From the Laboratoire Franco-Luxembourgeois de Recherche Biomédicale (CNRS and CRP-Santé), Centre Universitaire, 162A, avenue de la Faïencerie, L-1511 Luxembourg, Grand Duchy of Luxembourg
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ABSTRACT |
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We have investigated the structural requirements
of the 3 integrin subunit cytoplasmic domain
necessary for tyrosine phosphorylation of focal adhesion kinase (FAK)
and paxillin during
v
3-mediated cell
spreading. Using CHO cells transfected with various
3
mutants, we demonstrate a close correlation between
v
3-mediated cell spreading and tyrosine
phosphorylation of FAK and paxillin, and highlight a distinct
involvement of the NPLY747 and NITY759 motifs
in these signaling processes. Deletion of the NITY759
motif alone was sufficient to completely prevent
v
3-dependent focal contact
formation, cell spreading, and FAK/paxillin phosphorylation. The single
Y759A substitution induced a strong inhibitory phenotype, while the
more conservative, but still phosphorylation-defective, Y759F mutation
restored wild type receptor function. Alanine substitution of the
highly conserved Tyr747 completely abolished
v
3-dependent formation of
focal adhesion plaques, cell spreading, and FAK/paxillin
phosphorylation, whereas a Y747F substitution only partially restored
these events. As none of these mutations affected receptor-ligand
interaction, our results suggest that the structural integrity of the
NITY759 motif, rather than the phosphorylation status of
Tyr759 is important for
3-mediated
cytoskeleton reorganization and tyrosine phosphorylation of FAK and
paxillin, while the presence of Tyr at residue 747 within the
NPLY747 motif is required for optimal
3
post-ligand binding events.
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INTRODUCTION |
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Anchorage of cells to the extracellular matrix is mediated in part by integrins, a large family of heterodimeric cell surface receptors, that regulate numerous aspects of cell behavior, such as cell motility, proliferation, differentiation, and apoptosis (1). Cell engagement with extracellular matrix ligands induces integrin translocation to subcellular structures known as focal adhesion plaques that form at regions of close contact between the cell and its underlying substratum (2). Integrin clustering at focal contact sites in turn triggers major intracellular events, including cytoskeleton reorganization, intracellular ion transport, phosphoinositide turnover, kinase activation, and tyrosine phosphorylation of intracellular proteins (3). A large number of tyrosine-phosphorylated proteins have been identified within focal adhesion plaques. These include cytoskeletal proteins, kinases and adaptor proteins, growth factor receptors, and growth factor receptor-related signaling molecules, thus emphasizing the potential role of integrins as recruiting centers for molecules involved in various signaling pathways.
Although the link of integrins with focal adhesions is well
established, the precise mechanism by which integrins associate with
cytoskeletal proteins, regulate focal adhesion plaque assembly, and
participate in the activation of intracellular signaling cascades is
still unclear. There is convincing evidence that integrin subunits
are likely to play a major role in these processes: (i) truncation of
the
subunit cytoplasmic domain impairs integrin recruitment to
focal contacts (4-6), and (ii) information contained in
subunit
cytoplasmic tails coupled to the transmembrane and extracellular
domains of the interleukin-2 receptor is sufficient to target these
chimeric receptors to focal contacts (7) and to activate the focal
adhesion kinase (FAK)1
signaling pathway (8). Based on mutational analysis of the cytoplasmic
domain of the
1 integrin, three motifs have been identified that are important for the recruitment of integrins to
adhesion plaques; these motifs correspond to the highly conserved acidic membrane-proximal domain and to two C-terminal NPXY
motifs (6, 9), which constitute typical recognition sites for tyrosine kinases and adaptor proteins (10). Subsequent complementary studies
(based on a combination of deletion analysis, single amino acid
substitution, and the use of cytoplasmic domain synthetic peptides)
have provided evidence that these highly conserved cytoplasmic motifs
in the various integrin
subunits have similar functional properties
(11-16) and display overlapping binding sites for the structural
cytoskeletal proteins
-actinin and talin, the adaptor protein
paxillin, as well as regulatory proteins including FAK, integrin-linked
kinase-1 (ILK-1) (17),
3-endonexin (18), Shc, Grb2 (19),
and integrin cytoplasmic domain-associated protein-1 (ICAP-1) (20).
The importance of tyrosine phosphorylation of focal adhesion proteins
during focal contact formation is well established as tyrosine kinase
inhibitors prevent the organization of focal adhesion plaques and
stress fibers (21), and treatment of cells with cytochalasin B or D,
which block actin polymerization, inhibits tyrosine phosphorylation of
FAK and paxillin (22). In contrast, the precise mechanisms by which
integrin subunits trigger tyrosine phosphorylation of focal
adhesion proteins during integrin-dependent cell attachment
and spreading are less well understood. In an attempt to identify amino
acids of the
3 cytoplasmic domain involved in the
phosphotyrosine signaling cascade induced by
3
integrins, Tahiliani et al. (23) have expressed various
mutant
3 cytoplasmic domains as separate tails connected
to an extracellular reporter protein. Using this approach, they
deliberately excluded the role of upstream events, such as
integrin-dependent ligand binding, cell adhesion, and cell
spreading, in triggering the FAK signaling cascade (23). In the present
study, we have used an alternative approach to investigate the
structural requirements of the
3 subunit cytoplasmic
domain necessary to stimulate intracellular tyrosine phosphorylation
during cell spreading. By expressing various human
3
integrin cytoplasmic domain mutants, which either promote or inhibit
v
3-dependent CHO cell
spreading, we demonstrate a close correlation between a structurally
conserved
3 integrin cytoplasmic tail, cell spreading
and FAK/paxillin phosphorylation, as all C-terminal truncation mutants
unable to induce cell spreading, also failed to trigger tyrosine
phosphorylation. Our data further highlight major differences in the
involvement of the cytoplasmic domain tyrosine residues in
3-mediated post-ligand binding events. The presence of
residue Tyr759 in the membrane-distal NITY759
sequence is not necessary for
3-mediated focal contact
formation, cell spreading, and
3-triggered tyrosine
phosphorylation of FAK or paxillin, whereas residue Tyr747
of the membrane-proximal NPLY747 motif is required for
optimal
v
3 receptor function. And
finally, both the NPLY747 and NITY759 motifs
contribute in defining the appropriate
3 cytoplasmic domain conformation necessary for post-ligand binding signaling events.
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EXPERIMENTAL PROCEDURES |
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Cell Culture
The Chinese hamster ovary (CHO) cell line CRL 9096, defective in
the dihydrofolate reductase gene (CHO dhfr),
was purchased from the American Type Culture Collection (Rockville, MD). The cells were grown in Iscove's modified Dulbecco's medium (IMDM) (Life Technologies, Inc., Merelbeke, Belgium), supplemented with
glutamine, penicillin, and streptomycin, 10% heat-inactivated fetal
calf serum (complete IMDM), and, when required, hypoxanthine (100 µM) and thymidine (10 µM). The cells were
routinely passaged with EDTA buffer, pH 7.4 (1 mM EDTA, 126 mM NaCl, 5 mM KCl, 50 mM
Hepes).
Antibodies and Purified Adhesive Proteins
The following polyclonal or monoclonal antibodies were
purchased: anti-v from Life Technologies (24),
anti-phosphotyrosine (PY-20), anti-paxillin and anti-FAK from
Transduction Laboratories (Lexington, KY), and the polyclonal anti-FAK
antibody (C-903) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
The monoclonal antibody 4D10G3 (anti-human
3) was a
generous gift of Dr. D. R. Phillips (COR Therapeutics, South San
Francisco, CA). Monoclonal antibodies 13C2 (anti-human
v) and 23C6 (anti-
v
3) were
kindly provided by Dr. M. Horton (Bone and Mineral Centre, The
Middlesex Hospital, London, United Kingdom), monoclonal antibody P37
(anti-human
3) by Dr. J. Gonzalez-Rodriguez (Instituto
de Quimica Fisica, Madrid, Spain), and the blocking monoclonal antibody
MA-16N7C2 (anti-human
3) by Dr. M. Hoylaerts (Centre for
Molecular and Vascular Biology, University of Leuven, Leuven, Belgium).
Purified human fibrinogen and bovine serum albumin (BSA, fraction V)
were purchased from Sigma (Bornem, Belgium).
Construction of Mutant 3 Integrin cDNA
The full-length cDNA encoding wild type 3 was
inserted into the 5'-EcoRI/EcoRV-3' site of the
expression vector pBJ1 as described previously (25). The
3Y747A and
3Y747F mutations were
introduced in the full-length
3 cDNA by
site-directed mutagenesis using the Altered SitesTM in vitro
mutagenesis kit (Promega, Lyon, France). Briefly, full-length cDNA
encoding wild type
3 was cloned into the phagemid
pALTER-1, and the mismatched primers
5'-GCCAACAACCCACTGGCTAAAGAGGCCACG-3' (
3Y747A) and
5'-GCCAACAACCCACTGTTTAAAGAGGCCACGTCGACCTTC-3'
(
3Y747F) (Eurogentec, Seraing, Belgium) used
for the generation of the mutant constructs. Primer
3Y747F allowed the generation of a new SalI
restriction site (GTCGAC) in addition to the point mutation. Mutagenesis was performed according to the manufacturer's
instructions. The full-length mutated
3 cDNA was
finally excised from the pALTER phagemid with 5'
XbaI/HindIII 3' and inserted into the
XbaI/HindIII site of the pBJ1 mammalian cell
expression vector. The cDNAs encoding the mutant
3Y759A,
3Y759F,
3Y747A/Y759F,
3
754,
3
744, and
3
722 subunits were
generated by excision of the 3' end of the full-length
3
coding sequence, starting at the BamHI site at nucleotide
position 1501 of the published
3 cDNA sequence for mutant
3
722 and starting at the EcoRI site
at nucleotide position 2274 for the other mutants. The excision was
followed by an insertion of a BamHI-EcoRV or an
EcoRI-EcoRV cassette, obtained by
oligonucleotide-directed polymerase chain reaction (PCR) mutagenesis.
The nucleotides used to generate the cassette were purchased either
from Genset (Paris, France) or from Eurogentec. The upstream primer
(sense) for the
3Y759A,
3Y759F, and
3Y747A/Y759F mutant constructs was a 23-mer corresponding to the
3 nucleotide sequence 2023-2045:
5'-GTGAAAGAGCTTAAGGACACTGG-3'. The upstream primer (sense) for the
3
754 and
3
744 mutant constructs was
a 26-mer corresponding to the
3 nucleotide sequence
2264-2290: 5'-CGACCGAAAAGAATTCGCTAAATTTG-3' comprising an
EcoRI restriction site (GAATTC). The upstream primer (sense)
for the
3
722 mutant construct was a 22-mer
corresponding to the
3 nucleotide sequence 1497-1518:
5'-GCTGGGATCCCAGTGTGAGTGC-3' comprising a BamHI
restriction site (GGATCC). All downstream primers (antisense) contained
a stop codon followed by an EcoRV restriction site (GATATC).
The following downstream primers were used:
5'-CTTAAGCTTGATATCCTAGTTACTTAAGTGCCCCGGGCCGTGATATTGG-3' (
3Y759A);
5'-CTTAAGCTTGATATCCTAGTTACTTAAGTGCCCCGGAACGTGATATTGG-3' (
3Y759F and
3Y747A/Y759F);
5'-CTTAAGCTTGATATCCTAGTTACCTAGGTAGACGTGGCCTCTTTATAC-3' (
3
754);
5'-CTTAAGCTTGATATCCTAGTTACCTAGTTGGCTGTGTCCCATTTTGC-3' (
3
744); 5'-
CTTAAGCTTGATATCCTAGTTACCTAGATGGTGATGAGGAGTTTCCAG -3'
(
3
722). For
3Y759A,
3Y759F,
3
754,
3
744,
and
3
722 constructs, pBJ1
3wt plasmid
was used as a template for cDNA amplification, while the
3Y747A/Y759F mutant was generated using the plasmid pBJ1
3Y747A. For the
3
722 mutant construct,
the PCR-amplified fragment was purified, digested with BamHI
and EcoRV, and inserted into the pBJ1
3
plasmid from which the wild type BamHI-EcoRV
fragment had been removed. For all the other mutant constructs, the
PCRamplified fragments were digested with EcoRI and
EcoRV after purification and inserted into the pBJ1
3 plasmid from which the wild type
EcoRI-EcoRV fragment had been removed. Each
mutant
3 construct was verified by dideoxy sequencing
using the 26-mer corresponding to the
3 nucleotide
sequence 2264-2290 as a 5' primer.
Transfection and Selection of Stable Cell Clones
Full-length 3 cDNA in pBJ1 vector (20 µg)
and 1 µg of dihydrofolate reductase plasmid (pMDR901) were mixed with
40 µg of LipofectAMINE (Life Technologies, Inc.) in a final volume of
200 µl of IMDM and added to CHO dhfr
cells
grown to 60% confluence in 100-mm tissue culture plates. After 24 h, fetal calf serum was added to the culture medium and 48 h after
transfection, the cells were grown in nucleoside-free
-minimal
essential medium (Life Technologies, Inc.) used as selective medium.
Positive transfectants were analyzed for cell surface expression of the
recombinant human integrin
3 subunit using the
anti-
3 monoclonal antibody P37 and fluorescein
isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody
(Caltag Laboratories, Burlingame, CA). Stably transfected cells were
subcloned by limiting dilution and controlled for cell surface
expression of the transfected
3 integrin subunit.
Immunofluorescence and Flow Cytometry
Surface expression of the transfected human 3
integrins was analyzed by flow cytometry using the monoclonal
antibodies P37 (anti-human
3), 13C2 (anti-human
v), and 23C6 (anti-
v
3).
Selected transfectants were detached from culture plates with EDTA
buffer, pH 7.4, and washed twice in phosphate-buffered saline (PBS)
(136 mM NaCl, 2.7 mM KOH, 8 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.4). The cells (5 × 105) were then incubated for 30 min on ice with the primary
antibody, washed with PBS, and further incubated for 30 min on ice with a FITC-conjugated goat anti-mouse secondary antibody. Cells were washed
and resuspended in PBS and then analyzed on an Epics Elite ESP flow
cytometer (Coulter Corp., Hialeah, FL).
Reverse Transcriptase-PCR of mRNA and cDNA Sequencing
Total RNA was isolated from 5 × 106
transfected cells according to the method of Chomczynski and Sacchi
(26). First strand cDNA synthesis from 2 µg of total RNA was
performed with the Perkin-Elmer RNA-PCR kit using oligo(dT) as a
primer. The coding sequence, corresponding to the cytoplasmic domain of
the 3 integrin subunit was amplified using specific
primers. The amplified products were analyzed by agarose gel
electrophoresis and directly sequenced using the fmolTM DNA
sequencing kit (Promega).
Ligand Coating of Latex Beads and Cell-Bead Attachment Assay
For cell-bead attachment assay, 200 µl of polystyrene 3-µm beads (Sigma) were washed twice in distilled H2O, and resuspended in 1 ml of 0.1 M bicarbonate coating buffer, pH 9. Ligand coating was performed by adding fibrinogen or BSA to the beads at a final concentration of 100 µg/ml. The beads were rotated for 1 h at room temperature, washed once in PBS, and blocked with 0.1% BSA in IMDM for 2 h at room temperature. The beads were finally washed twice and resuspended in IMDM. For the cell-bead attachment assay, CHO cells were detached with EDTA buffer, washed twice, and resuspended in serum-free IMDM. After a preincubation of 45 min at room temperature in the presence or absence of either 500 nM echistatin or 1.5 µg of the monoclonal antibody MA-16N7C2, the cells (4 × 104) were added to individual wells of 96-well microtiter plates precoated overnight at 4 °C with poly-L-lysine (Sigma) at 100 µg/ml in IMDM, and allowed to settle for 1 h at 37 °C. The freshly prepared ligand-coated beads were then added to the wells at a 50:1 bead-to-cell ratio. After a further 45-min incubation at 37 °C with gentle shaking, the unbound beads were removed with six washes in IMDM. Microphotographs were then taken of the cells (magnification, ×300) using a Nikon invertoscope equipped with phase contrast.
Cell Adhesion Assay
Adhesion assays were carried out as described previously with minor modifications (27). Briefly, cultured cells were detached with EDTA buffer, washed twice, and resuspended in serum-free IMDM. The cells (3 × 104) were then added to individual wells of 96 well-microtiter plates coated with fibrinogen at 20 µg/ml in serum-free IMDM overnight at 4 °C, and cell attachment was allowed to occur at 37 °C. For time-course experiments, the cells in the individual microtiter wells were microphotographed at different time points without prior washing of the plates or discharge of nonadherent cells. Quantitation of spread fibroblastoid cells versus non-spread round cells was performed on the micrographs according to cell morphology. For each time point, approximately 200 cells were counted and the data reported as mean percent of three independent experiments performed in triplicate.
Cell Spreading and Immunofluorescence Staining of Focal Adhesion Plaques
Intracellular immunofluorescence staining of adherent cells
was performed using eight-well glass chamber slides (Lab-Tek, Nunc
International, Naperville, IL) precoated overnight at 4 °C with 20 µg/ml of fibrinogen in serum-free IMDM. The cultured cells were
detached with EDTA buffer, washed twice with IMDM, and incubated overnight in individual compartments of the chamber slides. The cells
were fixed for 15 min at 4 °C with 3% paraformaldehyde, 2% sucrose
in PBS, pH 7.4, rinsed twice with PBS, and permeabilized with labeling
buffer (0.5% Triton X-100, 0.5% BSA in PBS, pH 7.4) for 15 min at
room temperature. Immunofluorescent staining was performed by
incubating the glass slides for 30 min with a primary mouse monoclonal
antibody to human 3 (P37) or to the
v
3 complex (23C6) diluted in labeling
buffer. After three washing steps, the glass slides were incubated for
another 30 min with FITC-conjugated goat anti-mouse IgG in the presence
or absence of 0.5 µg/ml phalloidin conjugated to tetramethylrhodamine
isothiocyanate (TRITC, Molecular Probes, Eugene, OR). Negative controls
were stained in the absence of the primary antibody. The slides were
finally washed three times in labeling buffer and mounted in Mowiol
40-88/DABCO (Sigma). The specimens were examined with a Leica-DMRB
fluorescence microscope using a 63 × oil immersion objective.
Microphotograhs were taken using Ilford HP5 Plus 400 films (Ilford,
Mobberley, United Kingdom).
Tyrosine Phosphorylation Assay
Petri dishes (100 mm) were coated overnight at 4 °C with 100 µg/ml of purified human fibrinogen in serum-free IMDM. The dishes were then blocked with 5 mg/ml BSA in serum-free IMDM for 1 h at 37 °C and finally washed twice with serum-free IMDM. Cultured cells were detached with EDTA buffer, carefully washed twice with serum-free IMDM, resuspended in IMDM, and either kept in suspension or added to the coated dishes in the presence or absence of 5 µM cytochalasin B (Sigma). After a 2-h incubation at 37 °C, nonadherent cells were sedimented at 1000 rpm for 10 min and lysed with the following lysis buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 10 µg/ml pepstatin A, 3 mM phenylmethylsulfonyl fluoride. Adherent cells were lysed in situ with the same lysis buffer. Lysates were clarified by centrifugation at 12,000 rpm for 10 min at 4 °C, and the protein content determined with the Bio-Rad protein assay reagent (Bio-Rad, Nazareth, Belgium).
Immunoprecipitation and Western Blot Analysis
Preparation of Cell Lysates-- Cultured cells were detached with EDTA buffer, washed twice in cold PBS buffer, and lysed for 30 min in 300 µl of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM phenylmethylsulfonyl fluoride). Lysates were cleared by centrifugation at 12,000 rpm for 10 min at 4 °C, and the protein concentration was determined according to the method of Markwell (28).
Immunoprecipitation--
For each cell clone, equal amounts of
protein lysate (1-1.5 mg of protein) were incubated for 1 h at
4 °C with either monoclonal antibody P37 (to human
3), or, for tyrosine phosphorylation assays, with
polyclonal rabbit anti-FAK or monoclonal mouse anti-paxillin antibody.
Immune complexes were precipitated by a 30-min incubation at 4 °C
with protein A-Sepharose beads (75 µl of a 1:1 suspension in PBS).
The beads were then washed three times with lysis buffer, and the
precipitates recovered by boiling the beads in 30 µl of SDS sample
buffer (125 mM Tris-HCl, pH 6.8, 4.6% SDS, 20% glycerol, 0.5 mg/ml bromphenol blue) either in the presence or absence of 1.4 M
-mercaptoethanol.
Western Blot Analysis--
Immunoprecipitates or total cell
lysates (50 µg of protein) were resolved by SDS-polyacrylamide gel
electrophoresis (PAGE) and transferred onto nitrocellulose using a
semi-dry transblot apparatus (Amersham Pharmacia Biotech, Roosendaal,
The Netherlands). The membranes were blocked for 1 h in blocking
buffer (Tris-buffered saline (TBS) (20 mM Tris-HCl, pH 7.4, 137 mM NaCl) containing 0.1% Tween and either 1% BSA for
tyrosine phosphorylation assays or 5% nonfat dry milk) and incubated
overnight with the primary antibody diluted in blocking buffer. After
several 5 to 10 min washes in TBS-Tween (TBS, pH 7.4, 0.1% Tween), the
membranes were incubated for 1 h with sheep anti-mouse IgG
conjugated to horseradish peroxydase (Amersham Pharmacia Biotech,
Buckinghamshire, United Kingdom) in TBS-Tween containing 5% nonfat dry
milk at pH 7.4. The membranes were then washed in TBS and bound
antibody visualized using enhanced chemiluminescence (ECL) (Pierce)
according to the manufacturer's instructions. After exposure to
autoradiography films, the membranes prepared for tyrosine
phosphorylation assays were stripped by a 30-min incubation at 50 °C
in 50 ml of stripping buffer (62.5 mM Tris, pH 6.7, 2%
SDS, 100 mM -mercaptoethanol) and then reprobed with a
monoclonal antibody to either FAK or paxillin. For each experiment, the
level of antibody binding was quantified by scanning densitometry and
the results expressed as the ratio of phosphorylated FAK
versus total immunoprecipitated FAK. The data for each cell
clone were normalized to the ratio obtained for CHO
3wt
cells adherent on fibrinogen (expressed as 100%).
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RESULTS |
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In order to determine how the 3 integrin
cytoplasmic domain regulates integrin-dependent tyrosine
phosphorylation during cell spreading, a series of
3
integrin subunit mutants were generated that either promote or fail to
promote
3 integrin-dependent cell spreading
(Fig. 1). After stable transfection of
wild type or mutant
3 cDNA into CHO cells, cell
clones were analyzed by flow cytometry for surface expression of the
chimeric
v(hamster)
3(human) receptor,
using monoclonal antibodies specific to human
v (13C2), human
3 (P37), and the
v
3
complex (23C6). As shown in Fig. 2, all
the cell clones selected for the present study revealed similar levels
of cell surface expression of the chimeric
v
3 receptor, except mutant
3
722, for which only weak labeling could be observed,
despite several successive transfection attempts. Western blot analysis
of the expressed recombinant
3 subunit in each cell
clone essentially confirmed the immunofluorescence data (Figs.
3 and
4A). Interestingly however,
despite the weak surface expression of deletion mutant
3
722, a band even stronger in intensity to that
observed for wild type
3 could be demonstrated in CHO
3
722 cells. The slightly increased electrophoretic
mobility of deletion mutants
3
744 and
3
722 as compared with recombinant wild type
3 confirmed their smaller molecular size. Correct
heterodimerization of endogenous
v with the human
3 subunit was demonstrated for each deletion mutant by
immunoprecipitation experiments using the anti-human
3
antibody P37. As shown in Fig. 4B, two bands corresponding
to
v and
3 were coprecipitated with
similar intensities for all deletion mutants, including
3
722. Finally, to confirm that each selected cell
clone expressed the human
3 integrin subunit with the
expected cytoplasmic mutation, mRNA was isolated from the
transfected cell clones and transcribed into cDNA. The cDNA
segment encoding the cytoplasmic domain was amplified using
3 specific primers, and the amplified segment sequenced
(results not shown). Taken together, these data demonstrate that the
selected cell clones express on their cell surface the recombinant
3 subunit with the expected mutation, and that an almost
complete deletion of the cytoplasmic domain of the integrin
3 subunit (
3
722) interferes with
surface exposure of the preformed heterodimeric
v
3
722 integrin complex.
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v
3-mediated CHO Cell Binding to
Immobilized Fibrinogen Is Not Impaired by
3 Cytoplasmic
Domain Mutations--
In order to determine whether the selected
3 mutants retained the ability to interact with
immobilized fibrinogen, a cell binding assay was performed using
fibrinogen or BSA-coated polystyrene beads. When CHO
3wt
cells were tested, they were completely covered with fibrinogen-coated
beads and had a "morula" type appearance. In contrast, when the
cells were incubated with BSA-coated beads, no binding of the beads to
the cells could be observed. The binding of fibrinogen-coated beads was
RGD-dependent, since it could be specifically blocked with
the disintegrin echistatin or the blocking anti-human
3
monoclonal antibody MA-16N7C2 known to contain an RGD sequence in its
CDR3 domain (29) (Fig. 5A).
Interestingly, all the mutant cell clones studied bound the
fibrinogen-coated beads to a similar extent as CHO
3wt
cells, demonstrating that the
3 cytoplasmic domain
mutations did not impair
v
3
receptor-ligand interaction (Fig. 5B).
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Role of the Cytoplasmic Domain Tyrosine Residues in
3 Integrin-dependent Cell Spreading--
To
determine the functional role of the tyrosine residues in the
membrane-proximal NPLY747 and membrane-distal
NITY759 sequence in
v
3-mediated cell spreading on fibrinogen,
adherence of CHO cells expressing the
3 mutants
indicated in Fig. 1 was performed using a steady state adhesion assay.
The quantitative analysis of cell spreading is shown in Fig.
6 (A and B).
Spreading of CHO cells expressing wild type
3 was
essentially complete after a 2-h incubation at 37 °C, in contrast to
mock-transfected CHO cells that lacked the
v
3-dependent adhesive
phenotype on fibrinogen, demonstrating that CHO cell spreading on
fibrinogen completely relies on the transfected human
3
subunit. None of the three deletion mutants (
3
754,
3
744, and
3
722) underwent shape
change on fibrinogen, demonstrating that a minimal deletion of 9 C-terminal amino acids comprising the membrane-distal
NITY759 motif was already sufficient to completely prevent
3 integrin-dependent cell spreading. When
the single tyrosine residues 747 or 759 were mutated into alanine, a
complete inhibition of cell spreading on fibrinogen was observed with
mutant
3Y747A and a strong inhibition was observed with
mutant
3Y759A. Similarly, the double mutant
3Y747A/Y759F exhibited the same defective cell spreading
phenotype as mutant
3Y747A. On the other hand, when the
more conservative, but still phosphorylation-defective substitutions of
tyrosine by phenylalanine were tested (Y747F and Y759F), almost
complete restoration of cell spreading was observed for mutant
3Y759F, whereas only 50% of the cells expressing the
mutation Y747F underwent shape change, as compared with CHO
3wt cells. In order to determine whether this reduced
cell spreading was due to decreased cell spreading kinetics, a
time-course experiment was performed and cell spreading monitored over
12 h. As shown in Fig. 6B, spreading of CHO
3wt and CHO
3Y747F cells reached a
plateau at about 3 h. Interestingly however, only 50% of the CHO
3Y747F cells underwent cell spreading even after 12 h of incubation at 37 °C, although 100% of the cells expressed the
recombinant
3 receptor as monitored by
fluorescence-activated cell sorting analysis. To exclude the possibility that the observed differences in cell spreading depended on
clonal variation, additional cell clones that were independently isolated during the transfection procedure were analyzed. For each
mutation, up to three cell clones were tested and each exhibited the
same spreading phenotype (data not shown). Finally, no increase in cell
spreading was observed with increasing coating concentrations of
fibrinogen (data not shown). Taken together, these data provide evidence that the structural integrity of the
3 subunit
cytoplasmic tail is a prerequisite for
3-mediated cell
spreading and that the presence of residue Tyr747, but not
Tyr759, in the tandem NXXY motifs is required
for the normal spreading phenotype.
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Effect of Cytoplasmic Domain Mutations on 3 Integrin
Focal Contact Localization and Stress Fiber Formation--
We next
analyzed the ability of the
3 mutants to translocate to
focal contacts and to promote stress fiber formation. Immunofluorescent staining was performed after a 12-h incubation of transfected CHO cells
on fibrinogen-coated glass slides. The cells were then fixed,
permeabilized, and either stained with a monoclonal antibody to the
3 integrin subunit or
v
3
complex or costained with an anti-
3 antibody and
TRITC-labeled phalloidin to visualize actin stress fibers. As shown in
Fig. 7, none of the
3
deletion mutants were able to translocate to focal adhesion plaques.
The use of the complex-specific anti-
v
3
antibody 23C6 further demonstrated that all deletion mutants, including
3
722, formed heterodimeric complexes with endogenous
v. Fig. 8 displays the
costaining of
3 integrins and stress fibers in selected
CHO cell clones. The wild type human
3 subunit was
localized in focal contacts at the tips of well organized actin stress
fibers. In contrast, immunostaining of the transfected cells expressing
the point mutants
3Y747A or
3Y747A/Y759F
revealed the round morphology of firmly attached but unspread cells,
and the complete absence of
3 integrin-induced focal
adhesions or stress fibers, as visualized by the diffuse staining of
the cells with the anti-
3 antibody and phalloidin. An
identical result was obtained with the deletion mutants
3
754,
3
744, and
3
722 (data not shown). The cell clone expressing mutant
3Y759A exhibited strongly reduced stress fiber
formation and
3 focal contact recruitment in those cells
that were able to spread on fibrinogen, whereas cells expressing mutant
3Y759F had a wild type phenotype. Interestingly, with
mutant
3Y747F, an intermediate phenotype was observed;
in the cells that had undergone shape change,
3 integrin
was detectable in focal adhesion plaques, but the number of focal
adhesion plaques was reduced and the few actin stress fibers were
located predominantly at the cell periphery. Altogether, these results
essentially confirm the data described for cell spreading
experiments.
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Correlation between 3 Integrin-mediated Cell
Spreading and
3-triggered Tyrosine
Phosphorylation--
In an effort to determine how
3
integrin-dependent cell spreading correlated with tyrosine
kinase activation and intracellular phosphotyrosine signaling, we
investigated the effect of the cytoplasmic domain mutations on
3 integrin-triggered postreceptor occupancy events,
namely tyrosine phosphorylation of the intracellular proteins FAK and
paxillin. As tyrosine phosphorylation of FAK and paxillin is not only
an integrin-mediated response, but can also be stimulated by growth
factors, the transfected cells were carefully washed before plating, in
order to eliminate all traces of fetal calf serum. After a 2-h
incubation at 37 °C on immobilized fibrinogen, attached cells were
lysed in situ, and the lysate used for FAK or paxillin
immunoprecipitation. Immunoblots of the precipitates were first probed
with a monoclonal anti-phosphotyrosine antibody (PY-20), then stripped
and reprobed with a monoclonal anti-FAK or anti-paxillin antibody. In a
control experiment shown in Fig. 9,
stimulation of tyrosine phosphorylation of FAK was observed when
transfected CHO cells expressing wild type
3 were
allowed to spread on immobilized fibrinogen, whereas only background
tyrosine phosphorylation was observed when the same cells were kept in suspension for 2 h or when mock-transfected CHO cells were plated on fibrinogen, demonstrating that the observed increase in FAK tyrosine
phosphorylation could be specifically attributed to
3 integrin-triggered outside-in signaling. When the mutant cell clones
were tested, a strong correlation between
3-mediated
cell spreading and
3-triggered FAK phosphorylation was
observed (Fig. 10). All the cell clones
that were unable to spread on fibrinogen were also unable to trigger
FAK phosphorylation above background levels (CHO
3
754, CHO
3
744, CHO
3
722, as well as CHO
3Y747A and CHO
3Y747A/Y759F). The
3Y759A mutant failed
to signal tyrosine phosphorylation of FAK, consistent with the strongly
reduced spreading phenotype of CHO
3Y759A cells. In
contrast, the more conservative, but still phosphorylation-defective
phenylalanine substitution of Tyr759 restored FAK tyrosine
phosphorylation, while the Y747F substitution gave an intermediate
phenotype, suggesting that the presence of Tyr759, and
hence phosphorylation of this residue, is not strictly required to
signal FAK tyrosine phosphorylation. These data further indicate that
3 integrins with a structural modification of the
cytoplasmic tail, due to an alanine substitution of Tyr747
or Tyr759, fail to trigger FAK tyrosine
phosphorylation.
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DISCUSSION |
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Integrin cytoplasmic domains are key effectors in regulating
integrin-receptor function. In many cell types, both the and
subunit cytoplasmic domains modulate integrin affinity for
extracellular ligands, and hence play a role in inside-out signaling
(14, 30-32). Integrin-mediated cell spreading, in contrast, appears to
rely essentially on integrin
subunits, as the
subunit
cytoplasmic tail by itself contains sufficient information to target
integrins to focal adhesions (7) and to trigger tyrosine
phosphorylation of intracellular proteins (8). Following the initial
identification of three regions within the cytoplasmic tail of the
1 integrin subunit necessary for focal contact
recruitment of integrins (6), numerous studies on
1 and
3 subunits have focused on the functional role of two of
these highly conserved sequences, NPXY and NXXY, which constitute typical recognition sites for tyrosine kinases and are
encoded by a single exon known to undergo alternative splicing (33).
Both of these tandem domains appear to be crucial for integrin receptor
function, although studies of various recombinant mutant
3 integrin subunits expressed as heterodimers with
either
IIb as a fibrinogen receptor, or
v
as a major vitronectin receptor, have generated divergent results:
3 mutants with a deletion of the membrane-distal
NITY759 sequence up to amino acid 756 completely prevented
IIb
3 integrin-dependent cell
spreading on immobilized fibrinogen (13), while deletion of the same
C-terminal domain up to amino acid 751 allowed normal
v
3-dependent cell spreading
on vitronectin (12). Furthermore, by using cell-permeable peptides
carrying different linear
3 cytoplasmic domain
sequences, Liu and co-workers (15) identified the
3
C-terminal segment (residues 747-762) as a major cell adhesion regulatory domain capable of inhibiting the interaction of
IIb
3-expressing HEL cells or
v
3-expressing endothelial cells with
immobilized fibrinogen, while peptides with a Y759F substitution were
unable to induce this inhibitory effect. Differences concerning the
involvement of the membrane-proximal NPLY747 sequence in
signal transduction have also been reported; mutations in the
3 cytoplasmic domain that eliminate or disrupt the
membrane-proximal NPLY747 motif prevented
v
3-mediated cell attachment to
immobilized vitronectin, but did not perturb the ability of
v
3 to interact with soluble vitronectin
(12), while mutations in the NPLY747 sequence abolished
inside-out signaling of
IIb
3 (14).
The observations that the NPLY747 and NITY759
motifs in the 3 integrin subunit might differently
regulate
IIb
3 and
v
3 receptor function prompted us to
investigate the effect of
3 cytoplasmic domain
mutations, that either promote or inhibit cell spreading, on
v
3-mediated tyrosine phosphorylation of
two major focal adhesion proteins, the focal adhesion kinase FAK (34),
and the cytoskeleton-related "bridging" protein paxillin (35). Our
results demonstrate a close correlation between
v
3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759
sequences in these post-ligand binding events. Considering the membrane-distal NITY759 motif, deletion of this motif was
sufficient to completely prevent
v
3-dependent focal contact
formation, cell spreading, as well as FAK/paxillin tyrosine
phosphorylation. A Y759A substitution also resulted in a strong
inhibitory phenotype. In contrast, the more conservative, but still
phosphorylation-defective Y759F mutation was able to restore wild type
receptor function. These data suggest that the structural integrity of
the NITY759 motif, rather than the phosphorylation status
of Tyr759, is important for
3-mediated
cytoskeleton reorganization or tyrosine phosphorylation of FAK and
paxillin. Concerning the membrane-proximal NPLY747
sequence, our mutagenesis studies demonstrate that an alanine substitution of the highly conserved tyrosyl residue at 747 completely abolished
v
3-dependent
formation of focal adhesion plaques and cell spreading, and prevented
FAK and paxillin tyrosine phosphorylation, while a Y747F substitution,
compared with the Y759F substitution, only partially restored these
receptor functions, suggesting that phosphorylation of residue
Tyr747 might be required for optimal
3-mediated postreceptor signaling events. These data
could explain why the non-phosphorylated NPLY747-containing
peptides used by Liu et al. were unable to impair
v
3 integrin-mediated cell adhesion (15).
Our findings, together with previously reported results on the effect
of substitutions of Tyr747 on cell adhesion and spreading
(12, 13), strengthen the notion that the conformational organization of
the
3 cytoplasmic domain defined by the
NPLY747 and NITY759 motifs is essential for
3-mediated cytoskeletal organization and FAK/paxillin
phosphorylation. These data are in accordance with the results of
Tahiliani et al. (23) and with the findings obtained with
the alternative spliced variants
1B (36) and
3B (8), previously shown to be defective in
triggering FAK tyrosine phosphorylation. In contrast, our data seem to
differ from a previous report, showing that the membrane-distal
3 tail is not necessary for
IIb
3 integrin-triggered tyrosine
phosphorylation of FAK (37). Concerning cell spreading, our results are
also in good agreement with data on
IIb
3-mediated cell spreading (13), and
further demonstrate that the structural requirements of the
3 cytoplasmic domain necessary for
IIb
3- or
v
3-mediated cell spreading are
essentially the same. This conclusion is supported by the fact that the
naturally occurring S752P mutation, which is closely located to the
NITY759 motif and responsible for a variant Glanzmann's
thrombasthenia phenotype, renders
IIb
3
defective in both inside-out and outside-in signaling, while a S752A
mutation restores wild type receptor-mediated cell spreading for
IIb
3 (13) as well as
v
3 (25).
Recently, several novel subunit cytoplasmic domain-specific binding
proteins have been identified, which selectively interact with the
C-terminal region of
1 (ICAP-1),
2
(cytohesin-1), and
3 (
3-endonexin)
integrin cytoplasmic tails (18, 20, 38). It is quite interesting that
both
3-endonexin and ICAP-1, which display restricted
binding to the
3 and the
1 cytoplasmic
domain, respectively, rely on a structurally intact membrane-distal
NITY (
3) or NPKY (
1) motif for integrin
binding, as a Tyr
Ala substitution has been shown to completely
prevent these protein-protein interactions, while a Tyr
Phe
substitution has only minimal inhibitory effect (20, 39). ICAP-1, which
is a phosphoprotein and whose phosphorylation is regulated by
cell-matrix interaction, appears to play a major role during
1 integrin outside-in signaling processes, and could represent the missing adaptor protein necessary for linking the
1 integrin cytoplasmic tail to downstream signaling
events. In contrast, the functional role of
3-endonexin
appears to be restricted to inside-out signaling, as no strong
colocalization of
3-endonexin with
v
3 has been observed in
3-triggered focal adhesion plaques (40). Since
3-endonexin modulates the affinity state of
IIb
3, it has been suggested that this
protein might participate in integrin activation, and dissociate during
later stages of cell adhesion, allowing a
3-endonexin-independent interaction of the integrin cytoplasmic tail with cytoskeletal proteins (40), suggesting that
transient posttranslational modifications of the
3
subunit might be involved in modulating distinct
3
receptor functions.
Tyrosine phosphorylation of integrin subunits has been documented
in a number of different cell types. In Rous sarcoma virus-transformed fibroblasts, tyrosine phosphorylation of the
1 subunit
of the fibronectin receptor resulted in defective cytoskeletal
organization (41). Using an antiserum reacting specifically with the
phosphorylated cytoplasmic tail of
1, Johansson et
al. (42) were able to demonstrate a distinct subcellular
localization of tyrosine-phosphorylated
1 as compared
with non-phosphorylated
1. In vivo tyrosine
phosphorylation of the
3 subunit of
IIb
3 has been shown to occur during
thrombin-stimulated platelet aggregation (19), or after ligand-,
antibody- or Mn2+- stimulated clustering of
v
3 in erythroleukemic K562 cells and
ovarian carcinoma cells (43). Interestingly however, in the K562 cell
model coexpressing
v
3,
v
5, and
IIb
3, Mn2+ stimulation of the
cells in suspension only stimulated tyrosine phosphorylation of the
3 integrin subunit associated with
v, suggesting that inducible tyrosine phosphorylation of the
3 integrin requires the
v integrin
cytoplasmic tail. Data by Law et al. (19) have further shown
that in vitro tyrosine-phosphorylated
3
peptides associate with Grb2 as well as Shc, a phosphotyrosine-binding adaptor protein interacting through its PTB (phosphotyrosine binding) domain with phosphorylated NPXY motifs (44). A similar
direct in vivo association of
v
3 with Grb2 has also been reported by Blystone and co-workers (43). More recently, the same group has
provided evidence that the presence of residue Tyr747 of
the membrane-proximal NPLY747 motif is required for
3 tyrosine phosphorylation and for stimulated
v
3-mediated adhesion in K562 cells (45).
Our data are in good agreement with these findings, and further
underline the distinct involvement of the NPLY747 and
NITY759 sequences in triggering FAK/paxillin
phosphorylation, as they clearly demonstrate that Tyr759 is
not required for this process.
In summary, the results of this work provide evidence that modification
of the overall conformation of the 3 cytoplasmic domain,
due to deletion of the 9 C-terminal amino acids or to a structural
change within the membrane-proximal NPLY747 and to a lesser
extent within the membrane-distal NITY759 sequence, impairs
3-mediated cell spreading and actin stress fiber
formation as well as
3-triggered paxillin or FAK
tyrosine phosphorylation. Phosphorylation of residue Tyr759
of the membrane-distal NITY759 sequence is apparently not
necessary for the investigated
3 integrin receptor
functions, while phosphorylation of Tyr747 might be
required to optimize these functions. The presently described stable
CHO cell clones, expressing various
3 mutants, should
provide a valuable tool to further investigate interactions of the
3 subunit cytoplasmic tail with structural, regulatory or signaling proteins, and to dissect the involvement of distinct
3 cytoplasmic sequences in various
3
integrin-mediated signaling pathways.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. Gonzalez-Rodriguez, M. Horton, M. Hoylaerts, and D. R. Phillips for their generous gifts of monoclonal antibodies. We also thank Dr. Wim Ammerlaan (Department of Immunology, Laboratoire National de Santé, Luxembourg) for expert assistance with flow cytometry analysis.
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FOOTNOTES |
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* This work was supported by grants from Centre de Recherche Public-Santé (CRP-Santé, Luxembourg), CNRS (France), Fondation Luxembourgeoise Contre le Cancer (Luxembourg), and EC Biomed Project CT931685.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.
Recipient of a fellowship from the Ministère de l'Education
Nationale et de la Formation Professionnelle, Luxembourg. Data presented were obtained as part of a doctoral thesis to be submitted to
the University Paris XI.
§ To whom correspondence should be addressed. Tel.: 352-466644-440; Fax: 352-466644-442; E-mail: kieffer{at}cu.lu.
1 The abbreviations used are: FAK, focal adhesion kinase; BSA, bovine serum albumin; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; ICAP-1, integrin cytoplasmic domain-associated protein-1; IMDM, Iscove's modified Dulbecco's medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; TBS, Tris-buffered saline; TRITC, tetramethylrhodamine isothiocyanate.
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REFERENCES |
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