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
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ABSTRACT |
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The integrin
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 In this study we investigated the role of the cytoplasmic domain of the
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 Deletion of the Integrin 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%
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 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). 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.
Heterodimerization and Surface Expression of
Overexpression of integrin The Integrin The Integrin
The cell attachment assays presented in Fig. 2, showing equal
attachment of 293
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
Analysis of the cell morphology of migrating cells by microscopy
revealed that 293 The Integrin Integrin
Fig. 7 shows that p130CAS was not phosphorylated on
tyrosine residues when 293
The reduced tyrosine phosphorylation of p130CAS in
the 293 In the present study we have investigated the function of the
integrin Role of the Integrin
For ligand binding and signaling of integrins both cytoplasmic domains
and extracellular domains are involved. Ziober et al. have
shown that the Effect of the Integrin
In support of the results we obtained from our cell migration
experiments we observed fewer lamellipodia, cell polarization, and
stress fibers in 293 Influence of the Integrin
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 293
A mechanism explaining the failure of lamellipodia formation induced by
2937
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
7B as well as the
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
7 in the
laminin-induced signal transduction of
7
1
integrin regulating cell adhesion and migration. Deletion of the
cytoplasmic domain did not affect assembly of the mutated
7
cyt/
1 heterodimer on the cell surface
or adhesion of
7
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
7B-transfected cells. In
7B
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
7B integrin after cell adhesion to
laminin-1/E8 and imply
7B-controlled lamellipodia formation and cell migration through the p130CAS/Crk
protein complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
7 antibodies inhibit the migration of
myoblasts on laminin-1 and laminin-2, suggesting that
7
is responsible for myoblast migration on laminin (5, 29). Integrin
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
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
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
7 into
7-deficient cells induces cell migration specifically on
laminin-1 and -2 (5, 37, 38).
7 subunit in laminin-induced signaling. We deleted the
cytoplasmic domain of
7 and transfected 293 cells with a construct encoding the extracellular splice variant X2
(
7X2
cyt) to elucidate the role of the
7 cytoplasmic domain in terms of heterodimer formation,
surface expression, integrin
7-mediated cell attachment,
migration, and p130CAS/Crk coupling. Deletion of the
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
7 cytoplasmic
domain in signal transduction.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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
7 extracellular domain; Ref. 37)
was diluted 1:200. The monoclonal anti-
7 mAbs 3C12 and
6A11 have been described previously (38), and the monoclonal anti-
7 mAb 5A6 will be described
elsewhere.2
Anti-
1 mAb TS2/16 and anti-vinculin mAb 7F9 (40) were
generous gifts of Dr. Alexey Belkin, and anti-integrin
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).
7 Cytoplasmic
Domain--
The integrin
7X2A expression vector
pCEP4
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).
-mercaptoethanol, pH 2.5, neutralized extensively with TBST, and
blocked again.
7
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.
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-
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
7
1 Do Not Require the Cytoplasmic Domain
of Integrin
7X2--
We reported previously that
integrin
7X2B,3
7X2A, and
7X2C induce cell migration
specifically on laminin-1 and its E8 fragment (LN-1/E8) when expressed
ectopically in nonmotile, integrin
7-negative 293HEK
cells (38). To elucidate the role of the
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 293
7X2B and 293
7X2
cyt cells were
magnetically sorted with anti-
7 mAb 3C12 for high
surface expression levels (Fig. 1) and
scanned by FACS analysis with two different anti-
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
7 (Fig.
1A and Table I), indicating that deletion of the cytoplasmic domain of integrin
7X2
does not interfere with cell surface presentation.
293
7X2
cyt cells displayed reduced surface levels of
integrin
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
7 expression level was not reduced after deletion of the
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 293
7X2B and 293
7X2
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
7.
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Fig. 1.
Characterization of
7 protein expression in
293
7X2B and
293
7
cyt
cells. A, FACS analysis of mock-transfected
(Mock) and immunomagnetically sorted 293
7X2B
and 293
7X2
cyt cells. Cells were stained with a
nonblocking anti-
7 mAb (3C12), a blocking
anti-
7 mAb (6A11), with anti-integrin
6 mAb (GoH3) and with secondary antibodies
alone (controls).
7-transfected cells display
similar
7 surface expression levels and reduced
6 surface expression levels, whereas mock-transfected
cells express some
6 but no
7.
B, Western blot analysis of
7 integrin
expressed by untransfected (Mock), 293
7X2B,
and 293
7X2
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
7 (lanes
1-3) and with an antiserum (U4+) against the
cytoplasmic domain of
7 (lanes
4-9). Lanes 1, 4, and
7, untransfected cells. Lanes 2,
5, and 8, 293
7X2B cells;
lanes 3, 6, and 9,
293
7X2
cyt cells. Molecular mass positions (kDa) are
shown on the left. Positions of the unprocessed and
unreduced
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),
293
7X2B cells (lanes 4-7), and
293
7X2
cyt cells (lanes 8-11)
were subjected to immunoprecipitation with anti-integrin
1 mAb TS2/16 (lanes 3,
7, and 11), anti-
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, 293
7X2B, and 293
7X2
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
1 chain, the
7 N-terminal part, and the
7 C-terminal
part are indicated on the right. Arrows indicate
integrin
chains different from
7.
Integrin 7 surface expression
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
7
integrin.
7 leads to down-regulation of
6 and other integrin
subunits, but not the
1 chain, on the cell surface of 293 cells (38). As
integrins are being transported to the cell surface as
/
heterodimers (11), all integrin
7 on the cell surface
should be associated with the
1 chain. To verify that
both transfected
7 chains form heterodimers with the
endogenous
1 chain, cells were surface-biotinylated, and lysates were either adsorbed with LN-1/E8 fragment Sepharose or immunoprecipitated with
1 mAb TS2/16 or
anti-
7 mAb 3C12 (Fig. 1C). Transfected wild-type and
deleted integrin
7 chains were specifically retained by
LN-1/E8-Sepharose as complex with endogenous
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-
1 precipitated various other integrin chains
(Fig. 1C, lane 3,
arrowheads), but not
7. In
7-transfected cells, anti-
7
coprecipitated
1 and vice versa (Fig.
1C, lanes 6, 7, 10, and 11). This indicated that the integrin
7 cytoplasmic domain is not essential for
7 heterodimerization, processing, surface presentation,
or ligand binding.
7 Cytoplasmic Domain Is Not Essential
for Cell Attachment--
Comparison of mock-transfected cells and
293
7X2B and 293
7X2
cyt cells revealed a
dose-dependent cell attachment to LN-1/E8 fragment (Fig.
2A). Both
7
variants enhanced binding of 293 cells to LN-1/E8 fragment as compared
with the mock-transfected cells. Attachment of both
293
7X2B (38) and 293
7X2
cyt cells to
LN-1/E8 fragment, but not to PLL could be fully blocked with integrin
7 blocking mAb 6A11 (Fig. 2B), while attachment to LN-1
was only blocked by 50% owing to a
7-independent cell binding site
in the laminin-1 E1 fragment (4, 43). Thus, the truncated
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
6 and switches to
7 after overexpression of
7 due to
down-regulation of
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
cyt cells
attached similarly at these coating concentrations.
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Fig. 2.
Attachment of mock-transfected,
293 7X2B, and
293
7
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
7-transfected cells to
LN-1/E8 fragment is mediated solely by integrin
7.
Starved 293
7
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-
7
mAb (6A11; 10 µg/ml) (gray bars).
Bars represent the average of three wells ± S.D.
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Fig. 3.
Integrin
7X2-mediated cell migration depends on
the
7 cytoplasmic domain and on
serum. A, migration of integrin
7X2B-
and
7X2
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 293
7X2B cells
(gray bars) and 86 for
7X2
cyt
cells (white bars).
7X2B cells
migrated 300 µM/12 h and
7X2
cyt cells
migrated 171 µM/12 h. The difference is significant
(p < 0.01; *). B, the observed difference
in cell migration between 293
7X2B and
293
7X2
cyt cells is not due to differences in cell
attachment. 293
7X2B cells (gray
bars) and 293
7X2
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.
7 Cytoplasmic Domain Controls Integrin
7-mediated Cell Migration and
Polarization--
Although surface presentation of integrin
7 and cell adhesion to LN-1/E8 did occur independently
of the
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
7 cytoplasmic domain reduced
7-dependent cell motility significantly to
about 50% of the wild-type level (p < 0.01). Integrin
7-mediated cell migration required furthermore the
presence of serum because serum-starved cells did not migrate under
serum-free conditions (Fig. 3A).
7X2B and 293
7X2
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 293
7X2B and
293
7X2
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.
293
7X2B and 293
7X2
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.
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Fig. 4.
Spreading and polarization of
7X2-transfected 293 cells depends on
the cytoplasmic domain of integrin
7X2 and laminin-1 LN-1/E8 fragment in
the presence of serum. Video images of 293
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
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 293
7X2B (C) and
293
7X2
cyt (D) cells kept on laminin-1
LN-1/E8 fragment for 6.5 h. 293
7X2
cyt cells
spread (D; compare cells plated on PLL, A and
B) but polarized less than 293
7X2B cells (see
closed arrows in C). Open
arrow, nonpolarized 293
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. 293
7X2
cyt cells were less spread than
293
7X2B cells. 150-230 cells per field were
analyzed.
7X2
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
293
7X2
cyt cells spread after 30 min in contrast to
about 80% of 293
7X2B cells. After 16.5 h of
adhesion to LN-1/E8, 80% of 293
7X2B cells were still
spread, whereas spreading 293
7X2
cyt cells reached
only a level of 60% (Fig. 4E). Second, despite spreading as
compared with cells plated on PLL, 293
7X2
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 293
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).
293
7X2B cells showed extended lamellipodia, ruffled
their membranes, and organized stress fibers in contrast to
293
7X2
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
7 cytoplasmic domain controls a pathway regulating cytoskeletal architecture.
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Fig. 5.
Deletion of the
7 cytoplasmic domain affects
lamellipodia formation. 293
7X2B and
293
7X2
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. 293
7X2B cells plated on
LN-1/E8 develop lamellipodia and stress fibers, ruffle their membranes,
and polarize (arrows). 293
7X2
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.
7 Cytoplasmic Domain Controls
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
7. Adhesion of
293
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
293
7X2B cells and from serum-starved
293
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
7X2B and laminin-1 LN-1/E8 fragment
induce tyrosine phosphorylation of p130CAS, FAK, and
paxillin. 293
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
7 induces
tyrosine phosphorylation of FAK, p130CAS and paxillin
(black arrowheads). Molecular mass positions are
indicated on the left (kDa).
7-initiated p130CAS Tyrosine
Phosphorylation and p130CAS/Crk Coupling Are Dependent on
the Cytoplasmic Domain of
7--
Klemke and co-workers
(27) reported an essential role for p130CAS in cell
migration. The observation of integrin
7-dependent p130CAS tyrosine
phosphorylation prompted us to examine the role of the integrin
7 cytoplasmic domain in this event. Starved
293
7X2B and 293
7X2
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
7 cytoplasmic domain affects
serum-dependent tyrosine phosphorylation of
p130CAS and a 60-kDa protein. 293
7X2B
and 293
7X2
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.
7X2B cells were kept in
suspension or plated on poly-L-lysine. Tyrosine
phosphorylation of p130CAS was, however, induced by E8
fragment via integrin
7 and strongly enhanced after
addition of serum, paralleling the effect of serum on cell migration.
In contrast, 293
7X2
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
7 cytoplasmic domain is necessary for full p130CAS tyrosine phosphorylation in
this system. Furthermore, the cytoplasmic domain of integrin
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 293
7X2B cells but not from
293
7X2
cyt cells. We also examined the effect of the
deletion of the
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 293
7
cyt cells plated on
LN-1/E8 as compared with 293
7X2B cells.
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Fig. 8.
The integrin
7 cytoplasmic domain is involved in FAK
tyrosine phosphorylation. 293
7X2B and
293
7X2
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.
7X2
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, 293
7X2B and
293
7X2
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 293
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 293
7X2
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
7
cytoplasmic domain in p130CAS/Crk coupling.
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Fig. 9.
Integrin
7B-induced p130CAS/Crk
coupling depends on the
7
cytoplasmic domain. A, 293
7X2B and
293
7X2
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
7X2 cytoplasmic domain by comparing 293EBNA
cells expressing an integrin
7 wild-type receptor or an
integrin
7 lacking the cytoplasmic domain. The cells
were compared in terms of (i)
7 protein expression, (ii)
7 surface presentation, (iii) cell attachment and
migration conferred by
7, and (iv) initiation of
p130CAS/Crk signaling complexes. Both cell types assembled
7
1 heterodimers and attached equally well
on LN-1/E8. We showed that the integrin
7B cytoplasmic
domain is not required for heterodimer formation with
1
and not necessarily linked to
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.
7X2 Cytoplasmic Domain in
Receptor Assembly and Activity--
The function of the
cytoplasmic domains of various other
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
6
1 (46), and protein stability in the
case of
6
4 (47, 48). Moreover, deletion
of the cytoplasmic domains of
IIb and
1
including the GFFKR caused reduced surface expression and
heterodimerization but, on the other hand, resulted also in a high
integrin affinity state of
IIb
3 in K562
and
1
1 in LFA-1-deficient Jurkat cells (49, 50). In marked contrast, we show here that deletion of the
complete
7X2 cytoplasmic domain (including the conserved GFFKR motive) did not influence
7X2 heterodimerization
with the integrin
1 chain, correct processing of
7 or cell attachment conferred by
7
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
1
1 and
5
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
chains for the
chain as proposed previously
(47).
7X1 splice variant but not the X2 splice variant, required activation with the
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,
7X2B-
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
7 in inside-out signaling and ligand binding.
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
7 cytoplasmic
domain in cell migration and signaling events. The results of these
studies show that (a)
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
7 cytoplasmic domain, and (c)
293
7X2B and 293
7X2
cyt cells attached
similarly via integrin
7 to LN-1/E8 at coating
concentrations of 2 µg/ml. Taking these parameters into account, we
conclude that deletion of the
7 cytoplasmic domain
significantly affected cell migration.
7-mediated cell migration and cytoskeletal
reorganization was specific for the LN-1/E8 fragment.
293
7X2B and 293
7X2
cyt cells plated on
PLL did not display marked differences and did not migrate, even in the
presence of serum. Thus, integrin
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
7-mediated continuous cell
migration. This is consistent with the notion that sustained cell
migration requires the presence of growth factors (54). Thus, integrin
7 alone is not sufficient to provide enough signals for
cell migration but cooperates with soluble, so far by us unspecified serum factors.
7X2
cyt cells plated on LN-1/E8
than in 293
7X2B cells plated on LN-1/E8. In contrast,
filopodial extensions were remarkably increased in
293
7X2
cyt cells. Deletion of the
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
293
7X2
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.
7X2 Cytoplasmic Domain on
p130CAS/Crk Coupling--
The role of the adaptor protein
p130CAS and the p130CAS/Crk complex in
v
3 and
5
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
7 in our system. We observed less p130CAS
tyrosine phosphorylation and also less p125FAK tyrosine
phosphorylation in 293
7X2
cyt cells even in the
presence of serum, and thus we conclude that the integrin
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
(60), and
vascular endothelial growth factor (61), especially in the
p130CAS of cell migration (62). Our data strongly support
these observations because
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 293
7X2B and
293
7X2
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
7 cytoplasmic domain, is due to impaired p130CAS/Crk coupling and
impaired p130CAS tyrosine phosphorylation.
7X2
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
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
v reduced
Src activation induced by osteopontin as compared with the wild-type
v
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
293
7X2B and 293
7X2
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
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
(64).
7X2
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
7 cytoplasmic domain may reduce Rac
activation in comparison with the nonmutated
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.
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
2937X2B, and cells carrying the deletion are named
293
7X2
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.
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REFERENCES |
---|
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