Advertisement

E-cadherin Homophilic Ligation Directly Signals through Rac and Phosphatidylinositol 3-Kinase to Regulate Adhesive Contacts*

Open AccessPublished:December 13, 2001DOI:https://doi.org/10.1074/jbc.M109640200
      Classical cadherins mediate cell recognition and cohesion in many tissues of the body. It is increasingly apparent that dynamic cadherin contacts play key roles during morphogenesis and that a range of cell signals are activated as cells form contacts with one another. It has been difficult, however, to determine whether these signals represent direct downstream consequences of cadherin ligation or are juxtacrine signals that are activated when cadherin adhesion brings cell surfaces together but are not direct downstream targets of cadherin signaling. In this study, we used a functional cadherin ligand (hE/Fc) to directly test whether E-cadherin ligation regulates phosphatidylinositol 3-kinase (PI 3-kinase) and Rac signaling. We report that homophilic cadherin ligation recruits Rac to nascent adhesive contacts and specifically stimulates Rac signaling. Adhesion to hE/Fc also recruits PI 3-kinase to the cadherin complex, leading to the production of phosphatidylinositol 3,4,5-trisphosphate in nascent cadherin contacts. Rac activation involved an early phase, which was PI 3-kinase-independent, and a later amplification phase, which was inhibited by wortmannin. PI 3-kinase and Rac activity were necessary for productive adhesive contacts to form following initial homophilic ligation. We conclude that E-cadherin is a cellular receptor that is activated upon homophilic ligation to signal through PI 3-kinase and Rac. We propose that a key function of these cadherin-activated signals is to control adhesive contacts, probably via regulation of the actin cytoskeleton, which ultimately serves to mediate adhesive cell-cell recognition.
      Classical cadherin molecules are critical morphogenetic determinants in metazoan organisms (
      • Takeichi M.
      ,
      • Yap A.S.
      • Brieher W.M.
      • Gumbiner B.M.
      ). E-cadherin, the prototypical epithelial cadherin, participates in tissue patterning during development and preserves epithelial organization in postembryonic life. In addition to mediating cell-cell cohesion in mature epithelia, E-cadherin has a clear role in cell-to-cell recognition. E-cadherin-expressing cells sort out from tissue culture cells that express different cadherins, while changes in cadherin expression determine tissue segregation in the embryo (
      • Takeichi M.
      ). E-cadherin, like other classical cadherins, therefore plays a central role in determining how cells discriminate like from unlike.
      At the cellular level, there is increasing evidence that E-cadherin ligation initiates a cascade of molecular and cellular events that ultimately determine cellular recognition. Conversion of initial homophilic ligation to productive, stable cell-cell adhesion appears to constitute a key step in this recognition process. Studies in migrating cells have shown that E-cadherin-expressing cells make nascent contacts with one another through punctate cadherin contacts (
      • Adams C.L.
      • Nelson W.J.
      • Smith S.J.
      ,
      • Adams C.L.
      • Chen Y.-T.
      • Smith S.J.
      • Nelson W.J.
      ,
      • Abercrombie M.
      ,
      • Yonemura S.
      • Itoh M.
      • Nagafuchi A.
      • Tsukita S.
      ). Productive stabilization of adhesion is distinguished by the extension of these contact zones, leading cells to persist in coherent aggregates with altered migratory activity. Cells that fail to stabilize these initial contacts separate from one another and continue to migrate. Furthermore, a number of cellular mechanisms with the potential to mediate adhesive stabilization have been identified as downstream consequences of cadherin ligation. These include lateral clustering of cadherin molecules (
      • Yap A.S.
      • Brieher W.M.
      • Pruschy M.
      • Gumbiner B.M.
      ,
      • Angres B.
      • Barth A.
      • Nelson W.J.
      ), recruitment of cadherin-associated junction proteins (
      • Yokoyama S.
      • Tachibana K.
      • Nakanishi H.
      • Yamamoto Y.
      • Irie K.
      • Mandai K.
      • Nagafuchi A.
      • Moden M.
      • Takai Y.
      ), and reorganization of the actin cytoskeleton (
      • Adams C.L.
      • Nelson W.J.
      • Smith S.J.
      ,
      • Adams C.L.
      • Chen Y.-T.
      • Smith S.J.
      • Nelson W.J.
      ).
      Taken together, these observations raised the attractive possibility that E-cadherin binding may activate signaling pathways to coordinate the cellular cascade that leads from homophilic recognition to productive adhesion. In its simplest form, it is attractive to envisage a scenario where E-cadherin acts as a direct upstream receptor for intracellular signals that ultimately act on the effector mechanisms responsible for adhesive stabilization. Indeed, signaling by PI
      PI
      phosphatidylinositol
      CHO
      Chinese hamster ovary
      BSA
      bovine serum albumin
      GFP
      green fluorescent protein
      GST
      glutathione S-transferase
      PAK
      p21-activated kinase
      PLL
      poly-l-lysine
      PIP3
      phosphatidylinositol 3,4,5-trisphosphate
      PH
      pleckstrin homology
      1PI
      phosphatidylinositol
      CHO
      Chinese hamster ovary
      BSA
      bovine serum albumin
      GFP
      green fluorescent protein
      GST
      glutathione S-transferase
      PAK
      p21-activated kinase
      PLL
      poly-l-lysine
      PIP3
      phosphatidylinositol 3,4,5-trisphosphate
      PH
      pleckstrin homology
      3-kinase (
      • Pece S.
      • Chiariello M.
      • Murga C.
      • Gutkind J.S.
      ) as well as Rho family GTPases (
      • Noren N.K.
      • Niessen C.M.
      • Gumbiner B.M.
      • Burridge K.
      ,
      • Nakagawa M.
      • Fukata M.
      • Yamagawa M.
      • Itoh M.
      • Kaibuchi K.
      ,
      • Kim S.H., Li, Z.
      • Sacks D.B.
      ) has been observed to be activated as epithelial cells reestablish contacts after chelation of extracellular calcium. The use of cadherin-blocking antibodies demonstrated that such signals depended on functional E-cadherin.
      These studies could not, however, distinguish between signals that were activated as direct downstream consequences of cadherin ligation and juxtacrine signals that required cadherin adhesion to bring cell surfaces together but were not themselves direct downstream consequences of cadherin ligation (
      • Yap A.S.
      • Brieher W.M.
      • Gumbiner B.M.
      ,
      • Fagotto F.
      • Gumbiner B.M.
      ). This distinction has fundamental mechanistic implications for any model of cadherin signaling. For example, gap junction communication requires E-cadherin to bring cell surfaces into contact (
      • Musil L.S.
      • Cunningham B.A.
      • Edelman G.M.
      • Goodenough D.A.
      ), but connexins do not interact directly with cadherins. Ultimately, any mechanistic characterization of cadherin signaling must determine whether responses to cell-cell contact represent direct cadherin-activated signals or cadherin-dependent juxtacrine signals.
      Nor has it yet been determined whether cadherin-dependent signaling impacts on cell adhesion. Although PI 3-kinase and Rho GTPases have potent effects on the actin cytoskeleton, these signals exert diverse effects on cell behavior, including effects on cell growth and apoptosis, that are independent of adhesion.
      In the current report, we therefore aimed to test directly whether E-cadherin functions as an upstream receptor for Rac GTPase signaling and to define its functional significance for cell adhesion. To accomplish this, we developed an assay that allowed us to specifically engage E-cadherin homophilic ligation and determine its effects on cell signaling, adhesion, and cell shape.

      DISCUSSION

      Cadherin cell adhesion molecules are fundamental mediators of cell-cell recognition in metazoan organisms. Homophilic ligation between cadherin ectodomains presented on opposing cell surfaces is postulated to initiate a cascade of cellular responses that ultimately determine whether or not cells form cohesive associations with one another. While contact between cells undoubtedly activates intracellular signaling, the precise role of cadherins in this process is far from comprehensively understood. In this study, we sought to identify intracellular signals that were activated as direct downstream targets of E-cadherin homophilic ligation to control cadherin-based cellular adhesion. Using a recombinant E-cadherin adhesive ligand, we found that cadherin homophilic ligation to hE/Fc was sufficient to activate the Rac GTPase signaling pathway. Adhesion to PLL did not activate Rac, indicating that signaling was a specific response to cadherin homophilic ligation and not a nonspecific consequence of cell spreading. Recent reports that Rac signaling is activated as cultured cells form E-cadherin-dependent contacts with one another (
      • Nakagawa M.
      • Fukata M.
      • Yamagawa M.
      • Itoh M.
      • Kaibuchi K.
      ) could not exclude the possibility that Rac was activated by cadherin-dependent juxtacrine signals. Our current work now clearly demonstrates that E-cadherin is itself an upstream receptor for Rac, which signals upon homophilic ligation. Since XenopusC-cadherin has also recently been shown to directly activate Rac (
      • Noren N.K.
      • Niessen C.M.
      • Gumbiner B.M.
      • Burridge K.
      ), it will now be important to assess whether Rac is a downstream signal activated by all classical cadherins.
      Our data further identify PI 3-kinase as an intermediary component of the E-cadherin-activated Rac signaling pathway. 1) Inhibitors of PI 3-kinase blocked E-cadherin-based adhesion and lamellipodial extension as effectively as dominant-negative Rac1 mutants. This suggested that PI 3-kinase and Rac signaling were functionally connected elements of the cellular response that allows homophilic ligation of cadherin ectodomains to control cell shape and stabilize adhesive contacts. 2) Expression of a CA Rac1 mutant rescued the inhibition of cadherin-based lamellipodia induced by wortmannin. Therefore, activation of Rac signaling was capable of substituting for loss of PI 3-kinase signaling during cadherin-based adhesion. 3) E-cadherin-activated Rac signaling was significantly foreshortened in cells treated with wortmannin, indicating that PI 3-kinase activity was necessary for full activation of Rac by homophilic E-cadherin ligation. Furthermore, we found that homophilic ligation recruited PI 3-kinase to the cadherin complex and stimulated PI 3-kinase signaling at the plasma membrane. Taken together, these findings suggest that recruitment of PI 3-kinase to the cadherin adhesive complex facilitates maximal stimulation of Rac by E-cadherin.
      PI 3-kinase has been identified as an upstream regulator of Rac in models of growth factor signaling (
      • Hawkins P.T.
      • Eguinoa A.
      • Qiu R.-G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • Stephens L.
      ,
      • Cantrell D.A.
      ,
      • Reif K.
      • Nobes C.D.
      • Thomas G.
      • Hall A.
      • Cantrell D.A.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ). Our data, taken with a recent study of cell-cell contact formation in Madin-Darby canine kidney cells (
      • Nakagawa M.
      • Fukata M.
      • Yamagawa M.
      • Itoh M.
      • Kaibuchi K.
      ), indicate that this pathway also plays a role in E-cadherin-activated cell signaling. However, we also identified an early, previously unidentified (
      • Nakagawa M.
      • Fukata M.
      • Yamagawa M.
      • Itoh M.
      • Kaibuchi K.
      ), component of cadherin-activated Rac signaling that was not blocked by wortmannin and which may therefore be independent of PI 3-kinase. Consistent with this, the peak of pAkt accumulation at the plasma membrane occurred ∼30 min after cells adhered to hE/Fc and coincided with the wortmannin-sensitive phase of cadherin-activated Rac signaling. Potentially two distinguishable pathways may then link E-cadherin to Rac, the molecular details of which remain to be characterized. The p85 subunit of PI 3-kinase can interact with β-catenin both in vivo andin vitro (
      • Espada J.
      • Perez-Moreno M.
      • Braga V.M.M.
      • Rodriguez-Viciana P.
      • Cano A.
      ,
      • Carmeliet P.
      • Lampugnani M.G.
      • Moons L.
      • Breviario F.
      • Compernolle V.
      • Bono F.
      • Balconi G.
      • Spagnuolo R.
      • Oostuyse B.
      • Dewerchin M.
      • Zanetti A.
      • Angellilo A.
      • Mattot V.
      • Nuyens D.
      • Lutgens E.
      • Clotman F.
      • de Ruiter M.C.
      • Gittenberger-de Groot A.
      • Poelmann R.
      • Lupu F.
      • Herbert J.M.
      • Collen D.
      • Dejana E.
      ). It is therefore attractive to postulate that β-catenin may recruit PI 3-kinase to the cadherin-catenin complex upon ligation, leading to the generation of PIP3that we observed in early cadherin contacts. PIP3 has been reported to recruit exchange factors for GTPases (
      • Espada J.
      • Perez-Moreno M.
      • Braga V.M.M.
      • Rodriguez-Viciana P.
      • Cano A.
      ), such as Tiam-1 (
      • Hordijk P.L.
      • ten Klooster J.P.
      • van der Kammen R.A.
      • Michiels F.
      • Oomen L.C.
      • Collard J.G.
      ), a potential pathway for PI 3-kinase to potentiate Rac activity downstream of E-cadherin. Additionally, p120ctn has been reported to activate Rac signaling (
      • Noren N.K.
      • Liu B.P.
      • Burridge K.
      • Kreft B.
      ), making this protein a possible candidate to mediate PI 3-kinase-independent activation of Rac by E-cadherin.
      Given the pleotropic effects of PI 3-kinase and Rac (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Bishop A.L.
      • Hall A.
      ), what function may be served by these E-cadherin-activated signals? Our data implicate this signaling pathway in the cellular adhesive response to initial cadherin homophilic recognition. A characteristic feature of productive cadherin recognition is the expansion of the zones of contact between cells, from punctate initial contacts to continuous linear regions of contact (
      • Adams C.L.
      • Nelson W.J.
      • Smith S.J.
      ,
      • Adams C.L.
      • Chen Y.-T.
      • Smith S.J.
      • Nelson W.J.
      ,
      • Vasioukhin V.
      • Bauer C.
      • Yin M.
      • Fuchs E.
      ). This is replicated in our hE/Fc assay system, since initial adhesive binding is rapidly followed by cell spreading, a process that entails cadherin-based lamellipodial protrusion of the cell margins, which expand the zones of adhesive contact. Importantly, both PI 3-kinase inhibitors and expression of a DN Rac1 mutant blocked the formation of cadherin-based lamellipodia, preventing extension of the initial zones of adhesive contact. Similarly, PI 3-kinase inhibitors prevent cadherin-based cell-cell contacts from forming,
      R. G. Ali and A. S. Yap, unpublished results.
      while DN Rac mutants disrupt epithelial integrity (
      • Braga V.M.M.
      • Machesky L.M.
      • Hall A.
      • Hotchin N.A.
      ,
      • Braga V.M.
      • Del Maschio A.
      • Machesky L.
      • Dejana E.
      ,
      • Takaishi K.
      • Sasaki T.
      • Kotani H.
      • Nishioka H.
      • Takai Y.
      ). Taken together, we propose that PI 3-kinase and Rac form a signaling pathway that is activated by homophilic ligation of E-cadherin in nascent adhesive contacts, leading to extension of these zones of contact and ultimately to productive cell-cell recognition.
      The extension of nascent cadherin contacts to form stable regions of cell-cell cohesion is likely to involve coordinated changes in cadherin distribution and cytoskeletal organization at the cell surface. Of note, complex changes in actin cytoskeletal activity accompany the formation of cadherin-based cell-cell contacts (
      • Adams C.L.
      • Chen Y.-T.
      • Smith S.J.
      • Nelson W.J.
      ,
      • Vasioukhin V.
      • Bauer C.
      • Yin M.
      • Fuchs E.
      ). We recently found that E-cadherin can interact physically with the Arp 2/3 actin nucleation complex and identified the leading edges of cadherin-based lamellipodia as principal sites for actin assembly.2Insofar as Rac is well known to activate actin assembly, we propose that E-cadherin-activated Rac signaling serves to promote adhesive stabilization by stimulating actin assembly to convert limited nascent contacts into more expansive zones of adhesion (Fig.8).
      Figure thumbnail gr8
      Figure 8A model for local cadherin-activated cell signaling in adhesive contact formation. E-cadherin homophilic ligation activates Rac GTPase signaling through a pathway that involves PI 3-kinase. Cadherin-activated Rac signaling stimulates actin assembly (aa) to extend nascent adhesive contacts. In the cadherin-specific planar substrate assay (A), extension of adhesive contacts manifests in cadherin-based lamellipodia. In native cell-to-cell contacts (B), extension of contacts manifests in the apposition of cell surfaces, converting initial punctate adhesions into continuous zones of contact.
      Interestingly, several observations suggested that cadherin-directed signaling was restricted to specific regions of adhesive contact. Thus, we found that PIP3 was generated at the very leading edges of cadherin-based lamellipodia, where GFP-p85 co-localized with the cadherin-catenin complex. Similarly, although we did not image GTP-loading of Rac, we observed that at the cadherin adhesive interfaces Rac-1 co-accumulated with E-cadherin principally at leading edges. Strikingly, in regions of cadherin-based lamellipodia proximal to the leading edges, we observed relatively little PIP3, GFP-p85, or Rac, despite the presence of cadherin clusters at the adhesive interface. Since cadherins cluster upon homophilic engagement (
      • Yap A.S.
      • Brieher W.M.
      • Pruschy M.
      • Gumbiner B.M.
      ,
      • Angres B.
      • Barth A.
      • Nelson W.J.
      ,
      • Yap A.S.
      • Niessen C.
      • Gumbiner B.M.
      ), these observations suggest strongly that not all ligand-occupied cadherin molecules engage in signaling to PI 3-kinase or Rac. Instead, cadherin signaling in this pathway may be confined to regions of the cell where new contacts are forming and cadherin molecules are in the process of undergoing homophilic ligation. This would be consistent with increasing evidence for strict subcellular compartmentalization of 3-phosphoinositide (
      • Servant G.
      • Weiner O.D.
      • Herzmark P.
      • Balla T.
      • Sedat J.W.
      • Bourne H.R.
      ) and Rac signaling (
      • Kraynov V.S.
      • Chamberlain C.
      • Bokoch G.M.
      • Schwartz M.A.
      • Slabaugh S.
      • Hahn K.M.
      ). Identifying the molecular mechanisms responsible for such spatio-temporal restriction will be an interesting challenge for the future.
      In conclusion, we propose that the E-cadherin-activated Rac signaling pathway described in this report acts as a local, membrane-based regulator that controls the adhesive response to initial cadherin homophilic ligation (Fig. 8). We envisage that the spatial restriction of cadherin-activated Rac signaling allows actin assembly to be directed specifically to nascent adhesive sites. In the planar adhesion assay that we utilized, these sites are represented by the leading edges of cadherin-based lamellipodia. In native cell-cell contacts, such cadherin-activated signaling would occur in the punctate or filopodial contacts that cells first make with one another, thereby directing actin assembly to drive the extension of cell surfaces upon one another, expanding the zones of intercellular contact. This model does not exclude potential roles for other cell signals; indeed, it is probable that following initial homophilic ligation further signals may be initiated to reinforce adhesion and remodel the actin cytoskeleton (
      • Braga V.
      ,
      • Kaibuchi K.
      • Kuroda S.
      • Fukata M.
      • Nakagawa M.
      ). Instead, cadherin-activated Rac signaling is likely to be an initial point in a cascade of signaling events that are spatially and temporally regulated in the process of cadherin-based cell-cell recognition.

      Acknowledgments

      We thank our colleagues Carien Niessen and Barry Gumbiner for the generous gift of hE/Fc-expressing CHO cells and Cara Gottardi, Marc Symons, Masatoshi Takeichi, Peggy Wheelock, Mark Lemmon, Alan Hall, Kim Weber, John Hancock, Neil Hotchin, Bill Gullick, and Jon Chernoff for providing valuable reagents. We are grateful to Montse Jaumot for help with the Akt assays, to Marita Goodwin and Lucia Ha for adroit technical assistance, and all of the members of our laboratory for support and good cheer.

      REFERENCES

        • Takeichi M.
        Current Opin. Cell Biol. 1995; 7: 619-627
        • Yap A.S.
        • Brieher W.M.
        • Gumbiner B.M.
        Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146
        • Adams C.L.
        • Nelson W.J.
        • Smith S.J.
        J. Cell Biol. 1996; 135: 1899-1911
        • Adams C.L.
        • Chen Y.-T.
        • Smith S.J.
        • Nelson W.J.
        J. Cell Biol. 1998; 142: 1105-1119
        • Abercrombie M.
        Proc. R. Soc. Lond. B. 1980; 207: 129-147
        • Yonemura S.
        • Itoh M.
        • Nagafuchi A.
        • Tsukita S.
        J. Cell Sci. 1995; 108: 127-142
        • Yap A.S.
        • Brieher W.M.
        • Pruschy M.
        • Gumbiner B.M.
        Curr. Biol. 1997; 7: 308-315
        • Angres B.
        • Barth A.
        • Nelson W.J.
        J. Cell Biol. 1996; 134: 549-557
        • Yokoyama S.
        • Tachibana K.
        • Nakanishi H.
        • Yamamoto Y.
        • Irie K.
        • Mandai K.
        • Nagafuchi A.
        • Moden M.
        • Takai Y.
        Mol. Biol. Cell. 2001; 12: 1595-1609
        • Pece S.
        • Chiariello M.
        • Murga C.
        • Gutkind J.S.
        J. Biol. Chem. 1999; 274: 19347-19351
        • Noren N.K.
        • Niessen C.M.
        • Gumbiner B.M.
        • Burridge K.
        J. Biol. Chem. 2001; 276: 33305-33308
        • Nakagawa M.
        • Fukata M.
        • Yamagawa M.
        • Itoh M.
        • Kaibuchi K.
        J. Cell Sci. 2001; 114: 1829-1838
        • Kim S.H., Li, Z.
        • Sacks D.B.
        J. Biol. Chem. 2000; 275: 36999-37005
        • Fagotto F.
        • Gumbiner B.M.
        Dev. Biol. 1996; 180: 445-454
        • Musil L.S.
        • Cunningham B.A.
        • Edelman G.M.
        • Goodenough D.A.
        J. Cell Biol. 1990; 111: 2077-2088
        • Brieher W.M.
        • Yap A.S.
        • Gumbiner B.M.
        J. Cell Biol. 1996; 135: 487-489
        • Nobes C.D.
        • Hawkins P.
        • Stephens L.
        • Hall A.
        J. Cell Sci. 1995; 108: 225-233
        • Ridley A.J.
        • Paterson H.F.
        • Johnston C.L.
        • Diekmann D.
        • Hall A.
        Cell. 1992; 70: 401-410
        • Jaumot M.
        • Yan J.
        • Clyde-Smith J.
        • Sluimer J.
        • Hancock J.F.
        J. Biol. Chem. 2002; 277: 272-278
        • del Pozo M.A.
        • Price L.S.
        • Alderson N.B.
        • Ren X.-D.
        • Schwartz M.A.
        EMBO J. 2000; 19: 2008-2014
        • Hawkins P.T.
        • Eguinoa A.
        • Qiu R.-G.
        • Stokoe D.
        • Cooke F.T.
        • Walters R.
        • Wennstrom S.
        • Claesson-Welsh L.
        • Evans T.
        • Symons M.
        • Stephens L.
        Curr. Biol. 1995; 5: 393-403
        • Cantrell D.A.
        J. Cell Sci. 2000; 114: 1439-1445
        • Reif K.
        • Nobes C.D.
        • Thomas G.
        • Hall A.
        • Cantrell D.A.
        Curr. Biol. 1996; 6: 1445-1455
        • Vanhaesebroeck B.
        • Leevers S.J.
        • Ahmadi K.
        • Timms J.
        • Katso R.
        • Driscoll P.C.
        • Woscholski R.
        • Parker P.J.
        • Waterfield M.D.
        Annu. Rev. Biochem. 2001; 70: 535-602
        • Scheid M.P.
        • Woodgett J.R.
        Nat. Rev. Mol. Cell. Biol. 2001; 2: 760-768
        • Watton S.J.
        • Downward J.
        Curr. Biol. 1999; 9: 433-436
        • Andjelkovic M.
        • Alessi D.R.
        • Meier R.
        • Fernandez A.
        • Lamb N.J.
        • Frech M.
        • Cron P.
        • Cohen P.
        • Lucocq J.M.
        • Hemmings B.A.
        J. Biol. Chem. 1997; 272: 31515-31524
        • Downward J.
        Curr. Opin. Cell Biol. 1998; 10: 262-267
        • Gillham H.
        • Golding M.C.H.M.
        • Pepperkok R.
        • Gullick W.J.
        J. Cell Biol. 1999; 146: 869-880
        • Lemmon M.A.
        • Ferguson K.M.
        Biochem. J. 2000; 350: 1-18
        • Kavran J.M.
        • Klein D.E.
        • Lee A.
        • Falasca M.
        • Isakoff S.J.
        • Skolnik E.Y.
        • Lemmon M.A.
        J. Biol. Chem. 1998; 273: 30497-30508
        • Prior I.A.
        • Harding A.
        • Yan J.
        • Sluimer J.
        • Parton R.G.
        • Hancock J.F.
        Nat. Cell Biol. 2001; 3: 368-375
        • Espada J.
        • Perez-Moreno M.
        • Braga V.M.M.
        • Rodriguez-Viciana P.
        • Cano A.
        J. Cell Biol. 1999; 146: 967-980
        • Carmeliet P.
        • Lampugnani M.G.
        • Moons L.
        • Breviario F.
        • Compernolle V.
        • Bono F.
        • Balconi G.
        • Spagnuolo R.
        • Oostuyse B.
        • Dewerchin M.
        • Zanetti A.
        • Angellilo A.
        • Mattot V.
        • Nuyens D.
        • Lutgens E.
        • Clotman F.
        • de Ruiter M.C.
        • Gittenberger-de Groot A.
        • Poelmann R.
        • Lupu F.
        • Herbert J.M.
        • Collen D.
        • Dejana E.
        Cell. 1999; 98: 147-157
        • Hordijk P.L.
        • ten Klooster J.P.
        • van der Kammen R.A.
        • Michiels F.
        • Oomen L.C.
        • Collard J.G.
        Science. 1997; 278: 1464-1466
        • Noren N.K.
        • Liu B.P.
        • Burridge K.
        • Kreft B.
        J. Cell Biol. 2000; 150: 567-579
        • Bishop A.L.
        • Hall A.
        Biochem. J. 2000; 348: 241-255
        • Vasioukhin V.
        • Bauer C.
        • Yin M.
        • Fuchs E.
        Cell. 2000; 100: 209-219
        • Braga V.M.M.
        • Machesky L.M.
        • Hall A.
        • Hotchin N.A.
        J. Cell Biol. 1997; 137: 1421-1431
        • Braga V.M.
        • Del Maschio A.
        • Machesky L.
        • Dejana E.
        Mol. Biol. Cell. 1999; 10: 9-22
        • Takaishi K.
        • Sasaki T.
        • Kotani H.
        • Nishioka H.
        • Takai Y.
        J. Cell Biol. 1997; 139: 1047-1059
        • Yap A.S.
        • Niessen C.
        • Gumbiner B.M.
        J. Cell Biol. 1998; 141: 779-789
        • Servant G.
        • Weiner O.D.
        • Herzmark P.
        • Balla T.
        • Sedat J.W.
        • Bourne H.R.
        Science. 2000; 287: 1037-1040
        • Kraynov V.S.
        • Chamberlain C.
        • Bokoch G.M.
        • Schwartz M.A.
        • Slabaugh S.
        • Hahn K.M.
        Science. 2000; 290: 333-337
        • Braga V.
        Exp. Cell Res. 2000; 261: 83-90
        • Kaibuchi K.
        • Kuroda S.
        • Fukata M.
        • Nakagawa M.
        Curr. Opin. Cell Biol. 1999; 11: 591-596