Integrin regulation of epidermal growth factor (EGF) receptor and of ...

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Biochemical Society Transactions (2004) Volume 32, part 3

Integrin regulation of epidermal growth factor (EGF) receptor and of EGF-dependent responses S. Cabodi*1 , L. Moro†1 , E. Bergatto*, E. Boeri Erba*, P. Di Stefano*, E. Turco*, G. Tarone* and P. Defilippi*1 *Dipartimento di Genetica, Biologia e Biochimica, Universita` di Torino, Via Santena 5 bis, 10126 Torino, Italy, and †Dipartimento Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita` del Piemonte Orientale, Via Bovio 6, 28100 Novara, Italy

Abstract Integrin signalling co-ordinates with signalling originating from growth factor receptors in the co-operative control of cell proliferation, survival and migration. Increasing evidence suggests that integrins form physical complexes at the cell membrane with growth factor receptors, giving rise to signalling platforms at the adhesive sites. It is probable that at these sites integrins regulate adhesion and at the same time physically constrain and direct the response to soluble growth factors towards proliferation or survival stimuli. These cooperative effects might depend on integrin ability to activate growth factor receptors. In the present paper, we summarize our recent study showing that integrin-dependent adhesion triggers ligand-independent EGFR (epidermal growth factor receptor) activation to transduce downstream signalling. In addition, we also show that integrin-induced signalling pathways are necessary for EGF-dependent transcriptional response, demonstrating the requirement of the co-operation between cell–matrix adhesion and EGFR to achieve full biological responses.

Introduction Integrins are adhesive receptors formed by α and β subunits, which anchor extracellular matrix proteins to the actin cytoskeleton. Integrins also trigger multiple signalling pathways, which, on the basis of differential expression and specific localization of the receptors, are involved in cell migration, proliferation, differentiation and survival from apoptosis [1]. Integrin signalling includes Ca2+ influx, cytoplasmic alkalinization, activation of potassium channels, tyrosine phosphorylation of cytoplasmic proteins and activation of the MAPKs (mitogen-activated protein kinases) [2–6]. To promote intracellular signalling, integrins interact with transducing molecules such as the Fak (focal adhesion kinase) and Src kinase family. The N-terminal domain of Fak interacts with β1 and β3 integrins [7,8], whereas its C-terminal part binds SH2 (Src homology 2) and SH3 domains of several proteins involved in focal adhesion assembly and downstream signalling [9]. After integrin activation, Fak is phosphorylated on Tyr-397, which becomes an high-affinity binding site for the SH2 domain of c-Src. The Src kinase then phosphorylates focal adhesion components, such as the cytoskeletal adaptors talin, paxillin, p130Cas, and the Fak itself on the Tyr-925, leading to signalling functions [10,11].

Key words: adhesion, epidermal growth factor (EGF), extracellular matrix, growth factor receptor, integrin, signalling. Abbreviations used: EGF(R), epidermal growth factor (receptor); Egr-1, early growth response gene product 1; ERK, extracellular-signal-regulated kinase; Fak, focal adhesion kinase; Grb2, growth-factor-receptor-bound protein 2; MAPK, mitogen-activated protein kinase; PDGF, plateletderived growth factor; PI3K, phosphoinositide 3-kinase; RPTK, receptor protein tyrosine kinase; SH, Src homology; VEGFR, vascular endothelial growth factor receptor. 1 2

These authors contributed equally to this work. To whom correspondence should be addressed (email paola.defi[email protected]).

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Phosphorylated Fak interacts with the adaptor molecule Grb2 (growth-factor-receptor-bound protein 2), leading to MAPK activation [12], through a B-Raf-dependent pathway [13]. In addition to Fak, some β1 and αv integrins activate the Src family member Fyn and the adaptor Shc (Src-homology collagen). The assembly of this transduction complex involves caveolin, a transmembrane protein that co-operates with integrins to activate signalling pathways. After cell–matrix adhesion, integrin–caveolin–Fyn complexes associate with phosphorylated Shc, which in turn, interacts with the Grb2– Sos complex leading to the activation of the Ras-MAPK cascade [14]. Integrins can also associate with proteins belonging to the tetraspan family (CD9, CD63 and CD81) to modulate intracellular signalling [15]. In addition to the pathways described above, growth factor receptors are integrin partners in assembling a transduction machinery. Increasing evidence indicates that integrins can co-operate with RPTKs (receptor protein tyrosine kinases) in transducing proliferative signals and in regulating survival and migration [4,5]. The molecular mechanisms involved in such events include integrin-dependent activation of RPTKs [16], enhancement of growth factor signals [17,18], recruitment of crucial transducing proteins to membrane cytoskeletal complexes [19] and enhancement of nuclear translocation of transcriptional regulators [20,21]. In the present paper, we will discuss the ability of integrins to induce EGFR (epidermal growth factor receptor) activation and to regulate the responses of EGFR to its ligand EGF.

Integrin cross-talk with RPTKs Integrin–growth factor receptor co-operation has been extensively demonstrated [6], showing that integrins regulate

Molecular Environment of Integrins

RPTK functions including receptor transactivation, coordination and compartmentalization and downstream signalling. Regulation of the cell cycle is a prototypic event occurring by joint integrin/RPTKs signalling [22]. Strong evidence in fact, indicates that integrin- and RPTK-dependent signals need to be integrated at various levels to induce cell proliferation. Normal cells need to adhere to the matrix to progress through the mid-G1 phase of the cell cycle after mitogen treatment [23]. In adherent cells, soluble ligands, such as EGF or PDGF (platelet-derived growth factor), stimulate RPTK activity and a cascade of downstream events, which co-operates with those induced by integrins. Joint integrin/RPTKs signalling causes expression of cyclin D, activation of cyclin D-dependent kinases and degradation of Cdk (cyclin-dependent kinase) inhibitor p27 [24]. Reciprocal cross-talk between integrins and the RPTKs consists in at least three hierarchical mechanisms, the first consisting in integrin ability to trigger ligand-independent RPTKs activation. On the other hand, integrins are also required for propagation of ligand-mediated signalling, leading to a full repertoire of RPTK activities [25,26]. As a third possibility, integrin and growth factor receptor signalling can act on parallel pathways and synergize to reach a final biological response [22].

Figure 1 Integrin-dependent activation of the EGFR and transactivation of ERK1/ERK2 MAPK and AKT pathways (A) ECV304 cells were starved, detached and either kept in suspension (−) or allowed to adhere on 10 µg/ml fibronectin-coated dishes (+). Cell extracts, prepared as in [16], were immunoprecipitated with EGFR antibodies and the immunoprecipitates separated on an SDS/6% polyacrylamide gel. Western blots were analysed with phosphotyrosine mAb (monoclonal antibody) PY20 and re-blotted with EGFR antibodies. (B) ECV304 cells were plated for 15 min on mAb L230 (αv integrin)coated dishes with or without 5 µM c-Src kinase inhibitor PP1. mAb B212 to the β3 subunit was then added to the cells, which were further incubated for 30 min at 4◦ C before detergent extraction. Cell extracts were immunoprecipitated by addition of Protein A–Sepharose (IP anti-αvβ3). Immunoblotting was performed with antibodies to EGFR, p130Cas, p125Fak, c-Src or active c-Src (phosphorylated on Tyr-416). (C) ECV304 cells were kept in suspension (−) or seeded on 10 µg/ml fibronectin (+) with or without 250 nM tyrphostin AG1478 for 30 min. Cell extracts were separated on an SDS/10% polyacrylamide gel, blotted either with phospho AKT antibodies and re-blotted with AKT antibodies (upper panels) or with phospho ERK1/ERK2 antibodies and re-blotted with ERK1/ERK2 antibodies (lower panels).

Integrin-dependent activation of RPTKs: the EGFR Direct phosphorylation of growth factor receptors by integrin signalling represents a potential mechanism by which integrins can enhance signalling pathways emanating from growth factor receptors. It has been shown, in fact, that integrins stimulate phosphorylation, and at least partial activation, of several RPTKs, including the EGFR [16], HGF-R [27,28], PDGFβ-R [29], Ron kinase [30] and VEGFR (vascular endothelial growth factor receptor) [31], in the absence of any growth factor ligand. The number of RPTKs involved suggests that this activation scheme can be a broadly used mechanism in adhesion-mediated signalling. In other words, cell–matrix adhesion represents a priming event, and a limiting factor in addition to soluble ligand for RPTKs activation. Recently, we found that integrins induce EGFR tyrosine phosphorylation in the absence of EGFR ligands [16] (Figure 1A). Molecular mechanisms regulating integrin-dependent EGFR phosphorylation are distinct from the classical activation induced by mitogenic concentrations of soluble EGF. In the early phases of cell adhesion integrins associate with EGFR on the cell membrane in a macromolecular complex (Figure 1B), suggesting that a close proximity between integrins and RPTK could locally increase receptor densities and lead to oligomerization and transactivation. This simple mechanistic explanation is made complex by the fact that integrin-dependent EGFR phosphorylation requires Src kinase activity (Figure 1B), and the assembly of Src and the adaptor protein p130Cas in a multimeric complex with integrins and EGFR [32]. The requirement of Src kinase is in

line with the mechanism proposed for the integrin-induced activation of Ron [30]. In this model, in fact, integrins activate Src tyrosine kinase, which in turn mediates Ron activation. A second feature of integrin-dependent EGFR activation is that it leads to phosphorylation of EGFR on a specific subset of tyrosine residues, only partially overlapping with those phosphorylated by EGF. After adhesion, EGFR is phosphorylated on Tyr-845, Tyr-1068, Tyr-1086 and Tyr1173, but not on Tyr-1148, a major site of phosphorylation in response to EGF [32]. Interestingly, integrin-dependent phosphorylation of Tyr-845, which is located in the activation loop of the RPTK, could occur through Src kinase activity as a priming event for subsequent activation of the EGFR kinase activity, which in turn is required for phosphorylation of the other tyrosine residues. As an alternative mechanism, integrins can cause the recruitment of tyrosine phosphatases such as SHP-2 at the plasma membrane in close proximity to the PDGF-R. C 2004

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SHP-2 de-phosphorylates PDGF-R on specific tyrosine residues involved in Ras–GAP binding, thus decreasing Ras-GAP activation and potentiating Ras signalling [33], resulting in PDGF-R-downstream events, without involving the activation of the receptor tyrosine kinase. By treatment with the specific EGFR inhibitor tyrphostin AG1478 and by expression of a dominant-negative EGFR mutant, lacking the intracellular C-terminal domain (EGFR/ C), we have shown that integrin-dependent EGFR activation leads to adhesion-dependent ERK1/ERK2 (extracellular-signal-regulated kinase) MAPK (Figure 1C, lower panels and [16]) and AKT activation (Figure 1C, upper panels). These results indicate that integrins can utilize EGFR as a transducing element in the matrix-induced signalling pathways. To strengthen further the relevance of integrininduced EGFR activation, Marcoux and Vuori [34] have shown recently that it is instrumental for integrin-dependent Rac activation, through PI3K (phosphoinositide 3-kinase) activation and GTP loading on Vav2, a known exchange factor for Rac. A third feature of integrin-dependent EGFR activation is a lower apparent stoichiometry, which leads to a lower level of tyrosine phosphorylation compared with that observed with mitogenic doses of EGF [16]. Phosphorylation of EGFR on a specific subset of tyrosine residues [32] could account for this event. According to this characteristic, integrininduced EGFR activation is not sufficient for G1 –S cellcycle progression and proliferation [16], confirming that in untransformed cells anchorage-induced signalling is a key control step that co-operates with pathways activated by growth factors to induce cell proliferation. On the other hand, integrin-dependent EGFR activation is sufficient for adhesion-dependent cell survival, by an EGFR kinase-dependent mechanism [16]. It is well defined that cells plated on the matrix proteins activates signals that protect cells from anoikis [35]. In cells expressing EGFR, the ability of cells to survive on fibronectin is blocked by the specific EGFR kinase inhibitor AG1478 or by the expression of the dominant-negative form EGFR/C [16]. EGFR-mediated cell survival is blocked by the PI3K inhibitor wortmannin [16], suggesting that PI3K and AKT, well-defined players in adhesion-dependent survival to anoikis, are involved (see also Figure 1C). Integrin-EGFR signalling had also been recently implicated in the regulation of expression of the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death), a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment by concomitant lack of β1 integrin engagement, down-regulation of EGFR expression and inhibition of MAPK signalling [36]. The use of EGFR inhibitor AG1478 and of EGFR/C also demonstrated that integrin-dependent EGFR activation plays a crucial role in lamellipodia formation, cell spreading and migration [34]. Therefore, taken together, these results show that after adhesion, EGFR transactivation accounts for a specific repertoire of mechanisms, namely cell survival and actin cytoskeleton organization involved in cell migration (Figure 2). C 2004

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Figure 2 Model of integrin-dependent EGFR phosphorylation After cell–matrix adhesion, integrins associates with the EGFR, active c-Src kinase (PY416) and p130Cas. As a consequence of cell adhesion, EGFR is phosphorylated on Tyr-845, Tyr-1068, Tyr-1086 and Tyr-1173, but not on Tyr-1148 [16], and transduces signalling on ERK1/ERK2, AKT and Rac, leading to cell survival and actin cytoskeleton organization.

Integrin requirement for propagation of EGF-dependent signalling Integrins have been shown to potentiate signalling pathways in response to different growth factors, such as insulin, PDGF, EGF, FGF (fibroblast growth factor) and VEGF [17,37–45]. Many integrins form complexes with RPTKs and some appear to have preferred partners. For example, the αvβ3 integrin associates and synergizes with the insulin receptor, the PDGF-R and the VEGFR [38,41,45,46]. β1 integrins associate with the EGFR [17,47,48] and α6β4 combines with the EGFR [25], Erb-B2 [49] and the HGF-R [26]. Biochemical analysis revealed that αvβ3 integrin occupancy by its matrix ligand is required to get full tyrosine phosphorylation of insulin and PDGFβ receptors and their binding to signalling molecules, such as IRS-1 (insulin receptor substrate), PLCγ , Ras-GAP, p85-PI3K and the tyrosine phosphatases SHP-1 and SHP-2 [38,41,50–52]. In endothelial cells, moreover, αvβ3 integrin potentiates the activation of VEGFR and of p85-PI3K by its ligand [45]. Integrin-dependent adhesion is also required for EGFdependent activation of downstream signalling [17,18]. To dissect integrin-dependent pathways in EGF responses, we treated epithelial ECV304 cells with EGF in two distinct conditions, either attached to matrix proteins or kept in suspension, and we analysed the pattern and the extent of downstream signalling pathways. As shown in Figure 3(A), EGF treatment induces phosphorylation of the EGFR both in cells kept in suspension and in cells attached to the matrix, indicating that the receptor is activated by its ligand in suspension or in adherent conditions. When the immunoprecipitates were analysed for Shc and Grb2 binding

Molecular Environment of Integrins

Figure 3 Integrins are required for EGF-dependent transcriptional regulation ECV304 cells were kept in suspension (−) or allowed to adhere on fibronectin-coated dishes (+) with or without 50 ng/ml EGF for 30 min. (A) Cell extracts were immunoprecipitated with EGFR antibodies and the immunoprecipitates were separated on an SDS/6% polyacrylamide gel. Western blots were analysed with phosphotyrosine mAb PY20 and re-blotted with EGFR antibodies. (B) Cell extracts were separated on an SDS/10% polyacrylamide gel, and analysed with phospho ERK1/ERK2 antibodies (upper panel) and Egr-1 antibodies (lower panel).

to the receptor, no differences were observed in the ability of EGF to induce recruitment of these two signalling molecules (results not shown), indicating that the phosphorylation of the EGFR and its activation are sufficient to recruit early signalling molecules to the EGFR cytoplasmic domain. EGF was also capable of triggering phosphorylation of ERK1/ERK2 MAPK both in cells kept in suspension and in cells attached to the matrix (Figure 3B), indicating that EGF-dependent phosphorylation of EGFR and ERK1/ ERK2 MAPK, two early, membrane-proximal signalling events, are integrin independent. To investigate the role of cell–matrix adhesion in EGFdependent pathways, we analysed the ability of EGF to induce expression of the transcription factor Egr-1 (early growth response gene product 1) in adherent versus suspended cells. Egr-1, an immediate early gene induced by exposure of quiescent fibroblasts to serum, is a broadly expressed prototypical member of the zinc finger family of transcription factors [53]. Egr-1 is a specific target of cytokines and growth factors, such as EGF [54]. As shown in Figure 3(B), whereas EGF induced Egr-1 expression in adherent cells, any protein was detectable in cells treated by EGF in suspension, indicating that EGF triggers Egr-1 transcription only in adherent cells. Therefore integrins are required for EGF-dependent transcriptional control. Egr-1 expression is partially dependent on ERK1/ERK2 MAPK (unpublished results). Since ERK1/ERK2 MAPK, although phosphorylated by EGF in suspended cells, are not sufficient to trigger Egr-1 transcription, it is possible to speculate that either recruitment of additional transducing proteins or the enhancement of nuclear translocation of these transcriptional regulators [20] are required for transcriptional induction of Egr-1. Therefore integrin signalling makes an essential contribution to the regulation of early transcription factor expression by soluble growth factors, indicating that co-

stimulation of integrins and growth factor receptors is necessary for efficient transduction of EGF signalling to the nucleus.

Conclusions In conclusion, integrins exert prominent signalling functions through their co-operations with RPTKs. In different cell types, their association with RPTKs provides positional cues to control RPTK function, allowing the recruitment of signalling molecules in the proximity of the receptors. They induce RPTK activation in the absence of growth factors, thus priming the RPTKs towards distinct adhesiondependent biological processes, such as cell survival and migration. On the other hand, integrin signalling is essential for RPTK response to their specific ligands leading to control of transcriptional events and cell-cycle progression. Future studies will investigate the involvement of integrin/RPTKs co-operation in development and diseases, and will determine whether dysregulation of integrin/RPTKs cross-talk can contribute to the onset of pathological events.

This work was supported by grants from AIRC, Ministero dell’Istruzione, dell’Universita` e della Ricerca Scientifica, Special project ‘Oncology’, Compagnia San Paolo/FIRMS (Torino, Italy) and Progetti Regione Piemonte. S.C. is a fellowship of Fondazione Italiana Ricerca Cancro.

References 1 Hynes, R.O. (2002) Cell (Cambridge, Mass.) 110, 673–687 2 Defilippi, P., Olivo, C., Venturino, M., Dolce, L., Silengo, L. and Tarone, G. (1999) Microsc. Res. Tech. 47, 67–78 3 Howe, A.K., Aplin, A.E. and Juliano, R.L. (2002) Curr. Opin. Genet. Dev. 12, 30–35 4 Schwartz, M.A. and Ginsberg, M.H. (2002) Nat. Cell Biol. 4, E65–E68 5 Miranti, C.K. and Brugge, J.S. (2002) Nat. Cell Biol. 4, E83–E90 6 Giancotti, F.G. and Tarone, G. (2003) Annu. Rev. Cell Dev. Biol. 19, 173–206 7 Schaller, M.D., Borgman, C.A., Cobb, B.S., Vines, R.R., Reynolds, A.B. and Parsons, J.T. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5192–5196 8 Schaller, M.D., Otey, C.A., Hildebrand, J.D. and Parsons, J.T. (1995) J. Cell Biol. 130, 1181–1187 9 Malik, R.K. and Parsons, J.T. (1996) Biochim. Biophys. Acta 1287, 73–76 10 Frame, M.C. (2002) Biochim. Biophys. Acta 1602, 114–130 11 Frame, M.C., Fincham, V.J., Carragher, N.O. and Wyke, J.A. (2002) Nat. Rev. Mol. Cell Biol. 3, 233–245 12 Schlaepfer, D.D. and Hunter, T. (1997) J. Biol. Chem. 272, 13189–13195 13 Barberis, L., Wary, K.K., Fiucci, G., Liu, F., Hirsch, E., Brancaccio, M., Altruda, F., Tarone, G. and Giancotti, F.G. (2000) J. Biol. Chem. 275, 36532–36540 14 Wary, K.K., Mainiero, F., Isakoff, S.J., Marcantonio, E.E. and Giancotti, F.G. (1996) Cell (Cambridge, Mass.) 87, 733–743 15 Berditchevski, F. (2001) J. Cell Sci. 114, 4143–4151 16 Moro, L., Venturino, M., Bozzo, C., Silengo, L., Altruda, F., Beguinot, L., Tarone, G. and Defilippi, P. (1998) EMBO J. 17, 6622–6632 17 Miyamoto, S., Teramoto, H., Gutkind, J.S. and Yamada, K.M. (1996) J. Cell Biol. 135, 1633–1642 18 Short, S.M., Talbott, G.A. and Juliano, R.L. (1998) Mol. Biol. Cell 9, 1969–1980 19 Del Pozo, M.A., Kiosses, W.B., Alderson, N.B., Meller, N., Hahn, K.M. and Schwartz, M.A. (2002) Nat. Cell Biol. 4, 232–239 20 Aplin, A.E. and Juliano, R.L. (2001) J. Cell Biol. 155, 187–191 21 Hirsch, E., Barberis, L., Brancaccio, M., Azzolino, O., Xu, D., Kyriakis, J.M., Silengo, L., Giancotti, F.G., Tarone, G., Fassler, R. et al. (2002) J. Cell Biol. 157, 481–492 C 2004

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441

442


22 Assoian, R.K. and Schwartz, M.A. (2001) Curr. Opin. Genet. Dev. 11, 48–53 23 Assoian, R.K. (1997) J. Cell Biol. 136, 1–4 24 Carrano, A.C. and Pagano, M. (2001) J. Cell Biol. 153, 1381–1390 25 Mariotti, A., Kedeshian, P.A., Dans, M., Curatola, A.M., Gagnoux-Palacios, L. and Giancotti, F.G. (2001) J. Cell Biol. 155, 447–458 26 Trusolino, L., Bertotti, A. and Comoglio, P.M. (2001) Cell (Cambridge, Mass.) 107, 643–654 27 Rusciano, D., Lorenzoni, P. and Burger, M.M. (1996) J. Biol. Chem. 271, 20763–20769 28 Wang, R., Kobayashi, R. and Bishop, J.M. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8425–8430 29 Sundberg, C. and Rubin, K. (1996) J. Cell Biol. 132, 741–752 30 Danilkovitch-Miagkova, A., Angeloni, D., Skeel, A., Donley, S., Lerman, M. and Leonard, E.J. (2000) J. Biol. Chem. 275, 14783–14786 31 Wang, J.F., Zhang, X.F. and Groopman, J.E. (2001) J. Biol. Chem. 276, 41950–41957 32 Moro, L., Dolce, L., Cabodi, S., Bergatto, E., Erba, E.B., Smeriglio, M., Turco, E., Retta, S.F., Giuffrida, M.G., Venturino, M. et al. (2002) J. Biol. Chem. 277, 9405–9414 33 DeMali, K.A., Balciunaite, E. and Kazlauskas, A. (1999) J. Biol. Chem. 274, 19551–19558 34 Marcoux, N. and Vuori, K. (2003) Oncogene 22, 6100–6106 35 Frisch, S.M., Vuori, K., Ruoslahti, E. and Chan-Hui, P.Y. (1996) J. Cell Biol. 134, 793–799 36 Reginato, M.J., Mills, K.R., Paulus, J.K., Lynch, D.K., Sgroi, D.C., Debnath, J., Muthuswamy, S.K. and Brugge, J.S. (2003) Nat. Cell Biol. 5, 733–740 37 Cybulsky, A.V., McTavish, A.J. and Cyr, M.D. (1994) J. Clin. Invest. 94, 68–78 38 Vuori, K. and Ruoslahti, E. (1994) Science 266, 1576–1578

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39 Jones, P.L., Crack, J. and Rabinovitch, M. (1997) J. Cell Biol. 139, 279–293 40 Rusnati, M., Tanghetti, E., Dell’Era, P., Gualandris, A. and Presta, M. (1997) Mol. Biol. Cell 8, 2449–2461 41 Schneller, M., Vuori, K. and Ruoslahti, E. (1997) EMBO J. 16, 5600–5607 42 Guilherme, A., Torres, K. and Czech, M.P. (1998) J. Biol. Chem. 273, 22899–22903 43 Lee, Y.J. and Streuli, C.H. (1999) J. Biol. Chem. 274, 22401–22408 44 Li, J., Lin, M.L., Wiepz, G.J., Guadarrama, A.G. and Bertics, P.J. (1999) J. Biol. Chem. 274, 11209–11219 45 Soldi, R., Mitola, S., Strasly, M., Defilippi, P., Tarone, G. and Bussolino, F. (1999) EMBO J. 18, 882–892 46 Borges, E., Jan, Y. and Ruoslahti, E. (2000) J. Biol. Chem. 275, 39867–39873 47 Sieg, D.J., Hauck, C.R., Ilic, D., Klingbeil, C.K., Schaefer, E., Damsky, C.H. and Schlaepfer, D.D. (2000) Nat. Cell Biol. 2, 249–256 48 Yu, X., Miyamoto, S. and Mekada, E. (2000) J. Cell Sci. 113, 2139–2147 49 Falcioni, R., Antonini, A., Nistico, P., Di Stefano, S., Crescenzi, M., Natali, P.G. and Sacchi, A. (1997) Exp. Cell Res. 236, 76–85 50 Maile, L.A., Badley-Clarke, J. and Clemmons, D.R. (2003) Mol. Biol. Cell 14, 3519–3528 51 Baron, W., Decker, L., Colognato, H. and ffrench-Constant, C. (2003) Curr. Biol. 13, 151–155 52 Colognato, H., Baron, W., Avellana-Adalid, V., Relvas, J.B., Baron-Van Evercooren, A., Georges-Labouesse, E. and ffrench-Constant, C. (2002) Nat. Cell Biol. 4, 833–841 53 Thiel, G. and Cibelli, G. (2002) J. Cell. Physiol. 193, 287–292 54 Tsai, J.C., Liu, L., Guan, J. and Aird, W.C. (2000) Am. J. Physiol. Cell Physiol. 279, C1414–C1424 Received 3 December 2003