The Integrin alpha 7 Cytoplasmic Domain Regulates Cell Migration, Lamellipodia Formation, and p130CAS/Crk Coupling*

Dirk MielenzDagger, Sandra Hapke, Ernst Pöschl, Helga von der Mark, and Klaus von der Mark§

From Klinisch-molekular biologisches Forschungszentrum, Department for Experimental Medicine I, University of Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany

Received for publication, December 20, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The integrin alpha 7beta 1 is the major laminin-binding integrin in skeletal, heart, and smooth muscle and is a receptor for laminin-1 and -2. It mediates myoblast migration on laminin-1 and -2 and thus might be involved in muscle development and repair. Previously we have shown that alpha 7B as well as the alpha 7A and -C splice variants induce cell motility on laminin when transfected into nonmotile HEK293 cells. In this study we have investigated the role of the cytoplasmic domain of alpha 7 in the laminin-induced signal transduction of alpha 7beta 1 integrin regulating cell adhesion and migration. Deletion of the cytoplasmic domain did not affect assembly of the mutated alpha 7Delta cyt/beta 1 heterodimer on the cell surface or adhesion of alpha 7Delta cyt-transfected cells to laminin. The motility of these cells on the laminin-1/E8 fragment, however, was significantly reduced to the level of mock-transfected cells; lamellipodia formation and polarization of the cells were also impaired. Adhesion to the laminin-1/E8 fragment induced tyrosine phosphorylation of the focal adhesion kinase, paxillin, and p130CAS as well as the formation of a p130CAS-Crk complex in wild-type alpha 7B-transfected cells. In alpha 7BDelta cyt cells, however, the extent of p130CAS tyrosine formation was reduced and formation of the p130CAS-Crk complex was impaired, with unaltered levels of p130CAS and Crk protein levels. These findings indicate adhesion-dependent regulation of p130CAS/Crk complex formation by the cytoplasmic domain of alpha 7B integrin after cell adhesion to laminin-1/E8 and imply alpha 7B-controlled lamellipodia formation and cell migration through the p130CAS/Crk protein complex.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

During muscle repair, undifferentiated muscle precursor cells, so-called satellite cells, are activated and migrate to sites of damaged muscle along the basement membranes of pre-existing muscle fibers to close the wound by proliferating and fusing (1, 2). In vitro, skeletal myoblasts have been shown to migrate on laminin (LN)1 1 (3), the laminin-1-E8 fragment that is derived from laminin-1 by elastase digestion (4), and on laminin-2 (5), but not on fibronectin (4). The major component of muscle basement membranes supporting muscle cell migration is laminin-2 (6). The migration of fibroblast-like cells in culture involves polarization of cells, formation of filopodia, lamellipodia, stress fibers, and myosin-based contractility (7). Filopodia, lamellipodia and stress fiber formation are mediated by Cdc42, Rac, and Rho, respectively, which are members of the Rho family of small GTPases (8). Cdc42, Rac, and Rho can either be activated by soluble factors like growth factors, bioactive peptides, and hormones (8) or by integrins (9, 10), which can transduce signals from the extracellular matrix after clustering and ligand-induced conformational changes (11) in a hierarchical fashion (12).

Several proteins become tyrosine-phosphorylated after integrin-mediated cell attachment. Those are, among others, the focal adhesion kinase (FAK) (13), the adaptor protein p130CAS (Crk-associated Src substrate) (14, 15), and paxillin (13, 16). Activation of the nonreceptor FAK controls cell migration (13, 17-19). p130CAS is an adaptor protein, which was first identified as a highly tyrosine-phosphorylated protein in v-Src- and v-Crk-transformed cells (20-22). p130CAS contains an N-terminal SH3 domain, a substrate domain, a proline-rich region, and several tyrosine residues near the C terminus. p130CAS and paxillin are both Src substrates and bind to FAK with their SH3 domains (23). The adaptor protein Crk, which was first discovered as a highly tyrosine-phosphorylated protein in Rous sarcoma-transformed cells (24), forms a complex with tyrosine-phosphorylated p130CAS (25). Molecular cloning of c-Crk (26) revealed two isoforms, designated Crk I and Crk II, with molecular masses of 40 and 28 kDa, respectively. Crk II contains one N-terminal SH2 and two C-terminal SH3 domains (26). Tyrosine-phosphorylated p130CAS can exhibit up to 15 binding sites for the SH2 domain of Crk (24), and p130CAS/Crk binding serves as an integrin-induced switch promoting cytokine-induced migration of COS cells (27). Moreover, p130CAS has been shown to stimulate cell migration by overexpression in Chinese hamster ovary and tumor cells (28).

Blocking integrin alpha 7 antibodies inhibit the migration of myoblasts on laminin-1 and laminin-2, suggesting that alpha 7 is responsible for myoblast migration on laminin (5, 29). Integrin alpha 7 is mainly expressed in skeletal, smooth, and cardiac muscle (32), but also in some glioblastoma and melanoma cells (30, 31) and in nervous tissue (32, 33). The extracellular and the intracellular domains of integrin alpha 7 undergo developmentally regulated splicing (34-36); myoblasts express the cytoplasmic splice variant B and the extracellular splice variants X1 and X2. After myotube formation, the cytoplasmic splice variants A and C and the extracellular splice variant X2 become up-regulated. The alpha 7 chain is post-translationally cleaved into a 97-kDa fragment and a 35-kDa fragment (sizes for the B splice variant), which contains a large piece of the extracellular part (~25 kDa) (11). In the mature integrin, the fragments remain disulfide-linked. Transfection of integrin alpha 7 into alpha 7-deficient cells induces cell migration specifically on laminin-1 and -2 (5, 37, 38).

In this study we investigated the role of the cytoplasmic domain of the alpha 7 subunit in laminin-induced signaling. We deleted the cytoplasmic domain of alpha 7 and transfected 293 cells with a construct encoding the extracellular splice variant X2 (alpha 7X2Delta cyt) to elucidate the role of the alpha 7 cytoplasmic domain in terms of heterodimer formation, surface expression, integrin alpha 7-mediated cell attachment, migration, and p130CAS/Crk coupling. Deletion of the alpha 7 cytoplasmic domain did not affect receptor assembly or activity, as assessed by the ability of the mutant receptor to confer cell attachment. In contrast, cell migration, lamellipodia formation, and formation of the p130CAS signaling complex were reduced, highlighting a role for the alpha 7 cytoplasmic domain in signal transduction.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals-- Chemicals were from Sigma or Roth (Karlsruhe, Germany) if not stated otherwise.

Antibodies-- The affinity-purified polyclonal antibody U4+ directed against a peptide of the integrin alpha 7B cytoplasmic domain was kindly provided by Dr. Ulrike Mayer (39) and diluted 1:2000 for Western blotting. The rabbit antibody 242 E (directed against the alpha 7 extracellular domain; Ref. 37) was diluted 1:200. The monoclonal anti-alpha 7 mAbs 3C12 and 6A11 have been described previously (38), and the monoclonal anti-alpha 7 mAb 5A6 will be described elsewhere.2 Anti-beta 1 mAb TS2/16 and anti-vinculin mAb 7F9 (40) were generous gifts of Dr. Alexey Belkin, and anti-integrin alpha 6 mAb GoH3 was purchased from Immunotech (Marseille, France). Anti-p130CAS mAb, anti-Crk, anti-Erk, and anti-Shc mAbs and recombinant anti-phosphotyrosine Fab' fragment conjugated to peroxidase (RC20H) were from Signal Transduction Laboratories (distributed by Dianova, Hamburg, Germany) and diluted 1:1000 (p130CAS), 1:3000 (Crk), and 1:10,000 (RC20H), respectively. Monoclonal anti-phosphotyrosine antibody 2C8 (working concentration: 0.2 µg/ml) was purchased from Nanotools (Teningen, Germany). Secondary antibodies used for Western blotting (goat anti-rabbit-peroxidase and goat anti-mouse peroxidase) were from Bio-Rad and Jackson (West Grove, PA) and diluted 1:5000 and 1:20,000, respectively. FITC (fluorescein isothiocyanate)-labeled secondary antibodies were from Amersham Pharmacia Biotech (Braunschweig, Germany), and FITC-phalloidin was from Molecular Probes (Leiden, Netherlands).

Deletion of the Integrin alpha 7 Cytoplasmic Domain-- The integrin alpha 7X2A expression vector pCEP4alpha 7X2A (38) was digested with NheI and HindIII, which removed the cDNA segment encoding for the cytoplasmic domain except for the first two membrane-proximal amino acid residues (Lys-Leu). Ends were filled with Klenow polymerase, and the plasmid was religated, which resulted in a stop codon after the residues Lys-Leu. Plasmid DNA was purified according to the manufacturer's instructions (Qiagen, Hilden, Germany).

Cell Culture and Transfection-- 293HEK-EBNA cells were obtained from Invitrogen (Groningen, Netherlands) and cultured in DMEM/F-12 (Life Technologies, Inc.) containing 5% fetal calf serum (FCS; S0215-Lot 264S, Biochrom, Berlin, Germany), 50 µg of streptomycin, and 50 units of Penicillin/ml (Life Technologies, Inc.), 250 µg/ml G418 (Calbiochem, Bad Soden, Germany). Cells were kept in a humidified atmosphere containing 7.5% CO2. For certain experiments, cells were serum-starved by washing twice in serum-free medium and keeping them in serum-free medium for 20 h. The medium was replaced with serum-free medium 2 h before experiments, and for block of protein biosynthesis cycloheximide was added at a concentration of 25 µM and applied for 2 h. Trypsin was stopped with 1 mg/ml soybean trypsin inhibitor (Sigma) and 1% BSA (Sigma) in DMEM/F-12 under these conditions. For transfection, 106 HEK293-EBNA cells were seeded on 60-mm dishes and grown for 16 h. Cells were washed twice with PBS and once with OptiMEM (Life Technologies, Inc.). Cells were incubated with 600 µl of OptiMEM containing 10 µg of plasmid DNA and 15 µl of Lipofectin (Life Technologies, Inc.) for 6 h and then additionally 3 ml of DMEM/F-12 were added for 16 h. Medium was changed after 48 h, and cells were selected and maintained in culture medium containing 300 µg/ml hygromycin B (Roche Molecular Biochemicals, Mannheim, Germany).

FACS Analysis-- FACS analysis was performed as described previously (37). Briefly, cells were trypsinized, washed, and resuspended in FACS-PBS (5% FCS in PBS containing 0.02% sodium azide) (2 × 106 cells/ml). 2 × 105 cells were incubated with primary antibodies (GoH3, 2 µg/ml; TS2/16, 3C12, 5A6, and 6A11, hybridoma supernatant) for 30 min on ice. Cells were washed twice with FACS-PBS and incubated with FITC-labeled secondary antibodies (1:200) for 30 min on ice, washed twice with FACS-PBS and fixed in 1% p-formaldehyde in PBS. FACS analysis was performed with a Coulter cytometer.

Cell Migration Assay-- Flasks (25 cm2; Falcon) were coated with PBS-diluted PLL (20 µg/ml) or LN-1/E8 fragment (2 µg/ml) for 1 h at 37 °C with a volume of 1.5 ml/25-cm2 flask. Flasks were washed twice with PBS and blocked with 1% heat-denatured (30 min; 80 °C) BSA (Sigma; A7030) in PBS for 30 min at 37 °C and again washed twice with PBS. Cells were trypsinized, washed, and plated at a density of 2000/cm2 in 10 mM HEPES (pH 7.4)-buffered DMEM/F-12 containing antibiotics and 5% FCS. The flasks were allowed to equilibrate in a 7.5% CO2-containing atmosphere at 37 °C for 1 h, the lid was closed air-tight, and flasks were placed in a thermostatic chamber at 37 °C under a Zeiss ICM-405 microscope (Oberkochen, Germany). Migration was monitored by time-lapse video microscopy as described previously (37). Briefly, cells were filmed under low illumination with a CCD camera (JVC) connected to a time lapse video recorder triggered by an external timer. Pictures were taken every 2 min, and cells were recorded for >12 h. For analysis of cell migration, a set of 12 pictures in 1 h steps was imported in the McDraw program and cells were tracked manually by connecting the centers of the cell bodies of individual cells. Tracks of cells were digitalized and converted to pixels, which were converted to micrometers after calibration.

Immunofluorescence Microscopy-- Cells were washed quickly three times with ice-cold PBS and fixed in 3.7% p-formaldehyde in PBS for 15 min at 4 °C, washed three times in PBS and permeabilized with 0.5% Triton X-100 in PBS for 30 min at room-temperature. Samples were again washed three times with PBS and blocked for 30 min at room temperature with 3% BSA in PBS. FITC-phalloidin was diluted 1:1000 in 3% BSA in PBS and applied for 1 h at room temperature. Samples were washed three times for 5 min in PBS, mounted, and examined with a Zeiss Axiophot microscope equipped with a 63× oil immersion objective (numeric aperture = 1.24).

Cell Lysis, Immunoprecipitation, and Western Blot Analysis-- Cell lysis was performed in two ways, depending on the application. Cells were washed once with ice-cold PBS and lysed with 1 ml of buffer/107 cells. Condition A (LN-1/E8 chromatography) consisted of 50 mM N-octylglucopyranoside, 300 mM NaCl, 25 mM Tris/HCl, pH 7,4, 1 mM MnCl2, 1 mM CaCl2, 1 mM N-ethylmaleimide (Merck, Darmstadt, Germany), 1 mM PMSF (phenylmethylsulfonyl fluoride; Merck). Condition B (coimmunoprecipitations) was 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM sodium vanadate, 50 mM sodium fluoride, 1 mM PMSF, 5 mM EDTA. Cells were scraped into ice-cold lysis buffer and allowed to lyse for 30 min on a shaking platform at 4 °C, and the lysate was spun down at 10,000 × g for 15 min at 4 °C. The protein concentration of the supernatant was determined by Cu2+ complexation (Pierce). Samples were adjusted to equal protein concentrations with lysis buffer. For immunoprecipitations samples were adjusted to a volume of 1 ml with lysis buffer and precleared with equilibrated Protein G-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) for 30 min at 4 °C by rotation. After centrifugation appropriate antibodies were added to the supernatant, the mixture was rotated for 1 h, and 20-50 µl of equilibrated Protein-G-Sepharose were added for an additional 1 h. If not stated otherwise, antibodies were used at a concentration of 5 µg/mg of protein. Coimmunoprecipitations were performed the same way, but for 2 h. The immune complexes were retained by short pulse centrifugation, washed three times in lysis buffer (for condition A: lysis buffer containing 25 mM N-octylglucopyranoside), and finally boiled in 2× SDS sample buffer (41). If not otherwise indicated, SDS-PAGE was performed with 10% gels. Proteins were transferred to nitrocellulose (0.2 µm, Schleicher & Schuell, Dassen, Germany) for 1.5 h at 1 mA/cm2 by semidry blotting and a discontinuous buffer system. Blots were stained with Ponceau S and blocked with 3% BSA in TBST (0.1% Tween 20, 25 mM Tris/HCl, pH 7.4, 150 mM NaCl) for 1 h at room temperature. Antibodies were diluted in blocking solution, and incubated with the blot for 1 h at room temperature or for 16 h at 4 °C. Blots were washed four times for 10 min each with TBST, developed with peroxidase-conjugated secondary antibodies and chemiluminescence, and exposed to Kodak XAR-5 films (Eastman Co.). For stripping, blots were washed twice with distilled water, incubated two times for 10 min with 0.1 M glycine, 0.5 M NaCl, 0.1% Tween 20, 2% beta -mercaptoethanol, pH 2.5, neutralized extensively with TBST, and blocked again.

Cell Surface Biotinylation-- Cells were washed once in PBS containing 5 mM EDTA and released from plates with 5 mM EDTA in PBS. After washing three times with ice-cold PBS, pH 8.0, cells were adjusted to 25 × 106/ml and nonmembrane permeable NHS-LC-Sulfobiotin (Pierce) was added to a final concentration of 0.5 mg/ml. Surface biotinylation was carried out for 30 min at room temperature on a shaking platform, and the reaction was stopped by three washes with ice-cold PBS, 10 mM Tris/HCl, pH 8.0. Cells were lysed under condition A.

LN-1/E8-Sepharose Chromatography-- The LN-1/E8 fragment used throughout this study was a generous gift of Dr. Rainer Deutzmann (University of Regensburg, Regensburg, Germany). It was coupled to CNBr activated Sepharose CL 4-B according to the manufacturer's instructions (Amersham Pharmacia Biotech), which resulted in ~1 mg of LN-1/E8 fragment/ml of Sepharose. For purification of recombinant integrin alpha 7beta 1 complexes from transfected 293 cells, LN-1/E8-Sepharose was equilibrated in lysis buffer A by three washes and 200 µl of a 1:3 suspension was added to surface-biotinylated cell extracts from 107 cells. The mixture was rotated for 4 h at 4 °C and washed three times in lysis buffer A containing 25 mM N-octylglucopyranoside by short centrifugation at 200 × g and resuspension in 1 ml of wash buffer. The recombinant protein complexes were eluted in a volume of 50 µl with 5 mM EDTA in 25 mM N-octylglucopyranoside, 300 mM NaCl, 25 mM Tris/HCl pH 7,4, 1 mM N-ethylmaleimide, 1 mM PMSF by vortexing and centrifugation at 200 × g. Eluted proteins were subjected to Western blot analysis and probed with streptavidin-peroxidase complex (1:5000, Amersham Pharmacia Biotech)., LN-1/E8-Sepharose was recycled by repeated washes in 1 M NaCl, 5 mM EDTA, 20 mM Tris/HCl, pH 7.4 and stored at 4 °C in TBS containing 0.1% sodium azide.

Cell Attachment Assay-- Plates (96 wells; Nunc, Denmark) were coated with 100 µl of protein solution/well for 1 h at 37 °C with coating concentrations as indicated under "Results." Plates were then washed twice with PBS and blocked with 1% heat-denatured (30 min; 80 °C) BSA (Sigma; 7030). Cells were trypsinized and washed either in DMEM containing 1% BSA and 1 mg/ml trypsin inhibitor under serum-free conditions or in DMEM containing 5% FCS and kept in suspension for 30 min. 5 × 105 cells were seeded per well and allowed to attach for 1 h at 37 °C. Plates were washed three times with PBS under standardized conditions with an enzyme-linked immunosorbent assay washer (M96V; Merlin, Rotterdam, Netherlands), and the amount of attached cells was determined by measurement of lysosomal hexosaminidase activity (42). Background attachment on BSA was subtracted, and the percentage of attached cells was calculated using serially diluted cells (1:3) as standard. For antibody attachment inhibition assays, cells were incubated with 10 µg/ml purified antibody on ice prior to distribution in wells. The antibody concentration was kept at 10 µg/ml throughout the assay. Attachment assays were performed with internal controls, e.g. nontransfected 293 cells.

Magnetic Cell Sorting-- Sorting was performed over two rounds as described previously (38). alpha 7-transfected cells were trypsinized shortly, washed with culture medium, and adjusted to 3.3 × 107 cells/ml in 3C12 hybridoma supernatant diluted 1:1 with PBS (corresponding to approximately 25 µg/ml specific anti-alpha 7 antibody). The mixture was rocked at 4 °C for 30 min, and cells were washed twice with 4 ml of ice-cold culture medium. Cells were resuspended at a concentration of 108/ml in ice-cold culture medium. Sheep pan-anti-mouse coated magnetic beads (M-450; Dynal, Oslo, Norway) were washed three times in PBS containing 0.1% heat-denatured BSA, added to the cell suspension (1.3 × 108 beads/ml), and cells were rotated with the beads for 30 min at 4 °C. Cells bound to the beads were pulled out magnetically and washed three times with ice-cold medium. Cells were resuspended in warm culture medium, and beads were removed magnetically after trypsin treatment during passaging of the cells.

Analysis of Cell Spreading-- 5 × 104 cells were plated on laminin-1/E8-coated 30-mm dishes, and three pictures of each plate were taken with a Zeiss Axiovert microscope (Kodak TMax 100 film) by a second individual blinded for the experimental condition. Films were developed, and quantitation of cell spreading was performed by calculating the percentage of spread versus total cells by a blinded, second person. Data were analyzed statistically with a two-tailed Student's t test.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heterodimerization and Surface Expression of alpha 7beta 1 Do Not Require the Cytoplasmic Domain of Integrin alpha 7X2-- We reported previously that integrin alpha 7X2B,3 alpha 7X2A, and alpha 7X2C induce cell migration specifically on laminin-1 and its E8 fragment (LN-1/E8) when expressed ectopically in nonmotile, integrin alpha 7-negative 293HEK cells (38). To elucidate the role of the alpha 7B cytoplasmic domain, a truncation mutant lacking the cytoplasmic domain except of the two first membrane-proximal residues (Lys-Leu)3 was constructed and transfected in HEK293-EBNA cells. Transfected 293alpha 7X2B and 293alpha 7X2Delta cyt cells were magnetically sorted with anti-alpha 7 mAb 3C12 for high surface expression levels (Fig. 1) and scanned by FACS analysis with two different anti-alpha 7 mAbs, a nonblocking (3C12) as well as a blocking antibody (6A11). The wild-type and mutant cells were rather homogenous and displayed similar surface expression levels of integrin alpha 7 (Fig. 1A and Table I), indicating that deletion of the cytoplasmic domain of integrin alpha 7X2 does not interfere with cell surface presentation. 293alpha 7X2Delta cyt cells displayed reduced surface levels of integrin alpha 6, thus behaving similarly to the wild-type receptor (37). Immunoblot analysis confirmed deletion of the cytoplasmic domain (Fig. 1B, lanes 3, 6, and 9) and demonstrated that the total alpha 7 expression level was not reduced after deletion of the alpha 7 cytoplasmic domain (compare Fig. 1B, lanes 2 and 3). These data suggest that the truncated protein does not differ from the wild-type protein with respect to stability. However, some degradation products were observed in both 293alpha 7X2B and 293alpha 7X2Delta cyt cells (Fig. 1B, lanes 2, 3, and 8) and a large part of the transfected integrin was not processed (Fig. 1B, lane 8). Both findings are probably due to overexpression of alpha 7.


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Fig. 1.   Characterization of alpha 7 protein expression in 293alpha 7X2B and 293alpha 7Delta cyt cells. A, FACS analysis of mock-transfected (Mock) and immunomagnetically sorted 293alpha 7X2B and 293alpha 7X2Delta cyt cells. Cells were stained with a nonblocking anti-alpha 7 mAb (3C12), a blocking anti-alpha 7 mAb (6A11), with anti-integrin alpha 6 mAb (GoH3) and with secondary antibodies alone (controls). alpha 7-transfected cells display similar alpha 7 surface expression levels and reduced alpha 6 surface expression levels, whereas mock-transfected cells express some alpha 6 but no alpha 7. B, Western blot analysis of alpha 7 integrin expressed by untransfected (Mock), 293alpha 7X2B, and 293alpha 7X2Delta cyt cells. Total cell lysates (10 µg) were separated by 10% SDS-PAGE under either nonreducing (lanes 1-6) or reducing conditions (lanes 7-9) and transferred to nitrocellulose. Blots were probed with an antiserum (242AE) against the extracellular portion of alpha 7 (lanes 1-3) and with an antiserum (U4+) against the cytoplasmic domain of alpha 7 (lanes 4-9). Lanes 1, 4, and 7, untransfected cells. Lanes 2, 5, and 8, 293alpha 7X2B cells; lanes 3, 6, and 9, 293alpha 7X2Delta cyt cells. Molecular mass positions (kDa) are shown on the left. Positions of the unprocessed and unreduced alpha 7 chain (complete) and C-terminal part of the processed chain (C-term) are indicated on the right. C, immunoprecipitation and LN-1/E8-Sepharose chromatography of surface-biotinylated cell lysates. Surface-biotinylated cell lysates (condition B) of mock-transfected 293 cells (lanes 1-3), 293alpha 7X2B cells (lanes 4-7), and 293alpha 7X2Delta cyt cells (lanes 8-11) were subjected to immunoprecipitation with anti-integrin beta 1 mAb TS2/16 (lanes 3, 7, and 11), anti-alpha 7 mAb 3C12 (lanes 6 and 10) or to chromatography with LN-1/E8-Sepharose Cl 4B (lanes 2, 5, 9). Lanes 1, 4, and 8 represent 0.25% of the material used for immunoprecipitations and chromatography of 293, 293alpha 7X2B, and 293alpha 7X2Delta cyt cell lysate. Protein complexes were separated by 12% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with streptavidin-peroxidase. Molecular mass positions (kDa) are shown on the left. Positions of the beta 1 chain, the alpha 7 N-terminal part, and the alpha 7 C-terminal part are indicated on the right. Arrows indicate integrin alpha  chains different from alpha 7.

                              
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Table I
Integrin alpha 7 surface expression
Integrin alpha 7 expression of transfected 293 cells was measured after staining with two monoclonal antibodies (3C12, 5A6) and one polyclonal rabbit antibody 242 (IgG fraction) in a flow cytometer. Surface expression levels did not differ significantly between the wildtype and deletion mutant. For the analysis cell populations were used which were more than 95% positive for alpha 7 integrin.

Overexpression of integrin alpha 7 leads to down-regulation of alpha 6 and other integrin alpha  subunits, but not the beta 1 chain, on the cell surface of 293 cells (38). As integrins are being transported to the cell surface as alpha /beta heterodimers (11), all integrin alpha 7 on the cell surface should be associated with the beta 1 chain. To verify that both transfected alpha 7 chains form heterodimers with the endogenous beta 1 chain, cells were surface-biotinylated, and lysates were either adsorbed with LN-1/E8 fragment Sepharose or immunoprecipitated with beta 1 mAb TS2/16 or anti-alpha 7 mAb 3C12 (Fig. 1C). Transfected wild-type and deleted integrin alpha 7 chains were specifically retained by LN-1/E8-Sepharose as complex with endogenous beta 1 (Fig. 1C, lanes 5 and 9). No significant binding of proteins to LN-1/E8-Sepharose was detected in lysates from vector-transfected (mock-transfected) cells (Fig. 1C, lane 2). In the mock-transfected cells anti-beta 1 precipitated various other integrin chains (Fig. 1C, lane 3, arrowheads), but not alpha 7. In alpha 7-transfected cells, anti-alpha 7 coprecipitated beta 1 and vice versa (Fig. 1C, lanes 6, 7, 10, and 11). This indicated that the integrin alpha 7 cytoplasmic domain is not essential for alpha 7 heterodimerization, processing, surface presentation, or ligand binding.

The Integrin alpha 7 Cytoplasmic Domain Is Not Essential for Cell Attachment-- Comparison of mock-transfected cells and 293alpha 7X2B and 293alpha 7X2Delta cyt cells revealed a dose-dependent cell attachment to LN-1/E8 fragment (Fig. 2A). Both alpha 7 variants enhanced binding of 293 cells to LN-1/E8 fragment as compared with the mock-transfected cells. Attachment of both 293alpha 7X2B (38) and 293alpha 7X2Delta cyt cells to LN-1/E8 fragment, but not to PLL could be fully blocked with integrin alpha 7 blocking mAb 6A11 (Fig. 2B), while attachment to LN-1 was only blocked by 50% owing to a alpha 7-independent cell binding site in the laminin-1 E1 fragment (4, 43). Thus, the truncated alpha 7X2B receptor showed essentially the same behavior as the full-length receptor. Attachment of mock-transfected cells to the laminin-1 E8 fragment is mediated by integrin alpha 6 and switches to alpha 7 after overexpression of alpha 7 due to down-regulation of alpha 6 (see Fig. 1 and Ref. 38). A LN-1/E8 fragment coating concentration of 2 µg/ml was chosen for the following cell migration and biochemical experiments (Figs. 3-9) because wild-type and Delta cyt cells attached similarly at these coating concentrations.


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Fig. 2.   Attachment of mock-transfected, 293alpha 7X2B, and 293alpha 7Delta cyt cells to laminin-1 LN-1/E8 fragment. Experiments were performed with starved and cycloheximide-treated cells. A, dose-response curve. Cells were plated on laminin-1 LN-1/E8 fragment-coated 96-well plates under serum-free conditions. Symbols represent the average of three wells ± S.D. B, attachment of alpha 7-transfected cells to LN-1/E8 fragment is mediated solely by integrin alpha 7. Starved 293alpha 7Delta cyt cells were plated on 96-well plates coated with laminin-1 (10 µg/ml), laminin-1 LN-1/E8 fragment (2 µg/ml), or PLL (20 µg/ml) either without (white bars) or in the presence of a blocking anti-alpha 7 mAb (6A11; 10 µg/ml) (gray bars). Bars represent the average of three wells ± S.D.


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Fig. 3.   Integrin alpha 7X2-mediated cell migration depends on the alpha 7 cytoplasmic domain and on serum. A, migration of integrin alpha 7X2B- and alpha 7X2Delta cyt-transfected 293 cells on laminin-1 LN-1/E8 fragment (2 µg/ml). Bars, total cell tracks per 12 h expressed as average of five independent experiments ± S.D. The number of cells scored was 83 for 293alpha 7X2B cells (gray bars) and 86 for alpha 7X2Delta cyt cells (white bars). alpha 7X2B cells migrated 300 µM/12 h and alpha 7X2Delta cyt cells migrated 171 µM/12 h. The difference is significant (p < 0.01; *). B, the observed difference in cell migration between 293alpha 7X2B and 293alpha 7X2Delta cyt cells is not due to differences in cell attachment. 293alpha 7X2B cells (gray bars) and 293alpha 7X2Delta cyt cells (white bars) were allowed to attach to laminin-1 LN-1/E8 fragment (2 µg/ml) for 1 h either in the presence of serum or in the presence of 1% BSA. No significant difference in cell attachment was observed under either condition.

The Integrin alpha 7 Cytoplasmic Domain Controls Integrin alpha 7-mediated Cell Migration and Polarization-- Although surface presentation of integrin alpha 7 and cell adhesion to LN-1/E8 did occur independently of the alpha 7 cytoplasmic domain, its deletion affected significantly cell motility on LN-1/E8, i.e. the mean migration speed (Fig. 3A). Deletion of the integrin alpha 7 cytoplasmic domain reduced alpha 7-dependent cell motility significantly to about 50% of the wild-type level (p < 0.01). Integrin alpha 7-mediated cell migration required furthermore the presence of serum because serum-starved cells did not migrate under serum-free conditions (Fig. 3A).

The cell attachment assays presented in Fig. 2, showing equal attachment of 293alpha 7X2B and 293alpha 7X2Delta cyt cells to LN-1/E8, had been carried out under serum free-conditions, in contrast to cell migration assays. It seemed possible that the different migration rates of 293alpha 7X2B and 293alpha 7X2Delta cyt cells were due to different effects of serum upon their attachment cells. To rule this out, serum-starved cells were plated in the presence of serum or 1% BSA on LN-1/E8. 293alpha 7X2B and 293alpha 7X2Delta cyt cells attached similarly under either condition (Fig. 3B), indicating that the reduced cell migration was not a consequence of differences in cell attachment in the presence of serum.

To test whether serum alone accounted for the observed migration, cells were plated on PLL in the presence of serum and video images were taken 1 h after plating the cells and after 12 h (Fig. 4, A and B). Cells did neither spread nor move under these conditions (see Fig. 4, arrows), thus confirming serum alone does not induce cell migration


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Fig. 4.   Spreading and polarization of alpha 7X2-transfected 293 cells depends on the cytoplasmic domain of integrin alpha 7X2 and laminin-1 LN-1/E8 fragment in the presence of serum. Video images of 293alpha 7X2B cells (A and B) cells were taken during cell migration experiments. Pictures in C and D were taken under a light microscope. A and B show alpha 7X2B cells plated on PLL for 1 h (A) and 12 h (B). Cells did neither spread nor migrate on PLL but still divided (arrow; images show the same frame). C and D show 293alpha 7X2B (C) and 293alpha 7X2Delta cyt (D) cells kept on laminin-1 LN-1/E8 fragment for 6.5 h. 293alpha 7X2Delta cyt cells spread (D; compare cells plated on PLL, A and B) but polarized less than 293alpha 7X2B cells (see closed arrows in C). Open arrow, nonpolarized 293alpha 7X2B cell. Bar, 100 µm. E, quantification of spread cells plated on LN-1/E8 fragment. Bars represent the average percentage of cell spreading obtained by counting cells from three fields. 293alpha 7X2Delta cyt cells were less spread than 293alpha 7X2B cells. 150-230 cells per field were analyzed.

Analysis of the cell morphology of migrating cells by microscopy revealed that 293alpha 7X2Delta cyt cells displayed a different morphology on LN-1/E8 than cells expressing the wild-type receptor (Fig. 4). To analyze this in detail, we quantitated the percentage of spread cells plated on LN-1/E8 (Fig. 4). First, only about 40% of 293alpha 7X2Delta cyt cells spread after 30 min in contrast to about 80% of 293alpha 7X2B cells. After 16.5 h of adhesion to LN-1/E8, 80% of 293alpha 7X2B cells were still spread, whereas spreading 293alpha 7X2Delta cyt cells reached only a level of 60% (Fig. 4E). Second, despite spreading as compared with cells plated on PLL, 293alpha 7X2Delta cyt cells remained in a more or less round shape and extended filopodia, reflecting a different organization status of the actin cytoskeleton as compared with 293alpha 7X2B cells. These elongated and polarized (see Fig. 4, C and D). To examine changes in the actin cytoskeleton cells were fixed directly after cell migration experiments (i.e. after >12 h) and stained with FITC-phalloidin (Fig. 5). 293alpha 7X2B cells showed extended lamellipodia, ruffled their membranes, and organized stress fibers in contrast to 293alpha 7X2Delta cyt cells, which exhibited less lamellipodia, but showed a dramatic increase in number and size of filopodia (Fig. 5). The results from this experiment implicate that the integrin alpha 7 cytoplasmic domain controls a pathway regulating cytoskeletal architecture.


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Fig. 5.   Deletion of the alpha 7 cytoplasmic domain affects lamellipodia formation. 293alpha 7X2B and 293alpha 7X2Delta cyt cells were plated on LN-1/E8 (2 µg/ml) or PLL (20 µg/ml) in the presence of serum for 12 h, fixed, and stained with FITC-phalloidin. 293alpha 7X2B cells plated on LN-1/E8 develop lamellipodia and stress fibers, ruffle their membranes, and polarize (arrows). 293alpha 7X2Delta cyt cells plated on LN-1/E8 extend filopodia instead of forming lamellipodia (arrows) but can still spread, as compared with PLL. Bar, 10 µm.

The Integrin alpha 7 Cytoplasmic Domain Controls alpha 7-initiated Tyrosine Phosphorylation-- Activation of nonreceptor protein tyrosine kinases like FAK and Src and subsequent protein-protein interactions are rapid responses of cells to attachment to ECM molecules (44) and are believed to regulate cell adhesion as well as cell migration (45). We thus examined tyrosine phosphorylation events specifically induced by integrin alpha 7. Adhesion of 293alpha 7X2B cells to the LN-1/E8 fragment induced tyrosine phosphorylation of 60-80- and 120-140-kDa proteins already after 10 min (data not shown). To identify the tyrosine-phosphorylated proteins, Triton X-100 extracts from serum-starved, suspended 293alpha 7X2B cells and from serum-starved 293alpha 7X2B cells plated on PLL and LN-1/E8 fragment were subjected to immunoprecipitation with antibodies against tensin, p130CAS, FAK, vinculin, paxillin, Erk2, and Shc or anti-phosphotyrosine-agarose (Fig. 6). Immunoprecipitation with anti-phosphotyrosine-agarose revealed three major tyrosine-phosphorylated bands of 60-70, 120, and 130 kDa on LN-1/E8 fragment, which were not seen when cells were kept in suspension or plated on PLL. Three of these proteins were identified as p130CAS, FAK, and paxillin (Fig. 6B, lanes 5, 6, and 8). Tensin and vinculin were not tyrosine-phosphorylated (Fig. 6B, lanes 4 and 7), nor were Erk or Shc (data not shown).


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Fig. 6.   Integrin alpha 7X2B and laminin-1 LN-1/E8 fragment induce tyrosine phosphorylation of p130CAS, FAK, and paxillin. 293alpha 7X2B cells were serum-starved and then kept in suspension for 2 h (S), plated on PLL (PL) or LN-1/E8 fragment for 2 h under serum-free conditions. Cells were lysed in Triton X-100 buffer, and 1 mg of Triton X-100 lysate was immunoprecipitated with anti-phosphotyrosine-agarose (lanes 1-3), anti-tensin mAb (lane 4), anti-p130CAS mAb (lane 5), anti-FAK mAb (lane 6), anti-vinculin mAb (lane 7), and anti-paxillin mAb (lane 8). Precipitates were resolved by 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibody RC20H. Integrin alpha 7 induces tyrosine phosphorylation of FAK, p130CAS and paxillin (black arrowheads). Molecular mass positions are indicated on the left (kDa).

Integrin alpha 7-initiated p130CAS Tyrosine Phosphorylation and p130CAS/Crk Coupling Are Dependent on the Cytoplasmic Domain of alpha 7-- Klemke and co-workers (27) reported an essential role for p130CAS in cell migration. The observation of integrin alpha 7-dependent p130CAS tyrosine phosphorylation prompted us to examine the role of the integrin alpha 7 cytoplasmic domain in this event. Starved 293alpha 7X2B and 293alpha 7X2Delta cyt cells were plated on LN-1/E8 fragment in the absence or in the presence of serum for 30 min and lysed. Lysates were immunoprecipitated with anti-p130CAS, and immunoprecipitates were subjected to anti-phosphotyrosine blotting (Fig. 7).


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Fig. 7.   Deletion of the alpha 7 cytoplasmic domain affects serum-dependent tyrosine phosphorylation of p130CAS and a 60-kDa protein. 293alpha 7X2B and 293alpha 7X2Delta cyt cells were serum-starved for 16 h, trypsinized, and resuspended in 1% BSA-containing medium. Cells were recovered by centrifugation and resuspended in either 1% BSA-containing medium (-) or serum-containing medium (+). Cells were kept in suspension for 30 min and then either plated on LN-1/E8 (LN-1/E8) or PLL for 30 min, or kept in suspension for another 30 min (sus). Triton X-100 extracts were prepared, and 600 µg of total protein were subjected to anti-p130CAS IP. IPs were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine (PY; RC20H) or anti-p130CAS (p130CAS). Molecular mass positions are shown on the left. The black arrowhead marks the position of p130CAS. The open arrow marks a 60-kDa coprecipitating tyrosine-phosphorylated protein. Molecular mass positions are indicated on the left.

Fig. 7 shows that p130CAS was not phosphorylated on tyrosine residues when 293alpha 7X2B cells were kept in suspension or plated on poly-L-lysine. Tyrosine phosphorylation of p130CAS was, however, induced by E8 fragment via integrin alpha 7 and strongly enhanced after addition of serum, paralleling the effect of serum on cell migration. In contrast, 293alpha 7X2Delta cyt cells plated on LN-1/E8 in the presence of serum showed a markedly reduced p130CAS tyrosine phosphorylation with unaltered p130CAS protein levels. Thus, the presence of the alpha 7 cytoplasmic domain is necessary for full p130CAS tyrosine phosphorylation in this system. Furthermore, the cytoplasmic domain of integrin alpha 7 was essential for coprecipitation of a tyrosine-phosphorylated 60-kDa protein with p130CAS, which we could not identify so far. This protein coprecipitated with p130CAS from 293alpha 7X2B cells but not from 293alpha 7X2Delta cyt cells. We also examined the effect of the deletion of the alpha 7 cytoplasmic domain on FAK tyrosine phosphorylation (Fig. 8). According to the data obtained with CAS precipitates, we also found less tyrosine phosphorylation of FAK in 293alpha 7Delta cyt cells plated on LN-1/E8 as compared with 293alpha 7X2B cells.


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Fig. 8.   The integrin alpha 7 cytoplasmic domain is involved in FAK tyrosine phosphorylation. 293alpha 7X2B and 293alpha 7X2Delta cyt cells were serum-starved for 16 h, trypsinized, and resuspended in 1% BSA-containing medium. Cells were recovered by centrifugation and resuspended in serum-containing medium (+). Cells were kept in suspension for 30 min and then either plated on LN-1/E8 (LN-1/E8) for 30 min, or kept in suspension for another 30 min (sus). Triton X-100 extracts were prepared, and 800 µg of total protein were subjected to anti-p125FAK IP. IPs were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine (PY; clone 2C8) or anti-p125FAK (p125FAK). Molecular mass positions are shown on the left.

The reduced tyrosine phosphorylation of p130CAS in the 293alpha 7X2Delta cyt cells suggested that p130CAS/Crk coupling could be affected due to fewer Crk SH2-binding sites offered by p130CAS. For examination of p130CAS/Crk complexes, 293alpha 7X2B and 293alpha 7X2Delta cyt were plated on LN-1/E8 fragment for 1, 2, and 4 h in the presence of serum (conditions used for cell migration) or kept in suspension for 4 h in the presence of serum. Crk was immunoprecipitated from the standardized cell lysates and the precipitates were probed with anti-phosphotyrosine, anti-p130CAS, and anti-Crk antibodies (Fig. 9A). p130CAS coprecipitated with Crk in 293alpha 7X2B cells only after cell adhesion to LN-1/E8 but not when cells were kept in suspension. However, p130CAS was absent in Crk immunoprecipitates obtained from 293alpha 7X2Delta cyt cells although it was present in each lysate used for Crk immunoprecipitation (Fig. 9B). Crk itself was not tyrosine-phosphorylated (data not shown). Thus, our results point to an essential role for the integrin alpha 7 cytoplasmic domain in p130CAS/Crk coupling.


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Fig. 9.   Integrin alpha 7B-induced p130CAS/Crk coupling depends on the alpha 7 cytoplasmic domain. A, 293alpha 7X2B and 293alpha 7X2Delta cyt cells were plated either on dishes coated with LN-1/E8 fragment (2 µg/ml) or kept in suspension (sus) for the indicated times (t; 1, 2, and 4 h). 600 µg of cell lysate were used for anti-Crk IP. Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibody RC20H (PY), anti-p130CAS mAb (p130CAS), or anti-Crk mAb (Crk) as indicated on the right. Molecular mass positions (kDa) are shown on the left. B, 25 µg of total protein were separated by SDS-PAGE, transferred to nitrocellulose, and probed with p130CAS mAb (p130CAS) as indicated on the right. Molecular mass positions (kDa) are shown on the left.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we have investigated the function of the integrin alpha 7X2 cytoplasmic domain by comparing 293EBNA cells expressing an integrin alpha 7 wild-type receptor or an integrin alpha 7 lacking the cytoplasmic domain. The cells were compared in terms of (i) alpha 7 protein expression, (ii) alpha 7 surface presentation, (iii) cell attachment and migration conferred by alpha 7, and (iv) initiation of p130CAS/Crk signaling complexes. Both cell types assembled alpha 7beta 1 heterodimers and attached equally well on LN-1/E8. We showed that the integrin alpha 7B cytoplasmic domain is not required for heterodimer formation with beta 1 and not necessarily linked to alpha 7 protein stability, surface expression, or receptor activation but contributes to cell spreading, migration, and intracellular signaling via p130CAS/Crk complex formation in a serum-dependent manner.

Role of the Integrin alpha 7X2 Cytoplasmic Domain in Receptor Assembly and Activity-- The function of the alpha  cytoplasmic domains of various other beta 1 integrin receptors has been extensively studied by truncation and point mutation analysis. It has been reported that particularly the two phenylalanines in the conserved GFFKR motive regulate heterodimerization and surface transport, e.g. in the case of alpha 6beta 1 (46), and protein stability in the case of alpha 6beta 4 (47, 48). Moreover, deletion of the cytoplasmic domains of alpha IIb and alpha 1 including the GFFKR caused reduced surface expression and heterodimerization but, on the other hand, resulted also in a high integrin affinity state of alpha IIbbeta 3 in K562 and alpha 1beta 1 in LFA-1-deficient Jurkat cells (49, 50). In marked contrast, we show here that deletion of the complete alpha 7X2 cytoplasmic domain (including the conserved GFFKR motive) did not influence alpha 7X2 heterodimerization with the integrin beta 1 chain, correct processing of alpha 7 or cell attachment conferred by alpha 7beta 1. This is, on the other hand, in agreement with other studies showing that the GFFKR sequence does not influence inside-out signaling or surface presentation of alpha 1beta 1 and alpha 5beta 1 integrins (49, 51), as deletion of the GFFKR motive did not alter the activation state of the affected receptors. Thus, integrins differ in the requirement of the GFFKR motive for heterodimerization, surface transport, and inside-out activation. This may be due to different affinities of the alpha  chains for the beta  chain as proposed previously (47).

For ligand binding and signaling of integrins both cytoplasmic domains and extracellular domains are involved. Ziober et al. have shown that the alpha 7X1 splice variant but not the X2 splice variant, required activation with the beta 1 activating antibody TS2/16 to bind to laminin-1 in MCF7 cells (52), although both extracellular splice variants carried the same intracellular splice variant. In contrast, alpha 7X2B-beta 1 complexes are constitutively active and bind to laminin-1 and -2 when expressed in HEK293-EBNA cells and MCF-7 cells, independently of the cytoplasmic domain (Refs. 37, 38, and 52; this report). Thus, our results are in support of a key role of both intra- and extracellular domains of integrin alpha 7 in inside-out signaling and ligand binding.

Effect of the Integrin alpha 7B Cytoplasmic Domain on Cell Spreading and Migration-- Cell motility on the extracellular matrix is largely dependent on integrin surface expression levels, integrin activation state, and matrix concentration (53). Therefore, quantitative biochemical analysis of the truncated receptor and analysis of cell attachment was necessary to allow a direct functional description of the role of the integrin alpha 7 cytoplasmic domain in cell migration and signaling events. The results of these studies show that (a) alpha 7 surface and total protein levels in wild-type and mutant transfected cells were similar, (b) processing and heterodimer formation were independent of the integrin alpha 7 cytoplasmic domain, and (c) 293alpha 7X2B and 293alpha 7X2Delta cyt cells attached similarly via integrin alpha 7 to LN-1/E8 at coating concentrations of 2 µg/ml. Taking these parameters into account, we conclude that deletion of the alpha 7 cytoplasmic domain significantly affected cell migration.

alpha 7-mediated cell migration and cytoskeletal reorganization was specific for the LN-1/E8 fragment. 293alpha 7X2B and 293alpha 7X2Delta cyt cells plated on PLL did not display marked differences and did not migrate, even in the presence of serum. Thus, integrin alpha 7-mediated cell migration of transfected 293 cells is not due to endogenous proteins deposited as ligands like such as fibronectin. However, serum factors are required for integrin alpha 7-mediated continuous cell migration. This is consistent with the notion that sustained cell migration requires the presence of growth factors (54). Thus, integrin alpha 7 alone is not sufficient to provide enough signals for cell migration but cooperates with soluble, so far by us unspecified serum factors.

In support of the results we obtained from our cell migration experiments we observed fewer lamellipodia, cell polarization, and stress fibers in 293alpha 7X2Delta cyt cells plated on LN-1/E8 than in 293alpha 7X2B cells plated on LN-1/E8. In contrast, filopodial extensions were remarkably increased in 293alpha 7X2Delta cyt cells. Deletion of the alpha 7 cytoplasmic domain may lead to a block in transmission of signals from cdc42 to Rac, hence from Rac to Rho, and thus in accumulation of filopodia-inducing signals in 293 cells. This would explain the lack of membrane ruffling and stress fibers. Nobes and co-workers (55) reported that cdc42 is required for cell polarization by placing lamellipodia at the leading edge, but we did not observe polarization in 293alpha 7X2Delta cyt cells on LN-1/E8 yet observing the hallmark of cdc42 activation, filopodia. Cells may not be able to polarize due to lack of lamellipodial formations, despite extending filopodia and attaching.

Influence of the Integrin alpha 7X2 Cytoplasmic Domain on p130CAS/Crk Coupling-- The role of the adaptor protein p130CAS and the p130CAS/Crk complex in alpha vbeta 3 and alpha 5beta 1 integrin-mediated cell migration on fibronectin has previously been demonstrated (27, 28). p130CAS becomes tyrosine-phosphorylated after cell adhesion to fibronectin, and this allows formation of a p130CAS/Crk complex (25). Serum factors like platelet-derived growth factor, lysophosphatidic acid, and bombesin induce p130CAS tyrosine phosphorylation as well, leading to formation of a p130CAS/Crk complex (56). We showed for the first time that not only fibronectin, but also laminin can promote p130CAS tyrosine phosphorylation and p130CAS/Crk coupling, which occurs through integrin alpha 7 in our system. We observed less p130CAS tyrosine phosphorylation and also less p125FAK tyrosine phosphorylation in 293alpha 7X2Delta cyt cells even in the presence of serum, and thus we conclude that the integrin alpha 7 cytoplasmic domain is cooperatively involved in signals mediated by soluble factors. It is well known that integrins collaborate with growth factors and integrin signaling pathways converge with those of soluble factors and their receptors like epidermal growth factor (57), platelet-derived growth factor (58), fibroblast growth factor (59), transforming growth factor beta  (60), and vascular endothelial growth factor (61), especially in the p130CAS of cell migration (62). Our data strongly support these observations because alpha 7 and LN-1/E8 fragment-induced tyrosine phosphorylation of p130CAS in particular is strongly enhanced by serum. It has been reported that p130CAS expression levels correlate directly with cell migration (28). However, there were no different p130CAS or Crk protein levels in 293alpha 7X2B and 293alpha 7X2Delta cyt cells, and thus the difference in cell migration was not due to different p130CAS or Crk protein levels. Our results rather suggest strongly that the reduced cell migration, caused by deletion of the alpha 7 cytoplasmic domain, is due to impaired p130CAS/Crk coupling and impaired p130CAS tyrosine phosphorylation.

p130CAS can be tyrosine-phosphorylated by Src in a FAK-dependent and -independent manner after attachment of fibroblasts to fibronectin; the created phosphotyrosine residues provide binding sites for the SH2 domain of Crk (25). Lack of a p130CAS/Crk complex in 293alpha 7X2Delta cyt compared with wild-type cells could be due to less p130CAS tyrosine phosphorylation, which we did observe. Since p130CAS is a substrate for Src and Fyn (25), less p130CAS tyrosine phosphorylation induced by deletion of the alpha 7 cytoplasmic domain may be due to reduced Src or Fyn kinase activity induced by the mutant versus the wild-type receptor. Accordingly, deletion of the C-terminal 23 amino acids of integrin alpha v reduced Src activation induced by osteopontin as compared with the wild-type alpha vbeta 3 receptor (63). To identify the 60-kDa phosphoprotein coprecipitating with p130CAS, we performed coimmunoprecipitation analyses of p130CAS with Src and Lyn, both of which being expressed at same protein levels in 293alpha 7X2B and 293alpha 7X2Delta cyt cells (data not shown). However, we could not detect complexes of p130CAS with Src or Lyn, nor with FAK (data not shown), suggesting that the 60-kDa protein is neither Src nor Lyn, and that CAS tyrosine phosphorylation induced by integrin alpha 7 in 293 cells may occur in a FAK-independent manner, as is the case in p125FAK-/- cells. There, p130CAS can be phosphorylated on tyrosine by cell adhesion kinase beta  (64).

A mechanism explaining the failure of lamellipodia formation induced by 293alpha 7X2Delta cyt cells on LN-1/E8 may finally be through reduction of p130CAS/Crk induced signaling to Rac; Klemke and coworkers (27) have demonstrated that p130CAS/Crk coupling acts in a Rac-dependent manner on cell migration. It seems possible from our morphological and biochemical data that deletion of the alpha 7 cytoplasmic domain may reduce Rac activation in comparison with the nonmutated alpha 7 receptor as a consequence of reduced p130CAS tyrosine phosphorylation.

    ACKNOWLEDGEMENT

We thank Dr. Ulrike Mayer for providing the U4+ antibody, Helga Moch for providing 3C12 and 6A11 antibodies, and Drs. Guido Posern and Stefan Feller (University of Würzburg) for helpful discussions. We greatly acknowledge Dr. Victor Wixler for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant Sonderforschungsbereich (SFB) 263-B9 and by financial support from Dr. H. Jaeck (through Deutsche Forschungsgemeinschaft Grant SFB 466).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Medical Clinic III, Dept. of Molecular Immunology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.

§ To whom correspondence should be addressed. Tel.: 49-9131-8529104; Fax: 49-9131-8526341; E-mail: kvdmark@molmed.uni-erlangen.de.

Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M011481200

2 H. von der Mark, manuscript in preparation.

3 Wild-type transfected cells are named 293alpha 7X2B, and cells carrying the deletion are named 293alpha 7X2Delta cyt.

    ABBREVIATIONS

The abbreviations used are: LN, laminin; PLL, poly-L-lysine; IP, immunoprecipitation; mAb, monoclonal antibody; FAK, focal adhesion kinase; SH, Src homology; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TBST, Tris-buffered saline plus Tween 20.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Cossu, G., Zani, B., Coletta, M., Bouche, M., Pacifici, M., and Molinaro, M. (1980) Cell Differ. 9, 357-368[Medline] [Order article via Infotrieve]
2. Bischoff, R. (1986) Dev. Biol. 115, 129-139[Medline] [Order article via Infotrieve]
3. Öcalan, M., Goodman, S. L., Kuhl, U., Hauschka, S. D., and von der Mark, K. (1988) Dev. Biol. 125, 158-167[Medline] [Order article via Infotrieve]
4. Goodman, S. L., Risse, G., and von der Mark, K. (1989) J. Cell Biol. 109, 799-809[Abstract]
5. Yao, C. C., Ziober, B. L., Squillace, R. M., and Kramer, R. H. (1996) J. Biol. Chem. 271, 25598-25603[Abstract/Free Full Text]
6. Schuler, F., and Sorokin, L. M. (1995) J. Cell Sci. 108, 3795-3805[Abstract/Free Full Text]
7. Lauffenburger, D. A., and Horwitz, A. F. (1996) Cell 84, 359-369[Medline] [Order article via Infotrieve]
8. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
9. Clark, E. A., King, W. G., Brugge, J. S., Symons, M., and Hynes, R. O. (1998) J. Cell Biol. 142, 573-586[Abstract/Free Full Text]
10. Price, L. S., Leng, J., Schwartz, M. A., and Bokoch, G. M. (1998) Mol. Biol. Cell 9, 1863-1871[Abstract/Free Full Text]
11. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
12. Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo, P. D., Akiyama, S. K., and Yamada, K. M. (1995) J. Cell Biol. 131, 791-805[Abstract]
13. Burridge, K., Turner, C. E., and Romer, L. H. (1992) J. Cell Biol. 119, 893-903[Abstract]
14. Vuori, K., and Ruoslahti, E. (1995) J. Biol. Chem. 270, 22259-22262[Abstract/Free Full Text]
15. Petch, L. A., Bockholt, S. M., Bouton, A., Parsons, J. T., and Burridge, K. (1995) J. Cell Sci. 108, 1371-1379[Abstract/Free Full Text]
16. Sakai, R., Iwamatsu, A., Hirano, N., Ogawa, S., Tanaka, T., Mano, H., Yazaki, Y., and Hirai, H. (1994) EMBO J. 13, 3748-3756[Abstract]
17. Gilmore, A. P., and Romer, L. H. (1996) Mol. Biol. Cell 7, 1209-1224[Abstract]
18. Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., and Yamamoto, T. (1995) Nature 377, 539-544[CrossRef][Medline] [Order article via Infotrieve]
19. Sieg, D. J., Ilic, D., Jones, K. C., Damsky, C. H., Hunter, T., and Schlaepfer, D. D. (1998) EMBO J. 17, 5933-5947[Abstract/Free Full Text]
20. Reynolds, A. B., Kanner, S. B., Wang, H. C., and Parsons, J. T. (1989) Mol. Cell. Biol. 9, 3951-3958[Medline] [Order article via Infotrieve]
21. Kanner, S. B., Reynolds, A. B., Wang, H. C., Vines, R. R., and Parsons, J. T. (1991) EMBO J. 10, 1689-1698[Abstract]
22. Matsuda, M., Mayer, B. J., Fukui, Y., and Hanafusa, H. (1990) Science 248, 1537-1539[Medline] [Order article via Infotrieve]
23. Cary, L. A., and Guan, J. L. (1999) Frontiers Biosci. 4, D102-D113
24. Feller, S. M., Posern, G., Voss, J., Kardinal, C., Sakkab, D., Zheng, J., and Knudsen, B. S. (1998) J. Cell. Physiol. 177, 535-552[CrossRef][Medline] [Order article via Infotrieve]
25. Vuori, K., Hirai, H., Aizawa, S., and Ruoslahti, E. (1996) Mol. Cell. Biol. 16, 2606-2613[Abstract]
26. Matsuda, M., Tanaka, S., Nagata, S., Kojima, A., Kurata, T., and Shibuya, M. (1992) Mol. Cell. Biol. 12, 3482-3489[Abstract]
27. Klemke, R. L., Leng, J., Molander, R., Brooks, P. C., Vuori, K., and Cheresh, D. A. (1998) J. Cell Biol. 140, 961-972[Abstract/Free Full Text]
28. Cary, L. A., Han, D. C., Polte, T. R., Hanks, S. K., and Guan, J. L. (1998) J. Cell Biol. 140, 211-221[Abstract/Free Full Text]
29. Crawley, S., Farrell, E. M., Wang, W., Gu, M., Huang, H. Y., Huynh, V., Hodges, B. L., Cooper, D. N., and Kaufman, S. J. (1997) Exp. Cell Res. 235, 274-286[CrossRef][Medline] [Order article via Infotrieve]
30. Kramer, R. H., Vu, M. P., Cheng, Y. F., Ramos, D. M., Timpl, R., and Waleh, N. (1991) Cell Regul. 2, 805-817[Medline] [Order article via Infotrieve]
31. von der Mark, H., Durr, J., Sonnenberg, A., von der Mark, K., Deutzmann, R., and Goodman, S. L. (1991) J. Biol. Chem. 266, 23593-23601[Abstract/Free Full Text]
32. Velling, T., Collo, G., Sorokin, L., Durbeej, M., Zhang, H., and Gullberg, D. (1996) Dev. Dyn. 207, 355-371[CrossRef][Medline] [Order article via Infotrieve]
33. Pinkstaff, J. K., Detterich, J., Lynch, G., and Gall, C. (1999) J. Neurosci. 19, 1541-1556[Abstract/Free Full Text]
34. Ziober, B. L., Vu, M. P., Waleh, N., Crawford, J., Lin, C. S., and Kramer, R. H. (1993) J. Biol. Chem. 268, 26773-26783[Abstract/Free Full Text]
35. Collo, G., Starr, L., and Quaranta, V. (1993) J. Biol. Chem. 268, 19019-19024[Abstract/Free Full Text]
36. Song, W. K., Wang, W., Sato, H., Bielser, D. A., and Kaufman, S. J. (1993) J. Cell Sci. 106, 1139-1152[Abstract/Free Full Text]
37. Echtermeyer, F., Schöber, S., Pöschl, E., von der Mark, H., and von der Mark, K. (1996) J. Biol. Chem. 271, 2071-2075[Abstract/Free Full Text]
38. Schöber, S., Mielenz, D., Echtermeyer, F., Hapke, S., Pöschl, E., von der Mark, H., Moch, H., and von der Mark, K. (2000) Exp. Cell Res. 255, 303-313[CrossRef][Medline] [Order article via Infotrieve]
39. Cohn, R. D., Mayer, U., Saher, G., Herrmann, R., van der Flier, A., Sonnenberg, A., Sorokin, L., and Voit, T. (1999) J. Neurol. Sci. 163, 140-152[CrossRef][Medline] [Order article via Infotrieve]
40. Belkin, A. M., Ornatsky, O. I., Glukhova, M. A., and Koteliansky, V. E. (1988) J. Cell Biol. 107, 545-553[Abstract]
41. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
42. Landegren, U. (1984) J. Immunol. Methods 67, 379-388[CrossRef][Medline] [Order article via Infotrieve]
43. Aumailley, M., Gerl, M., Sonnenberg, A., Deutzmann, R., and Timpl, R. (1990) FEBS Lett. 262, 82-86[CrossRef][Medline] [Order article via Infotrieve]
44. Yamada, K. M., and Miyamoto, S. (1995) Curr. Opin. Cell Biol. 7, 681-689[CrossRef][Medline] [Order article via Infotrieve]
45. Schoenwaelder, S. M., and Burridge, K. (1999) Curr. Opin. Cell Biol. 11, 274-286[CrossRef][Medline] [Order article via Infotrieve]
46. De Melker, A. A., Kramer, D., Kuikman, I., and Sonnenberg, A. (1997) Biochem. J. 328, 529-537[Medline] [Order article via Infotrieve]
47. De Melker, A. A., and Sonnenberg, A. (1996) Eur. J. Biochem. 241, 254-264[Abstract]
48. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H. (1994) J. Cell Biol. 124, 1047-1059[Abstract]
49. Briesewitz, R., Epstein, M. R., and Marcantonio, E. E. (1993) J. Biol. Chem. 268, 2989-2996[Abstract/Free Full Text]
50. Lu, C. F., and Springer, T. A. (1997) J. Immunol. 159, 268-278[Abstract]
51. Bauer, J. S., Varner, J., Schreiner, C., Kornberg, L., Nicholas, R., and Juliano, R. L. (1993) J. Cell Biol. 122, 209-221[Abstract]
52. Ziober, B. L., Chen, Y., and Kramer, R. H. (1997) Mol. Biol. Cell 8, 1723-1734[Abstract]
53. Palecek, S. P., Loftus, J. C., Ginsberg, M. H., Lauffenburger, D. A., and Horwitz, A. F. (1997) Nature 385, 537-540[CrossRef][Medline] [Order article via Infotrieve]
54. Ware, M. F., Wells, A., and Lauffenburger, D. A. (1998) J. Cell Sci. 111, 2423-2432[Abstract/Free Full Text]
55. Nobes, C. D., and Hall, A. (1999) J. Cell Biol. 144, 1235-1244[Abstract/Free Full Text]
56. Casamassima, A., and Rozengurt, E. (1997) J. Biol. Chem. 272, 9363-9370[Abstract/Free Full Text]
57. Moro, L., Venturino, M., Bozzo, C., Silengo, L., Altruda, F., Beguinot, L., Tarone, G., and Defilippi, P. (1998) EMBO J. 17, 6622-6632[Abstract/Free Full Text]
58. Miyamoto, S., Teramoto, H., Gutkind, J. S., and Yamada, K. M. (1996) J. Cell Biol. 135, 1633-1642[Abstract]
59. Plopper, G. E., McNamee, H. P., Dike, L. E., Bojanowski, K., and Ingber, D. E. (1995) Mol. Biol. Cell 6, 1349-1365[Abstract]
60. Sastry, S. K., Lakonishok, M., Thomas, D. A., Muschler, J., and Horwitz, A. F. (1996) J. Cell Biol. 133, 169-184[Abstract]
61. Soldi, R., Mitola, S., Strasly, M., Defilippi, P., Tarone, G., and Bussolino, F. (1999) EMBO J. 18, 882-892[Abstract/Free Full Text]
62. Klemke, R. L., Cai, S., Giannini, A. L., Gallagher, P. J., de Lanerolle, P., and Cheresh, D. A. (1997) J. Cell Biol. 137, 481-492[Abstract/Free Full Text]
63. Chellaiah, M., Fitzgerald, C., Filardo, E. J., Cheresh, D. A., and Hruska, K. A. (1996) Endocrinology 137, 2432-2440[Abstract]
64. Ueki, K., Mimura, T., Nakamoto, T., Sasaki, T., Aizawa, S., Hirai, H., Yano, S., Naruse, T., and Nojima, T. (1998) FEBS Lett. 423, 192-201[CrossRef]


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