoA control many of the changes in the actin cytoskeleton that are triggered when growth factor receptors and integrins bind their ligands [1] [2]. Rac1 and Cdc42 stimulate the formation of protrusive structures such as membrane ruffles, lamellipodia and filopodia. RhoA regulates contractility and assembly of actin stress fibers and focal adhesions. Although prolonged integrin engagement can stimulate RhoA [3] [4] [5], regulation of this GTPase by early integrin-mediated signals is poorly understood. Here we show that integrin engagement initially inactivates RhoA, in a c-Src-dependent manner, but has no effect on Cdc42 or Rac1 activity. Additionally, early integrin signaling induces activation and tyrosine phosphorylation of p190RhoGAP via a mechanism that requires c-Src. Dynamic modulation of RhoA activity appears to have a role in motility, as both inhibition and activation of RhoA hinder migration [6] [7] [8]. Transient suppression of RhoA by integrins may alleviate contractile forces that would otherwise impede protrusion at the leading edge of migrating cells.     

Eph/ephrin signaling in morphogenesis, neural development and plasticity

Ephrins are cell-surface-tethered ligands for Eph receptors, the largest family of receptor tyrosine kinases. During development, the Eph/ephrin cell communication system appears to influence cell behavior such as attraction/repulsion, adhesion/de-adhesion and migration, thereby influencing cell fate, morphogenesis and organogenesis. During adulthood, the Eph/ephrin system continues to play roles in tissue plasticity, for example in shaping dendritic spines during neuronal plasticity. Mechanistically, Eph-ephrin repulsive behavior appears to require ligand-receptor internalization and signaling to Rho GTPases.     

Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.     

The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via alpha(5)beta(1)- or beta(3) integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110gamma isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110gamma. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110gamma mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110gamma PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion.