An ephrin-A-dependent Signaling Pathway Controls Integrin Function and Is Linked to the Tyrosine Phosphorylation of a 120-kDa Protein

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, have been implicated in the development of the retinotectal projection. Here, glycosylphosphatidylinositol-anchored A-ephrins are not only expressed in the tectum but also on retinal axons, raising the possibility that they function in this context as receptors. We now show that activation of ephrin-A2 or ephrin-A5 by one of their receptors, ephA3, results in a β1-integrin-dependent increased adhesion of ephrin-A-expressing cells to laminin. In the search for an ephrin-A-dependent signaling pathway controlling integrin activation, we identified a 120-kDa raft membrane protein that is tyrosine-phosphorylated specifically after ephrin-A activation. Tyrosine phosphorylation of this protein is not seen after stimulating ephrin-A2-expressing cells with basic fibroblast growth factor, epidermal growth factor, insulin growth factor, or fetal calf serum containing a large set of different growth factors. The role of p120 as a mediator of an ephrin-A-integrin coupling is supported by the finding that inhibiting tyrosine phosphorylation of p120 correlates with an abolishment of the β1-dependent cell adhesion. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, have been implicated in the development of the retinotectal projection. Here, glycosylphosphatidylinositol-anchored A-ephrins are not only expressed in the tectum but also on retinal axons, raising the possibility that they function in this context as receptors. We now show that activation of ephrin-A2 or ephrin-A5 by one of their receptors, ephA3, results in a β1-integrin-dependent increased adhesion of ephrin-A-expressing cells to laminin. In the search for an ephrin-A-dependent signaling pathway controlling integrin activation, we identified a 120-kDa raft membrane protein that is tyrosine-phosphorylated specifically after ephrin-A activation. Tyrosine phosphorylation of this protein is not seen after stimulating ephrin-A2-expressing cells with basic fibroblast growth factor, epidermal growth factor, insulin growth factor, or fetal calf serum containing a large set of different growth factors. The role of p120 as a mediator of an ephrin-A-integrin coupling is supported by the finding that inhibiting tyrosine phosphorylation of p120 correlates with an abolishment of the β1-dependent cell adhesion. glycosylphosphatidylinositol phosphate-buffered saline basic fibroblast growth factor epidermal growth factor insulin growth factor fetal calf serum alkaline phosphatase During development of the retinotectal projection, members of the Eph family of receptor tyrosine kinases and their “ligands”, the ephrins, are strongly involved in guiding retinal axons to, and in, the tectum (for review see Refs. 1Drescher U. Bonhoeffer F. Müller B.K. Curr. Opin. Neurobiol. 1997; 7: 75-80Crossref PubMed Scopus (168) Google Scholar, 2Flanagan J.G. Vanderhaeghen P. Annu. Rev. Neurosci. 1998; 21: 309-345Crossref PubMed Scopus (939) Google Scholar, 3O'Leary D.D.M. Yates P.A. McLaughlin T. Cell. 1999; 98: 255-269Abstract Full Text Full Text PDF Scopus (102) Google Scholar). Besides the graded expression of Eph receptors on retinal axons and of ephrins in the tectum, the ephrin-As are also differentially expressed on retinal axons themselves (4Hornberger M.R. Dütting D. Ciossek T. Yamada T. Handwerker C. Lang S. Weth F. Huf J. Wessel R. Logan C. Tanaka H. Drescher U. Neuron. 1999; 22: 731-742Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 5Dütting D. Handwerker C. Drescher U. Dev. Biol. 1999; 216: 297-311Crossref PubMed Scopus (70) Google Scholar). Gain of function and loss of function analyses suggest that here the ephrin-As modulate the function of the coexpressed receptors in that coexpression of ligands and receptors runs in parallel to a decrease in sensitivity for the repellent activity of the tectally expressed ephrins (4Hornberger M.R. Dütting D. Ciossek T. Yamada T. Handwerker C. Lang S. Weth F. Huf J. Wessel R. Logan C. Tanaka H. Drescher U. Neuron. 1999; 22: 731-742Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 5Dütting D. Handwerker C. Drescher U. Dev. Biol. 1999; 216: 297-311Crossref PubMed Scopus (70) Google Scholar). These findings fit to subsequent results ofin vivo analyses of ephrin-A5 single and ephrin-A5;ephrin-A2 double knockout mice (6Feldheim J. Kim Y.-I. Bergemann A.D. Frisén J. Barbacid M. Flanagan J.G. Neuron. 2000; 25: 563-574Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). A hint of how ephrin-A functions on retinal axons may be provided by the concept that these ligands are localized, because of their membrane attachment by a glycosylphosphatidylinositol (GPI)1 anchor, to so-called rafts (7Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (7946) Google Scholar, 8Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1711) Google Scholar, 9Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2529) Google Scholar, 10Varma R. Mayor S. Nature. 1998; 394: 798-801Crossref PubMed Scopus (1016) Google Scholar, 11Friedrichson T. Kurzchalia T.V. Nature. 1998; 394: 802-805Crossref PubMed Scopus (475) Google Scholar, 12Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2035) Google Scholar). rafts are small dynamic microdomains in the membrane with a special glycosphingolipid and cholesterol composition, which have been proposed, besides having other functions, to serve as localized platforms for signal transduction (13Harder T. Simons K. Curr. Opin. Cell Biol. 1997; 9: 534-542Crossref PubMed Scopus (714) Google Scholar). GPI-anchored proteins are bound to the outer leaflet of the raft membranes (with no direct contact to the cytosol), whereas signaling molecules such as members of the src family are localized to the inner leaflet of rafts. In fact, “activated” GPI-anchored proteins can transduce signals to the cytosol, leading, for example, to changes in intracellular Ca2+ concentrations and activation of specific signaling pathways. This signaling might occur through formation of larger raft domains triggered by clustering of GPI-anchored proteins, which allows an interaction of signaling molecules normally separated through direct interactions between raft lipids or through (regulated) association of GPI-anchored molecules with appropriate transmembrane (co) receptors (9Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2529) Google Scholar, 14Jacobson K. Dietrich C. Trends Cell Biol. 1999; 9: 87-91Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). For example, the GPI-anchored cell adhesion molecule contactin/F11 associates with the transmembrane protein CASPR (15Peles E. Nativ M. Lustig M. Grumet M. Schilling J. Martinez R. Plowman G.D. Schlessinger J. EMBO J. 1997; 16: 978-988Crossref PubMed Scopus (350) Google Scholar), representing a coreceptor involved in signal transduction after contactin/F11 stimulation. Other examples of GPI-anchored proteins capable of mediating signals include Thy-1 (16Doherty P. Singh A. Rimon G. Bolsover S.R. Walsh F.S. J. Biol. Chem. 1993; 122: 181-189Google Scholar), CNTF (17Economides A.N. Ravetch J.V. Yancopoulos G.D. Stahl N. Science. 1995; 270: 1351-1353Crossref PubMed Scopus (25) Google Scholar), GDNF (18Massague J. Nature. 1996; 382: 29-30Crossref PubMed Scopus (37) Google Scholar), and neurturin (19Buj-Bello A. Adu J. Pinon L.G. Horton A. Thompson J. Rosenthal A. Chinchetru M. Buchman V.L. Davies A.M. Nature. 1997; 387: 721-724Crossref PubMed Scopus (256) Google Scholar). Simons and coworkers (7Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (7946) Google Scholar) have put forward the idea that a fundamental principle guiding the way raft microdomains exert their function in signal transduction is the (regulated) separation of different membrane proteins (7Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (7946) Google Scholar, 20Rodgers W. Rose J.K. J. Cell Biol. 1996; 135: 1515-1523Crossref PubMed Scopus (285) Google Scholar). According to this concept, GPI-anchored molecules and other specific sets of membrane proteins have a strong tendency to associate with these domains, whereas others, such as molecules with a transmembrane domain, rarely appear in these areas. Recently, it was shown that activation of ephrin-A5 results in an increase in adhesion of the ligand-expressing cells and in the level of tyrosine phosphorylation of molecules with molecular masses of 75–80 and 60 kDa (21Davy A. Gale N.W. Murray E.W. Klinghofer R.A. Soriano P. Feuerstein C. Robbins S.M. Genes Dev. 1999; 13: 3125-3135Crossref PubMed Scopus (247) Google Scholar). These changes, however, were not specific for an ephrin-A activation and were also seen after incubation of these cells with, for example, basic fibroblast growth factor (bFGF) (22Davy A. Feuerstein C. Robbins S.M. J. Neurochem. 2000; 74: 676-683Crossref PubMed Scopus (31) Google Scholar). Here we show that binding of the ephA3 receptor to ephrin-A2 and ephrin-A5 leads to an activation of the integrin system, as seen in a β1-integrin-dependent increase in adhesion of the ephrin-A-expressing cells. Activation of ephrin-As and changes in adhesion correlate well with the tyrosine phosphorylation of a protein with a molecular mass of 120 kDa, which is observed only after ephrin-A activation. p120 thus might represent a component of a new ephrin-A-dependent signaling pathway functionally linking ephrin-As to integrins. HEK293 cell clones stably expressing chick ephrin-A2 or the long or the short form of mouse ephrin-A5 were established according to standard protocols. In brief, about 2 × 106 HEK293 cells were transfected with 25 μg of the respective pClNeo-based expression plasmids and were selected for about 2 weeks in standard medium (Dulbecco's modified Eagle's medium with 10% fetal calf serum and antibiotics) containing 400 μg/ml G418 (PAA Laboratories). Then individual clones were isolated, expanded, and analyzed for ephrin-A expression using ephA3-AP staining and later, Western blots. Those cell clones most strongly expressing ephrin-As were further expanded and used in this study. ephA3-AP and AP were produced as described previously (23Monschau B. Kremoser C. Ohta K. Tanaka H. Kaneko T. Yamada T. Handwerker C. Hornberger M.R. Löschinger J. Pasquale E.B. Siever D.A. Verderame M.F. Müller B.K. Bonhoeffer F. Drescher U. EMBO J. 1997; 16: 1258-1267Crossref PubMed Scopus (212) Google Scholar, 24Ciossek T. Monschau B. Kremoser C. Löschinger J. Lang S. Müller B.K. Bonhoeffer F. Drescher U. Eur. J. Neurosci. 1998; 10: 1574-1580Crossref PubMed Scopus (54) Google Scholar); protein G-agarose is from Roche Molecular Biochemicals. Antibodies against chicken ephrin-A2 were described by Hornberger et al. (4Hornberger M.R. Dütting D. Ciossek T. Yamada T. Handwerker C. Lang S. Weth F. Huf J. Wessel R. Logan C. Tanaka H. Drescher U. Neuron. 1999; 22: 731-742Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar), monoclonal antibodies to phosphotyrosine (clone 4G10), the β1-integrin-neutralizing antibody (clone DE9), and anti-human fyn (rabbit whole serum, used for immunoprecipitation) were from Upstate Biotechnologies. Monoclonal antibodies to fyn and were from and the antibodies were from HEK293 cell clones expressing the ephrin-As were for in from the with for with and in medium containing ephA3-AP or Then the cells were for the on and with containing the cells were for in of 2 This was used for The cell was to in of this was with of in of 10% in of and was for in an were from the The containing the between and 10% was as and the was as the also A. Gale N.W. Murray E.W. Klinghofer R.A. Soriano P. Feuerstein C. Robbins S.M. Genes Dev. 1999; 13: 3125-3135Crossref PubMed Scopus (247) Google Scholar). The were with for to and with 10% on for and for The was with for and for The was and were for further such as Western The was with containing and was with appropriate of protein G-agarose for 2 were by the with protein G-agarose for the was and with and were according to standard protocols. were with to μg/ml in or and with binding were for using serum The cells were from the with and in ephA3-AP or AP was to a of about 2 × cells in were well and for Then the medium was and cells were with the cells were with for and with in for The cells were with and were with was shown in and were on the of to src family kinases the increase in cell adhesion seen after stimulating ephrin-A2-expressing cells with ephrin-A2 expressing cells were for with or Then the cells were from the and in the of ephA3-AP or AP on with concentrations of cells were The of cells was using a of cells in The increase in ephA3-AP of cells can be by cells with src family but not with a but functionally to For see and a receptor or signaling function of the GPI-anchored ephrin-A ligands, we established a HEK293 cell stably expressing the been shown to be involved for in the of cell Rev. 2000; PubMed Google Scholar), we of these cells with their receptor changes the of these cells to laminin. For an activation we used ephA3-AP or AP as a ephA3-AP is a protein of the of the ephA3 receptor to strongly to ephrin-A2 (23Monschau B. Kremoser C. Ohta K. Tanaka H. Kaneko T. Yamada T. Handwerker C. Hornberger M.R. Löschinger J. Pasquale E.B. Siever D.A. Verderame M.F. Müller B.K. Bonhoeffer F. Drescher U. EMBO J. 1997; 16: 1258-1267Crossref PubMed Scopus (212) Google and alkaline alkaline phosphatase is in an form T. Monschau B. Kremoser C. Löschinger J. Lang S. Müller B.K. Bonhoeffer F. Drescher U. Eur. J. Neurosci. 1998; 10: 1574-1580Crossref PubMed Scopus (54) Google Scholar) and thus might represent the form of the shown in cells with but not to a increase in adhesion to but not to which an activation of the integrin that the increase in adhesion is on the of ephrin-A ligands, we this also using the cell Here we not increase in cell adhesion after ephA3-AP not the role of we the adhesion in the of a monoclonal antibody to used a of μg/ml Here we observed a strong in the increase in whereas a antibody used the not this increase activation of ephrin-As by their receptors leads to an activation of the integrin the signaling function of ephrin-A ligands we were in proteins that ephrin-A ligands to the integrin ephrin-A molecules are they no direct to the and thus might a for such a signaling GPI-anchored molecules are in which are to represent platforms in which molecules involved in signal transduction are thus a coreceptor might here with In we ephrin-A2 is with rafts. A for the association of a protein with rafts is the of that protein in domains, which can be because of their by R. London E. D. U. S. A. PubMed Scopus Google Scholar). ephrin-A2-expressing cells were to an and individual were to Western using monoclonal antibodies to ephrin-A2 and Curr. Opin. Cell Biol. 1996; PubMed Scopus Google Scholar) are proteins that are with these rafts and might function in the raft domains (13Harder T. Simons K. Curr. Opin. Cell Biol. 1997; 9: 534-542Crossref PubMed Scopus (714) Google Scholar). These analyses that ephrin-A2 is in the as that ephrin-A2 is localized to rafts. containing proteins ephrin-A2 A and This increased the sensitivity of as the of cell proteins was in the not proteins that might be of an ephrin-A2 signaling we the raft from cells after incubation with ephA3-AP or with Western we in on changes in tyrosine phosphorylation using monoclonal activation, cells were for to the level of tyrosine phosphorylation of membrane proteins, which is for cells standard with shown in we such changes in the tyrosine phosphorylation of proteins in the raft after ephrin-A2-expressing cells with ephA3-AP but not with In a protein of 120 kDa, which is only tyrosine in the strongly after and later, after about 60 to a to phosphorylation This protein was not in the containing proteins but in the as that it a molecule specifically with raft proteins with a molecular mass of kDa, which were tyrosine-phosphorylated after ephA3-AP with an after 60 (for this set of proteins be to in the as These proteins also were specifically in the raft are proteins tyrosine phosphorylation were in after activation of we can not the possibility that the increase in p120 tyrosine phosphorylation is because of a of p120 to the raft domains after ephrin-A2 stimulation. the of phosphorylation might be because of a of in rafts after ephrin-A2 activation. In we observed a of strongly tyrosine-phosphorylated proteins, phosphorylation not after ephA3-AP with an antibody in the of a the as after the with the that one of these to the tyrosine fyn This was in an set of Here the raft of cells was using an serum and analyzed in a Western using an antibody and after of the an monoclonal antibody 2 analyses that the the phosphorylation level of fyn was after activation of ephrin-A2 A A of the with an antibody against as shown in 2 that of raft proteins have been we in the changes that in the level of tyrosine of p120 and after cells with shown in p120 tyrosine-phosphorylated only after about thus with an of about The tyrosine phosphorylation a level for about and to a signal 60 after the of ephrin-A2 activation. The of that is after ephA3-AP a in the level of tyrosine phosphorylation is and with leads to an The expression level and tyrosine phosphorylation level of fyn was not the We also after a incubation with the of ephrin-A2 with the raft is not the changes in cell adhesion after ephrin-A2 activation we that the increase in adhesion was observed only after a of that is the increase in adhesion is seen only after the increase in p120 tyrosine phosphorylation In an to the of we have the cells using or and we have analyzed the raft with selected results from these the possibility that p120 to an receptor or adhesion not we the changes in phosphorylation observed were specific for interactions or other proteins such as growth also these or changes in tyrosine For this we analyzed a of different signaling molecules to the tyrosine phosphorylation of proteins, such as epidermal growth factor insulin growth factor and We also fetal calf serum in which a large of different growth factors. We observed a decrease in the phosphorylation of after however, of these to changes in the tyrosine phosphorylation of p120 with by ephA3-AP We to the observed changes in tyrosine phosphorylation can be observed also for other ephrin-A We established HEK293 cells stably expressing the long or the short form of ephrin-A5 A.M. Gale N.W. Yancopoulos G.D. Dev. Biol. 1996; PubMed Scopus Google Scholar) and the as described for after activation by ligands the of phosphorylation and the of p120 phosphorylation was to the between ephrin-As and integrins. A of molecules are to be involved in the of integrin and we were in signaling used in ephrin-A and integrin signaling We on src family which are to be involved in integrin signaling and are for an adhesion but appear not to be for these D.A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Curr. Opin. Cell Biol. 1997; 9: PubMed Scopus (350) Google Scholar, S.M. Annu. Rev. Cell Dev. 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We the possibility that the increase in tyrosine phosphorylation of p120 is because of a of tyrosine-phosphorylated p120 to the raft we this is because the increase in p120 tyrosine phosphorylation can be by inhibiting src family to be a of p120 can be observed also after clustering of other GPI-anchored proteins expressed in HEK293 cells. is that the strong increase in p120 tyrosine phosphorylation only after a of about the of other signaling the is with changes in the or of raft with ephA3-AP might to a clustering of ephrin-A2 and a of raft which by an in an activation of src family p120 tyrosine and integrin activation. In with p120 a role in coupling ephrin-A activation to integrin activation, we have shown here a specific src family that p120 tyrosine phosphorylation correlates with an abolishment of the increase in cell adhesion. 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Genes Dev. 1999; 13: 3125-3135Crossref PubMed Scopus (247) Google Scholar) identified a of proteins tyrosine phosphorylation level increased after binding of to an cell most a which after ephrin-A2 with the tyrosine phosphorylation of the which the activation of a but not signaling The tyrosine phosphorylation level of the protein we identified was after ephrin-A activation and was also not specific for an ephrin-A signaling it is these sets of proteins are the of a molecular the of et al. (21Davy A. Gale N.W. Murray E.W. Klinghofer R.A. Soriano P. Feuerstein C. Robbins S.M. Genes Dev. 1999; 13: 3125-3135Crossref PubMed Scopus (247) Google Scholar) and appear to be are in the the of and these might the different results A molecular for example, the this In the of tyrosine phosphorylation of p120 was specific for the receptor interaction the of molecules it was not seen after of cells with growth such as or or with fetal calf serum, which a large of different growth factors. A of a 120-kDa protein was not by et al. (21Davy A. Gale N.W. Murray E.W. Klinghofer R.A. Soriano P. Feuerstein C. Robbins S.M. Genes Dev. 1999; 13: 3125-3135Crossref PubMed Scopus (247) Google Scholar). The role of p120 in the linking ephrin-A activation to integrin activation is thus further be a of this We F. Bonhoeffer for and We also and Drescher for on the