J Biol Chem, Vol. 274, Issue 40, 28575-28583, October 1, 1999
Identification of a Talin-binding Site in the Integrin
3 Subunit Distinct from the NPLY Regulatory Motif
of Post-ligand Binding Functions
THE TALIN N-TERMINAL HEAD DOMAIN INTERACTS WITH THE
MEMBRANE-PROXIMAL REGION OF THE 
3 CYTOPLASMIC TAIL*
Sonali
Patil
§,
Arom
Jedsadayanmata§,
June D.
Wencel-Drake¶,
Wei
Wang,
Irina
Knezevic, and
Stephen
C.-T.
Lam
From the Department of Pharmacology and ¶ School
of Biomedical and Health Information Sciences, the University of
Illinois, Chicago, Illinois 60612
 |
ABSTRACT |
Following platelet aggregation,
integrin 
IIb3 becomes associated
with the platelet cytoskeleton. The conserved NPLY sequence represents
a potential -turn motif in the 3 cytoplasmic tail and
has been suggested to mediate the interaction of 3
integrins with talin. In the present study, we performed a double
mutation (N744Q/P745A) in the integrin 3 subunit to test
the functional significance of this -turn motif. Chinese hamster
ovary cells were co-transfected with cDNA constructs encoding
mutant 3 and wild type IIb. Cells
expressing either wild type (A5) or mutant (D4)
IIb3 adhered to fibrinogen; however, as
opposed to control A5 cells, adherent D4 cells failed to spread, form
focal adhesions, or initiate protein tyrosine phosphorylation. To
investigate the role of the NPLY motif in talin binding, we examined
the ability of the mutant IIb3 to
interact with talin in a solid phase binding assay. Both wild type and
mutant IIb3, purified by RGD affinity chromatography, bound to a similar extent to immobilized talin. Additionally, purified talin failed to interact with peptides containing the AKWDTANNPLYK sequence indicating that the talin binding
domain in the integrin 3 subunit does not reside in the NPLY motif. In contrast, specific binding of talin to peptides containing the membrane-proximal HDRKEFAKFEEERARAK sequence of the
3 cytoplasmic tail was observed, and this interaction
was blocked by a recombinant protein fragment corresponding to the 47-kDa N-terminal head domain of talin (rTalin-N). In addition, RGD
affinity purified platelet IIb3 bound
dose-dependently to immobilized rTalin-N, indicating that
an integrin-binding site is present in the talin N-terminal head
domain. Collectively, these studies demonstrate that the NPLY -turn
motif regulates post-ligand binding functions of
IIb3 in a manner independent of talin
interaction. Moreover, talin was shown to bind through its N-terminal
head domain to the membrane-proximal sequence of the 3
cytoplasmic tail.
| |
INTRODUCTION |
Integrins are transmembrane · receptor complexes involved
in numerous physiological processes such as embryogenesis,
angiogenesis, immune response, and hemostasis (1). It is generally
agreed that binding of adhesive proteins to integrins initiates a
series of post-ligand occupancy events that are dependent on an intact cytoskeleton (2). On blood platelets, integrin
IIb3 is the most prominent adhesion
receptor and plays an essential role in platelet aggregation and the
retraction of platelet-rich fibrin clots (3, 4). At sites of vascular
injury, stimulation of platelets with physiological agonists is thought
to result in a change of the native IIb3
conformation from an inactive to an active state which is competent to
bind soluble fibrinogen. Following fibrinogen binding to
IIb3 and platelet aggregation, this
integrin becomes associated with the cytoskeleton (5-7). Increasing
evidence suggests that the interaction between
IIb3 and the platelet cytoskeleton is
crucial for IIb3-dependent post-ligand occupancy events such as clot retraction, protein tyrosine
phosphorylation, and receptor clustering and redistribution (8-12). In
this regard, it has been shown that cytochalasins, which block actin
polymerization, have profound inhibitory effect on these processes (10,
12, 13).
Biochemical and functional studies revealed that the 3
cytoplasmic tail modulates the ligand binding affinity state of the receptor (14-16) and serves as an assembly site for cytoskeletal proteins and signaling molecules (17-20). Thus, truncation of the cytoplasmic sequence of 3, but not IIb,
completely abolished the ability of IIb3
to initiate cell spreading and focal adhesion formation upon
IIb3-mediated cell adhesion to fibrinogen
(21). Moreover, deletion of the 3 cytoplasmic tail also
rendered the 3 integrins in transfected Chinese hamster
ovary (CHO)1 cells incapable
of supporting clot retraction (21) and phosphorylation of focal
adhesion kinase (22, 23). In addition, site-directed mutagenesis of
integrin 1 and 3 cytoplasmic domains
defined the membrane-proximal region (cyto-1) and two highly homologous NXXY motifs (cyto-2 and cyto-3) as being important in the
regulation of integrin affinity states, cell spreading, and receptor
recruitment into adhesion plaques (24, 25) (Fig.
1). Recently, it has been shown that the
tyrosine residues in the NXXY sites of the 3
cytoplasmic tail are phosphorylated upon thrombin-induced platelet aggregation, and their phosphorylation state may regulate the binding
of signaling molecules such as SHC and GRB2 (19), as well as
cytoskeletal proteins including myosin (20).
View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
The amino acid sequences of the cytoplasmic
domains of integrin 1 and 3 subunits. A comparison of
integrin 1 (59) and 3 (60) cytoplasmic
tails with the cyto-1, cyto-2, and cyto-3 sequences (24) in
boldface letters.
|
|
Prediction of protein secondary structures by the Chou and Fasman
algorithm (26) indicate that the N744PLY sequence forms a
tight -turn while the N756ITY sequence resides within a
-sheet structure. In addition, a N744Q/P745A double mutation would
greatly diminish the probability of forming the -turn within the
3 cytoplasmic tail. To examine the functional
significance of the N744PLY -turn motif, we generated
this N744Q/P745A 3 mutant construct and co-transfected
it with a wild type IIb construct into CHO cells. The
abilities of cells expressing mutant 3 integrins to support cell spreading, focal adhesion formation, and protein tyrosine
phosphorylation were determined. Since the corresponding region in the
1 cytoplasmic tail has been implicated in talin binding
function (27, 28), we also examined the ability of the purified mutant
receptor to interact with talin in a solid phase binding assay. In
these experiments, we found that the N744Q/P745A mutation in
3 blocked post-ligand binding functions but did not affect IIb3 binding to talin. This led us
to investigate further the talin-binding site within the
3 cytoplasmic tail as well as the integrin-binding site
within talin. Results of our study show that the N-terminal head domain
of talin interacts with the membrane-proximal region of the
3 cytoplasmic sequence.
| |
MATERIALS AND METHODS |
Antibodies, Peptides, and Reagents--
The monoclonal
antibodies mAb15 (29) and AP-2 (30) were generous gifts of Dr. M. H. Ginsberg and Dr. T. J. Kunicki, respectively, of the Scripps
Research Institute, La Jolla, CA. The anti-talin monoclonal antibody
8d4 (31) and streptavidin were obtained from Sigma. The 6× His
monoclonal antibody was from CLONTECH Laboratories, Inc. Human fibrinogen (grade L) was purchased from KabiVitrum, Inc. For
flow cytometry studies, AP-2, was conjugated with fluorescein isothiocyanate (FITC) using FITC-celite (Sigma) as described (32). For
solid phase binding studies, mAb15, 8d4, and streptavidin were labeled
with carrier-free Na125I (Amersham Pharmacia Biotech) using
the IODO-BEADS iodination reagent (Pierce) to a specific activity of
approximately 3 µCi/µg for the antibodies and 0.5 µCi/µg for
streptavidin. Peptides were synthesized by solid phase synthesis using
an Applied Biosystems model 431 peptide synthesizer or were obtained
from Research Genetics, Inc. Synthetic peptides were represented by the
single letter amino acid code corresponding to their sequences (33).
Table I shows the amino acid sequences of the 3
cytoplasmic domain peptides used in the present study.
cDNA Constructs--
The generation of wild type
IIb (CD2b) and 3 (pc3A) constructs has
been described previously (34, 35). The N744Q/P745A mutation in
3 was generated by splice-overlap extension mutagenesis (36). Overlapping fragments containing this mutation were first generated by polymerase chain reaction (PCR) amplifications using the
oligonucleotide pairs 5'-CGAGGCTGATCAGCGAGCTC-3',
5'-CAAAATGGGACACAGCCAACcAggCACTGTATAAAGAGGCCACG-3' and
5'-CGTGGCCTCTTTATACAGTGccTgGTTGGCTGTGTCCCATTTTGC-3',
5'-GGCCAGTGCAGCTGTGGGGAC-3' as primers and pc3A as template. The
overlapping fragments were combined, denatured by heating at 85 °C
for 10 min, and reannealed by cooling to 22 °C. The ends were filled
in with Sequenase, and the double-stranded fragments were then
amplified by PCR using the oligonucleotide 5'-CGAGGCTGATCAGCGAGCTC-3',
5'-GGCCAGTGCAGCTGTGGGGAC-3' pair. The amplified product was digested
with AflII and XbaI and ligated into an
AflII-XbaI-digested pc3A vector fragment. The mutant construct was verified by automated DNA sequencing (Applied Biosystems model 373A DNA sequenator) and purified by chromatography on
Qiagen Tip-100.
Cell Culture and Transfection--
CHO-K1 cells were obtained
from American Type Culture Collection (ATCC, Manassas, VA) and
maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma)
supplemented with 10% fetal bovine serum (Sigma), 1% non-essential
amino acids, 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin.
The N744Q/P745A mutant 3 construct was co-transfected
with the wild type IIb construct (CD2b) into CHO cells
by liposome-mediated transfection. Briefly, 2 µg of each construct
were incubated with 20 µl of LipofectAMINE reagent (Life Technologies
Inc.) in 180 µl of unsupplemented DMEM at 22 °C for 20 min, and
3.8 ml of unsupplemented DMEM was then added. The DNA-liposome
complexes were overlaid onto CHO cells and incubated for 6 h at
37 °C. The cells were washed with phosphate-buffered saline (PBS, 10 mM sodium phosphate, pH 7.4, 0.15 M NaCl) and
incubated in complete medium at 37 °C for 48 h with a change of
medium at 24 h. The cells were analyzed for transient expression
of mutated IIb3 by flow cytometry using FITC-conjugated AP-2, an anti-IIb3
complex specific monoclonal antibody (30). Stable cell lines were
selected in medium containing 0.75 mg/ml G418 (Sigma), and single cell
sorting was performed to obtain stable clonal lines that were high
expressors of the mutant IIb3. The
production of the control A5 cell line expressing wild type
IIb3 has been described previously
(37).
Phase Contrast and Immunofluorescence Microscopy--
Coverslips
were coated with fibrinogen (100 µg/ml in PBS) overnight at 4 °C
and then blocked with 1% BSA for 1 h at 22 °C. CHO cells were
harvested and seeded onto the fibrinogen-coated coverslips in a
serum-free medium for 3 h. Adherent cells were fixed with 2%
paraformaldehyde for 10 min at 4 °C and subsequently neutralized
with NH4Cl/Tris-buffered saline, pH 7.4. For phase contrast
microscopy, the specimens were examined with a Leitz microscope using a
10× dry objective and photographed with Eastman Kodak
Tmax 400 film.
For indirect immunofluorescent staining of focal contacts, fixed
adherent cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min. Following incubation with the anti-3 monoclonal antibody mAb15 for 1 h, cells were stained with FITC-conjugated goat anti-mouse IgG (Sigma) for 1 h. After washing, the samples were mounted on a droplet of FITC-GuardTM (Testog Inc.) and
viewed with a Jenaval phase/fluorescence microscope equipped with an
HBO 50-watt mercury lamp, and an IVF1 epifluorescence condenser with BP
485 and 546 nm excitation filters and BP 520-560 and LP 590 barrier
filters. CHO cells were photographed with Eastman Kodak
Tmax 400 film.
Protein Tyrosine Phosphorylation--
Tissue culture plates (100 mm, Falcon) were coated with 100 µg/ml fibrinogen in PBS overnight at
4 °C and then blocked with 0.5% BSA for 2 h at 37 °C.
Harvested cells (1 × 107 cells), suspended in 1 ml of
20 mM HEPES, pH 7.4, containing 137 mM NaCl,
2.7 mM MgCl2, 5.6 mM glucose, and
3.3 mM NaH2PO4, were added to the
fibrinogen-coated plates and allowed to adhere for 90 min at 37 °C.
Non-adherent cells on control BSA-coated plates and adherent cells on
fibrinogen-coated plates were lysed with RIPA buffer (10 mM
Tris-HCl, 158 mM NaCl, 1% sodium deoxycholate, 1% Triton
X-100, 0.1% SDS, 1 mM Na2EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, 100 KIU/ml aprotinin, pH 7.2). The
lysates were clarified by centrifugation at 12,500 × g
at 4 °C for 30 min, and their protein concentrations were determined
by the BCA protein assay (Pierce). Proteins were separated by
electrophoresis on 7% polyacrylamide gels under reducing conditions
and transferred onto nitrocellulose membranes. In some experiments, the
cell lysates were subjected to immunoprecipitation using an anti-FAK
monoclonal antibody (Transduction Laboratories) coupled to protein
G-Sepharose (GammaBind Plus Sepharose, Amersham Pharmacia Biotech).
Protein tyrosine phosphorylation was analyzed by immunoblotting using the anti-phosphotyrosine monoclonal antibody PY20 (ICN) and detected by
enhanced chemiluminescence (Pierce) as described (10, 23).
Solid-phase Binding Assays--
For the binding of
IIb3 to talin, wild type and mutant
IIb3 were RGD affinity purified from A5
and D4 cells, respectively. In some experiments,
IIb3 was purified from octyl glucoside lysates of outdated human platelets as described (18). Briefly, 6 × 108 A5 and D4 cells were harvested, washed, and
solubilized in lysis buffer containing 10 mM HEPES, pH 7.5, 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM leupeptin, 10 mM N-ethylmaleimide, 1 mM
phenylmethylsulfonyl fluoride, and 50 mM octyl glucoside.
The cell lysates were applied to 0.5 ml of GRGDSPK-coupled Sepharose 4B
and incubated overnight at 4 °C. After washing unbound proteins,
IIb3 was eluted with 2 mM
HHLGGAKQAGDV peptide. Talin was purified from detergent lysates of
human platelets by a combination of DEAE-cellulose anion exchange chromatography, gel filtration on Sepharose CL-6B, and hydroxyapatite column chromatography (38) (Fig. 2,
lane 1). The concentrations of
IIb3 and talin were determined using the
BCA protein assay (Pierce) with BSA as the standard. In the binding
studies, purified talin (100 µg/ml, 50 µl/well) was coated onto
microtiter wells (Immulon 2 Removawell strips, Dynex Technologies,
Inc.) for 48 h at 4 °C. After blocking with 3% BSA, purified
IIb3 was added and incubated for 4 h
at 37 °C. Following extensive washing, bound receptor was detected
with 125I-labeled mAb15 as described (18).
View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2.
SDS-PAGE analyses of purified talin and its
N-terminal recombinant fragment. Purified talin was
electrophoresed on 6.5% SDS-polyacrylamide gels and detected by
Coomassie Blue staining (lane 1). The production of the
recombinant talin N-terminal fragment was described under "Materials
and Methods." Lanes 2 and 3 represent the purified
fragment before and after calpain cleavage, respectively. Proteins were
resolved on 10% SDS-polyacrylamide gels and detected by Coomassie Blue
staining. Positions of molecular mass markers in kDa are indicated on
the left of the gel lanes.
|
|
For the binding of talin to synthetic peptides encompassing partial
sequences of the 3 cytoplasmic tail, the peptides (0.5 mM, 50 µl/well) were coated onto microtiter wells
(Immulon 2, Removawell strips) overnight at 22 °C. After blocking
the peptide-coated wells with 3% BSA, purified talin was added and
incubated for 4 h at 37 °C. Bound talin was detected with
125I-labeled 8d4, a monoclonal antibody directed against
the 190-kDa C-terminal tail domain of talin (31). To determine the
amounts of immobilized peptides, the peptide-coated wells were blocked with 1% gelatin and incubated overnight at 22 °C with 10 mM EZ-linkTM PEO-maleimide activated biotin
(Pierce), which reacts with the free sulfhydryl group of the N-terminal
cysteine residue of the peptides. After extensive washing, the amounts
of coupled biotin were quantitated with 0.1 µM
125I-labeled streptavidin.
In some experiments, talin binding to peptides with a biotinylated
N-terminal lysine residue (Research Genetics, Inc., Table I) was
examined. The biotinylated peptides (0.5 mM, 50 µl/well) were coupled onto Reacti-BindTM NeutrAvidin-coated
polystyrene microtiter wells (Pierce) at 37 °C for 2 h, and the
binding of soluble talin to the immobilized peptides was performed as
described above. Alternatively, talin (50 µg/ml, 50 µl/well) was
immobilized onto Immulon 2 microtiter wells overnight at 22 °C.
After blocking with 3% BSA, biotinylated peptides were allowed to bind
to the immobilized talin for 3 h at 37 °C. Bound peptides were
detected with 0.5 µM 125I-labeled streptavidin.
Generation of a Recombinant Protein Fragment Corresponding to the
47-kDa N-terminal Head Domain of Human Talin (rTalin-N)--
At the
time of our experiments, the human talin sequence was not available.
Thus, based on the mouse sequence (GenBankTM accession
number L46861), we performed 5'-RACE reaction to determine the
nucleotide sequence encoding the N terminus of human talin (39).
Briefly, total cellular RNA was extracted from human skin fibroblasts
(CCD 1064Sk, ATCC, Rockville, MD) using the RNeasyTM mini
kit (Qiagen) and reversed-transcribed using primer a,
5'-CACCTGGAAATTCTCAGGACCAGAGGC-3', derived from the mouse sequence.
5'-RACE was performed using the 5'/3'-RACE Kit (Mannheim Boehringer)
according to the manufacturer's instructions. The coding sequence of
the human talin N terminus was determined to be
5'-ATGGTTGCACTTTCACTGAAGATCAGCATTGGG-3', and a forward primer
b corresponding to this sequence was synthesized. To create
the rTalin-N fragment, PCR was performed using Pfu and the
oligonucleotide pair b and a with the
NdeI and XhoI restriction sites added to the
5'-ends of these primers, respectively. The rTalin-N fragment was
cloned into a pET-30-a(+) vector (Novagen) as a fusion protein with a
hexahistidine tag at its C terminus and transformed into BL21(DE3)
competent cells (Novagen). DNA sequencing revealed that the human talin
N-terminal domain nucleotide sequence was 91.6% identical to the mouse
sequence. As compared with the mouse talin protein (40), there are 6 changes out of 465 amino acid residues in this region: M31I, L40P,
N45S, D139E, G141I, and S352N (mouse to human). The expression of
rTalin-N histidine-tagged fusion protein was induced in transformed
bacteria with isopropylthio--D-galactoside, and the
histidine-tagged fusion protein was purified by chromatography on
Ni2+ resin (Novagen). The molecular mass of the purified
fusion protein was ~52 kDa, and it contained 32 amino acid residues
downstream of the calpain cleavage site plus the histidine tag (Fig. 2,
lane 2). To produce the 47-kDa rTalin-N fragment, the
isolated fusion protein was digested with m-calpain (Sigma), and the
released ~5-kDa fragment was removed by extensive dialysis against 20 mM Tris acetate, pH 7.6, 20 mM NaCl, 0.1 mM EDTA, 1 mM EGTA, 0.1% -mercaptoethanol
(Fig. 2, lane 3).
| |
RESULTS |
The NPLY Motif in the 3 Cytoplasmic Tail Is
Essential for IIb3-mediated Post-ligand
Binding Functions--
By using the Chou and Fasman and Gor II methods
to predict protein secondary structures, it has previously been
reported that the N744PLY sequence represents a potential
-turn motif in the cytoplasmic tail of the integrin 3
subunit (41). To examine the functional significance of this -turn
motif, we performed site-directed mutagenesis and replaced the NPLY
sequence with QALY which was predicted to alter the secondary structure
of this region. The N744Q/P745A 3 mutant construct was
co-transfected with a wild type IIb construct into
CHO-K1 cells, and stable clonal lines expressing the mutant receptor
were developed for functional studies. All experiments described below
were performed with at least three clonal cell lines yielding similar
results. For simplicity, only those obtained with the D4 cell line are
presented. Additionally, the control A5 cell line bearing wild type
IIb3 in CHO-K1 cells (37) was used for
comparison. In preliminary experiments, we determined the levels of
surface expression of the 3 integrins on A5 and D4 cells
by flow cytometry. Both A5 and D4 cells bound similar amounts of
FITC-conjugated AP-2, a complex-specific
anti-IIb3 monoclonal antibody (30),
indicating similar expression of IIb3 on
these cell lines (mean fluorescence intensity: A5, 78.0 ± 4.9; D4, 76.0 ± 5.0, mean ± S.D., n = 3). By
using these two cell lines, we examined the effect of mutation on
IIb3-mediated cell adhesion and
spreading, as well as focal adhesion formation. As shown in Fig.
3, both A5 and D4 cells bound to
fibrinogen-coated coverslips. In the presence of 1 mM
GRGDSP, adhesion of both cell types was inhibited by >80% (data not
shown). Examination of the morphology of the adherent cells revealed
that most of the adherent A5 cells became fully spread (Fig. 3A,
left) with IIb3 clustered into punctate focal adhesion plaques (Fig. 3B, left). In
contrast, D4 cells remained round (Fig. 3A, right) with a
diffused distribution of IIb3 (Fig.
3B, right).
View larger version (121K):
[in this window]
[in a new window]
|
Fig. 3.
The ability of adherent A5 and D4 cells to
spread and form focal contacts. Cells were allowed to adhere to
fibrinogen-coated coverslips for 3 h at 37 °C and fixed with
2% paraformaldehyde. A, for the determination of cell
spreading, fixed cells were photographed by phase contrast microscopy.
B, for immunofluorescent staining of focal contacts, fixed
cells were permeabilized with 0.5% Triton X-100 and immunostained with
mAb15 followed by FITC-conjugated goat anti-mouse IgG. Figure is
representative of three experiments.
|
|
Cell adhesion through integrins initiates signaling events such as
protein tyrosine phosphorylation, and this process is thought to be
dependent on the interaction between integrins and the cytoskeleton (42). Therefore, we examined whether the NPLY sequence in
3 integrins modulates protein tyrosine phosphorylation.
In these experiments, A5 or D4 cells were allowed to adhere to
fibrinogen- or BSA-coated tissue culture plates. Non-adherent and
adherent cells were recovered, lysed, and subjected to immunoblotting
with PY20, an anti-phosphotyrosine antibody. As shown in Fig.
4A, similar basal levels of
protein tyrosine phosphorylation were observed in non-adherent A5
(lane 1) and D4 cells (lane 3) recovered from BSA-coated plates. Following adhesion to fibrinogen, A5 cells spread
and demonstrated an increase in tyrosine phosphorylation of protein
bands at 120-130 and 70-90 kDa (lane 2). In contrast, adherent D4 cells remained round and did not exhibit an increase in
protein tyrosine phosphorylation (lane 4). To confirm that the observed difference in protein tyrosine phosphorylation in adherent
A5 and D4 cells was not due to variations in protein concentrations
among samples, immunoblotting with 125I-labeled mAb15
directed against 3 was performed. Fig. 4B
shows that similar amounts of 3 integrins were present
in all samples.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of N744Q/P745A mutation of the
integrin 3 cytoplasmic tail
on IIb3-mediated
protein tyrosine phosphorylation. A, harvested A5 and
D4 cells were incubated for 90 min at 37 °C on BSA- or
fibrinogen-coated plates. Lysates of cells not adherent to BSA
(lanes 1 and 3) or adherent to fibrinogen (lanes 2 and
4) were subjected to SDS-PAGE on 7% polyacrylamide gels under
reducing conditions. Protein tyrosine phosphorylation was analyzed by
immunoblotting with PY20 and detected by enhanced chemiluminescence.
B, to determine the amounts of 3 integrins in
the samples of A, identical samples were subjected to
SDS-PAGE under non-reducing conditions and immunoblotted with
125I-labeled mAb15. C, FAK in the cell lysates
described in A was isolated by immunoprecipitation with an
anti-FAK mAb, subjected to SDS-PAGE under reducing conditions, and
immunoblotted with PY20. D, the amounts of
immunoprecipitated FAK were analyzed by immunoblotting with an anti-FAK
monoclonal antibody. Figure is representative of two experiments.
|
|
Since focal adhesion kinase (FAK or pp125FAK) becomes
tyrosine-phosphorylated following
IIb3-mediated platelet aggregation and A5
cell adhesion to fibrinogen (10, 23), we determined the phosphorylation
state of FAK in adherent D4 cells. In these experiments, FAK in cell
lysates was isolated by immunoprecipitation and subjected to
immunoblotting with PY20. As shown in Fig. 4C, FAK in
adherent A5 cells (lane 2), but not D4 cells (lane
4), was phosphorylated on tyrosine residues. As controls, parallel samples were subjected to immunoblotting with an anti-FAK monoclonal antibody, confirming that similar amounts of FAK were being
immunoprecipitated from adherent A5 and D4 cells (Fig. 4D).
Collectively, these results indicate that the structural
integrity of the NPLY motif in the 3 cytoplasmic tail is
essential for mediating cell spreading, focal adhesion formation, and
protein tyrosine phosphorylation.
The N744Q/P745A Mutation Does Not Affect the Binding of
IIb3 to Immobilized Talin--
By using
a solid phase binding assay, we previously demonstrated direct binding
of RGD affinity purified IIb3 to
immobilized talin (18). Since the WDTGENPIYK peptide derived from the
1 cytoplasmic tail containing the NPXY motif
has been shown to block talin binding to the avian integrin complex
(27), we examined the effect of the N744Q/P745A mutation on the
interaction of IIb3 with talin. In these
studies, IIb3 was purified from lysates of A5 and D4 cells by RGD affinity chromatography and allowed to bind
to immobilized talin for 4 h at 37 °C. Following washing, bound
receptor was detected with 125I-labeled mAb15. Fig.
5A (top) shows that
similar amounts of wild type and mutant
IIb3 bound to talin-coated wells but not
to control wells coated with BSA. As a control, we examined the binding of IIb3 with truncations of both
IIb (
991) and 3 (728) cytoplasmic sequences (43). As expected, the
IIb991/3728 truncation mutant
failed to bind to immobilized talin. To ascertain that equal
concentrations of IIb3 were added to the
microtiter wells, the samples were subjected to immunoblotting with
125I-labeled mAb15 and found to contain similar amounts of
integrin 3 subunit (Fig. 5A,
bottom). To examine further whether the N744Q/P745A mutation
affects the affinity of IIb3-talin
interaction, we performed binding isotherms with varying concentrations
of IIb3. As shown in Fig. 5B,
at all input concentrations of the receptor, similar extents of binding
of wild type and mutant IIb3 to
immobilized talin were observed. Thus, these results demonstrated that
the cytoplasmic domain of recombinant
IIb3 mediates its interaction with talin;
however, the N744Q/P745A mutation of the 3 cytoplasmic tail has no effect on this process.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Binding of purified recombinant IIb3
to immobilized talin. A, integrin
IIb3 was purified from A5, D4, and
IIb991/3728 cells by RGD affinity
chromatography. In the top panel, purified
IIb3 was added to microtiter wells coated
with talin (200 µg/ml, 50 µl/well) or BSA, and binding proceeded
for 4 h at 37 °C. Bound receptor was detected with
125I-mAb15 (50 nM). Data shown represent means
of triplicate determinations from two experiments, and error
bars represent standard deviations. In the bottom panel, the amounts of integrin 3 subunit in
the samples were analyzed by immunoblotting with
125I-labeled mAb15. B, varying concentrations of
wild type and mutant IIb3, isolated from
A5 and D4 cells, respectively, were added to microtiter wells coated
with talin or BSA. Binding proceeded as described above. Results are
means of triplicate determinations of one experiment, and error
bars represent standard deviations.
|
|
Talin Binds to the Membrane-proximal Region of the 3
Cytoplasmic Tails--
To further investigate the role of the NPLY
sequence in talin binding, we examined whether talin binds to synthetic
peptides encompassing partial sequences of the 3
cytoplasmic tail. In the integrin 1 cytoplasmic
sequence, three distinct regions, namely cyto-1, cyto-2, and cyto-3,
have been implicated in mediating the localization of 1
integrins in focal adhesions (24). In the present study,
Cys-3-(722-738) and Cys-3-(737-748)
peptides with partial 3 cytoplasmic sequences containing
the corresponding cyto-1 and cyto-2 regions, respectively, were
synthesized and coated onto microtiter wells (Fig. 1). Since these
peptides contain a free sulfhydryl group in their N-terminal cysteine
residue, their coating efficiencies were estimated by the binding of
PEO-maleimide-activated biotin, followed by detection with
125I-labeled streptavidin. As shown in Table
I, similar amounts of both peptides were
coated onto microtiter wells. To examine the binding of talin to the
immobilized peptides, purified talin was incubated with peptide-coated
wells at 37 °C for 4 h, followed by detection with
125I-labeled 8d4, an anti-talin monoclonal antibody. Fig.
6 shows that talin bound in a
dose-dependent manner to
Cys-3-(722-738)-coated wells but not to wells coated
with Cys-3-(737-748) or BSA. Since Cys-3-(737-748) contains the N744PLY
sequence, these results indicate that this motif is by itself not
sufficient in mediating talin binding to 3 integrins. To demonstrate the specificity of talin binding to
Cys-3-(722-738), we used a scrambled
Cys-3-(722-738) peptide that was found to be much less
effective in supporting talin binding at the highest input talin
concentration of 200 µg/ml (Fig. 6, open square).
View this table:
[in this window]
[in a new window]
|
Table I
Amino acid sequences of synthetic peptides containing partial sequences
of the integrin 3 cytoplasmic tail
|
|
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6.
Binding of talin to immobilized peptides
containing partial sequences of the integrin 3 cytoplasmic tail. Microtiter
wells were coated with the indicated peptides (0.5 mM, 50 µl/well), and blocked with 3% BSA; the amounts of peptides coated
onto the wells were shown in Table I. Varying concentrations of
purified talin were added and binding proceeded for 4 h at
37 °C. After washing, bound talin was detected with
125I-labeled 8d4 (50 nM). Data shown are means
of triplicate determinations, and error bars represent
standard deviations. Figure is representative of three
experiments.
|
|
To eliminate the possibility that talin failed to bind to the
Cys-3-(737-748) peptide was due to obstruction of the
NPLY motif resulting from peptide immobilization, we examined talin binding to biotinylated peptides that have been coupled to
NeutrAvidin-coated microtiter wells (Table I). In control experiments,
successful coupling of the biotin-Lys-3-(737-748)
peptide to NeutrAvidin-coated wells was monitored by an enzyme-linked
immunosorbent assay (ELISA) using an anti-peptide polyclonal antibody
raised against Cys-3-(737-748) (data not shown). Also,
since the biotin group was conjugated onto the N-terminal lysine
residue of the peptide, this would allow the C-terminal NPLY motif to
be protruded from the NeutrAvidin-coated wells. Consistent with the
above observations, talin bound efficiently and
dose-dependently to wells coated with
biotin-Lys-3-(716-738) but poorly to wells coated with
biotin-Lys-3-(737-748) (Fig. 7A). Conversely, using
125I-labeled streptavidin to detect the binding of
biotinylated peptides to immobilized talin, we found that
biotin-Lys-3-(716-738), but not
biotin-Lys-3-(737-748), bound in a
dose-dependent manner to talin-coated wells (Fig.
7B). As expected, neither peptide bound to control wells
coated with BSA. Together, these findings indicate that talin interacts
with the 3 cytoplasmic tail at its membrane-proximal
region which does not contain the NPLY regulatory motif of post-ligand
binding functions.
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Interaction of talin with biotinylated
peptides containing partial sequences of the integrin 3 cytoplasmic tail. A,
biotin-Lys-3-(716-738) and
biotin-Lys-3-(737-748) peptides were coupled to
NeutrAvidin-coated microtiter wells as described under "Materials and
Methods." Varying concentrations of talin were added to wells
conjugated with the indicated peptide and binding proceeded for 4 h at 37 °C. Bound talin was detected with 125I-labeled
8d4 (50 nM). Data shown are means of triplicate
determinations, and error bars represent standard
deviations. This is representative of two experiments. B,
varying concentrations of the biotinylated peptides were added to
microtiter wells coated with talin or BSA and incubated for 3 h at
37 °C. Bound peptides were detected with 125I-labeled
streptavidin (0.5 µM). Results shown are means of
triplicate determinations, and error bars represent standard
deviations.
|
|
The 47-kDa N-terminal Head Domain of Talin Contains a Binding Site
for IIb3--
The 47-kDa N-terminal head
domain of talin contains a homologous domain with members of the
protein 4.1 superfamily (40, 44). Ezrin/Radixin/Moesin (ERM) in this
protein family have been shown to bind to basic amino acid clusters in
the juxtamembrane cytoplasmic domains of membrane proteins (45). Thus,
we postulated that talin may bind through its 47-kDa head domain to the
3-(722-738) sequence. To test this possibility, we
examined the inhibitory effect of a recombinant 47-kDa fragment of
human talin N-terminal domain (rTalin-N) on the binding of intact talin
to immobilized Cys-3-(722-738) peptide. Fig.
8A shows that preincubation of Cys-3-(722-738)-coated wells with 15 µg/ml (0.3 µM) rTalin-N completely blocked talin binding to the
peptide, suggesting that the interaction is mediated through the head
domain of talin. To ascertain that the rTalin-N fragment binds directly
to the Cys-3-(722-738) peptide, we performed an ELISA
using the His-tagged rTalin-N fusion protein and a detecting monoclonal
antibody directed against the hexahistidine tag. As shown in Fig.
8B, the recombinant talin head fusion protein bound directly
to wells coated with Cys-3-(722-738), whereas minimal
binding to control wells coated with BSA was observed.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 8.
Interaction of rTalin-N with immobilized 3-(722-738). A,
inhibition of talin binding to Cys-3-(722-738) by
rTalin-N. Microtiter wells coated with Cys-3-(722-738)
or BSA were preincubated with or without rTalin-N (15 µg/ml) for
2 h at 37 °C as indicated. After removing rTalin-N, purified
intact talin (200 µg/ml) or its carrier buffer was added and binding
proceeded as described in the legend of Fig. 6. B, binding
of His-tagged rTalin-N to Cys-3-(722-738). The fusion
protein (5 µg/ml) was added to the peptide-coated wells and incubated
for 2 h at 37 °C. After washing, bound protein was detected by
an ELISA using the 6× His monoclonal antibody, followed by a goat
anti-mouse IgG conjugated to horseradish peroxidase. Data shown are
means of triplicate determinations, and error bars represent
standard deviations.
|
|
To examine further the role of the talin N-terminal head domain in
binding to integrin, we performed direct binding of purified platelet
IIb3 to immobilized rTalin-N as described
(18). Fig. 9 shows that
IIb3 bound saturably to wells coated with
rTalin-N but not to control wells coated with BSA. Moreover,
half-saturation binding was observed at approximately 12 nM
IIb3, which is similar to that observed
for IIb3 binding to intact talin (18).
Thus, these results indicated that the talin head domain contains a binding site for integrin IIb3 and
interacts with the membrane-proximal region of the 3
cytoplasmic tail.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 9.
Binding of purified platelet IIb3
to rTalin-N. Varying concentrations of purified platelet
IIb3 were added to microtiter wells
coated with rTalin-N (50 µg/ml, 50 µl/well) or BSA. Incubation
proceeded for 3 h at 37 °C, and bound receptor was detected
with 125I-mAb15. Data shown are means of triplicate
determinations, and error bars represent standard
deviations. Figure is representative of two experiments.
|
|
| |
DISCUSSION |
Fibrinogen binding to integrin IIb3
on activated platelets results in platelet aggregation which is
accompanied by a series of post-ligand binding events dependent on the
interaction between IIb3 and the platelet
cytoskeleton. In the present study, we investigated the functional role
of the putative NPLY -turn motif in the 3 cytoplasmic
domain by site-directed mutagenesis. The major findings of these
studies are as follows: 1) the N744Q/P745A double mutation in
3 does not affect cell attachment to fibrinogen but
blocks post-ligand binding functions of
IIb3 including cell spreading, focal
contact formation, and protein tyrosine phosphorylation; 2) the NPLY
sequence in the integrin 3 cytoplasmic tail is not by
itself sufficient to mediate the interaction between
IIb3 and talin; and 3) talin binds
through its N-terminal head domain to the membrane-proximal region of
the 3 cytoplasmic sequence. Collectively, these findings
indicate that the NPLY motif mediates post-ligand binding functions of
IIb3 in a manner independent of
IIb3-talin interaction.
The NPXY motif is highly conserved among the cytoplasmic
sequences of integrin subunits and is present in several
non-integrin receptors (46). Previous mutational studies on the
N744PLY sequence in 3 integrins focused on
the tyrosine residue since its phosphorylation state may regulate
receptor interaction with cytoskeletal proteins and/or signaling
molecules (19, 20). However, the secondary structure of this sequence
may also be important in mediating protein-protein interaction(s). In
this regard, NMR analysis of synthetic peptides encompassing the NPVY internalization sequence of the LDL receptor confirmed its predicted secondary structure as a type I -turn (47). Moreover, asparagine and
proline are strong -turn promoters and are frequently found in the
first two positions of type I -turns in proteins (48). Similarly,
the aromatic side chain of the tyrosine residue has been suggested to
contribute to the overall structural stability of the -turn motif
(47). To address the structural importance of the N744PLY
sequence in the integrin 3 subunit without modifying the tyrosine residue, we mutated asparagine to glutamine and proline to
alanine. These amino acid substitutions were predicted to alter the
type I -turn into an -helical structure.
It has been shown that replacement of Asn744 or
Tyr747, but not Pro745, with alanine in the
NPLY sequence of the integrin 3 subunit resulted in a
complete loss of v3-mediated cell
adhesion to vitronectin (14). However, comparable adhesion of A5 and D4 cells bearing wild type and mutant IIb3
to fibrinogen was observed, indicating that the N744Q/P745A mutation
does not affect extracellular ligand binding activities of the
receptor. One possible explanation of the difference in cell adhesion
between our N744Q/P745A mutant and the N744A mutant in the earlier
study is that glutamine for asparagine is a much more conservative
substitution than alanine for asparagine. Thus, N744Q, in conjunction
with the permissible P745A substitution, has no effect on cell
attachment to fibrinogen mediated by the mutant
IIb3. Nonetheless, in agreement with earlier studies that demonstrated that Y747A and N744A single mutations
in 3 abolished cell spreading (14, 25), we found that
adherent D4 cells remained round and were not able to induce focal
contact formation. Interestingly, point mutations of the corresponding
-turn motif in the integrin 1 cytoplasmic tail had
similar inhibitory effects on cell spreading and focal adhesion (24).
These results suggest a role for the conserved NPXY -turn motif in mediating interaction(s) with cytoskeletal proteins and/or signaling molecules necessary for these cellular processes.
A major outside-in signaling event following cell adhesion through
integrins is protein tyrosine phosphorylation. In this regard, it has
been shown that a number of proteins including FAK becomes
tyrosine-phosphorylated in aggregated platelets, and this process is
blocked by cytochalasins indicating an involvement of
integrin-cytoskeleton interaction (10). By using single subunit chimeric receptors containing the interleukin-2 receptor extracellular and transmembrane domains connected to mutated 3
cytoplasmic sequences, Tahiliani et al. (22) reported that
the NPLY motif is essential for the chimeric receptor to induce FAK
phosphorylation. Our present finding that N744Q/P745A mutation in the
native integrin 3 subunit abolished
IIb3-dependent FAK
phosphorylation in D4 cells adherent to fibrinogen is in agreement with
these results. Furthermore, tyrosine phosphorylation of other proteins
with molecular masses of 70-90 kDa was similarly affected, suggesting
that the NPLY motif in 3 is crucial for this
integrin-mediated signaling event.
Talin and -actinin have been suggested to serve as linkage proteins
between integrins and actin filaments (17, 49). Mutational studies have
identified three amino acid clusters (i.e. cyto-1, cyto-2,
and cyto-3, see Fig. 1) in the 1 integrin cytoplasmic sequence that are important for integrin localization to focal adhesions (24). An -actinin-binding site has been mapped to the
cyto-1 region (50). The NPIY motif at cyto-2 is generally believed to
mediate talin binding since a synthetic peptide encompassing this
sequence was found to inhibit talin interaction with the avian integrin
complex (27); however, it is unclear whether this inhibitory effect was
due to a direct interaction between talin and the 1
peptide. In a more recent study, talin has been shown to bind to
affinity matrices containing 1A or 1D
cytoplasmic sequences, and the binding was abolished by a Y788A
substitution at the NPIY788 motif of the 1A
sequence (28). Although these findings suggest that the NPXY
motif plays a regulatory role in talin binding to the 1
peptides, whether talin interacts directly with this sequence in
integrin receptors has not been conclusively demonstrated. In fact, it
has been reported that talin bound to antibody-captured integrin
51 with either Y788S or Y800S point
mutation in the 1A cytoplasmic tail, suggesting that
these motifs do not constitute the talin-binding site of the
51 integrin (51). Consistent with the
latter mutational study, we found that both wild type and
N744Q/P745A-mutated IIb3 bound equally
well to immobilized talin in a solid phase binding assay. Thus, in
intact integrin receptors, the NPXY structural motif is not
essential in mediating talin binding.
In accord with the results of the mutational studies, we found that
talin failed to bind to synthetic peptides encompassing the
3-(737-748) sequence that contains the NPLY -turn
motif. In contrast, specific interaction of talin with peptides
containing the 3-(722-738) membrane-proximal sequence
of the 3 cytoplasmic tail was observed. These findings
indicate that a talin-binding site resides in the cyto-1 rather than
the cyto-2 region of the 3 cytoplasmic domain.
Similarly, we found that talin also binds to a synthetic peptide
containing the cyto-1 region of the 1 cytoplasmic
domain.2 Thus, these results
suggest that the conserved cyto-1 sequence in different integrin subunits contains a common recognition site for this cytoskeletal protein.
Talin is a single chain polypeptide consisting of a 47-kDa N-terminal
globular head domain and a 190-kDa C-terminal tail domain that can be
separated be proteolytic cleavage with calpain (38). Whereas vinculin-
and actin-binding sites have been localized to the talin C-terminal
tail domain (52-54), the integrin recognition site(s) in talin has not
been identified. Sequence analyses of the primary structures of a
number of membrane-cytoskeleton linkers including talin, band 4.1, Ezrin, Radixin, and Moesin reveal the presence of a homologous FERM
domain near the N termini of these proteins (40, 44). Our findings that
a recombinant fragment of the talin head domain bound to the
Cys-3-(722-738) peptide and blocked talin binding to
this sequence are consistent with the suggestion that the FERM domain
provides an attachment site to transmembrane proteins. In addition, we
found that purified IIb3 bound saturably
to the recombinant talin head fragment, reinforcing the notion that an
integrin-binding site in talin resides within its N-terminal head domain.
Recently, Ezrin, Radixin, and Moesin have been shown to bind to
clusters of three basic amino acid residues in the juxtamembrane cytoplasmic domains of CD44, CD43, and ICAM-2 (45). It is interesting to note that basic amino acid residues are also found in the
juxtamembrane regions of most integrin and cytoplasmic tails.
Furthermore, it has been shown that the cytoplasmic sequences of
IIb and 3 interact with each other to
form defined tertiary structures (41, 55, 56). Thus, it is conceivable
that such interaction results in the formation of basic amino acid
clusters to which the FERM domain of talin interacts. It has previously
been reported that both in vitro and in vivo
talin interaction with integrin 51 is
dependent on ligand occupancy of the receptor (51, 57), suggesting that
the talin-binding site in the cytoplasmic domains of integrins is
dynamically regulated. In this regard, we recently showed that
extracellular ligand binding induces a transmembrane conformational
change in integrin IIb3 (58). It is
therefore an intriguing possibility that such conformational change in
the receptor cytoplasmic domain may expose cryptic basic amino acid clusters in its membrane-proximal region, thereby modulating the affinity of integrin-talin interaction. Thus, whether the juxtamembrane basic amino acid residues in integrin cytoplasmic domains play a role
in talin binding merits further investigation.
The observation that mutations of the NPLY motif in the integrin
3 subunit block post-ligand binding functions but not
talin binding suggests that IIb3-talin
interaction is not sufficient for inducing outside-in signaling of
IIb3. At present, the mechanisms by which
the NPLY motif regulates post-ligand binding functions of
IIb3 remain elusive. Recently, it has
been shown that the 3 cytoplasmic tail becomes
phosphorylated on tyrosine residues upon ligand binding and cell
aggregation, and several cytoskeletal proteins (e.g. myosin)
and signaling molecules (e.g. GRB2 and SHC) interact with
the NXXY motifs in a phosphorylation-dependent manner (19, 20). Thus, the interaction of these proteins, rather than
talin, with the phosphorylated NPLY motif in the 3 cytoplasmic tail may be important for inducing
IIb3-dependent post-ligand
binding events.
| |
ACKNOWLEDGEMENTS |
We thank Drs. M.H. Ginsberg, T. J. Kunicki, and T. E. O'Toole for providing the monoclonal
antibodies and CHO cell lines expressing wild type and truncated
IIb3. We also thank Dr. J. C. Loftus for
his assistance in producing the N744Q/P745A mutant 3 construct.
| |
Note Added in Proof |
In this issue of The Journal of
Biological Chemistry, a Communication by Dr. M. H. Ginsberg and
his associates (Calderwood, D. A., Zent, R., Grant, R., Rees, D. J. G.,
Hynes, R. O., and Ginsberg, M. H. (1999) J. Biol. Chem.
274, 28071-28074) reports direct interaction of the
N-terminal head domain of talin with integrin cytoplasmic tails,
resulting in integrin activation.
| |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants HL-41793 (to S. C.-T. L.) and HL-52755 (to
J. D. W.-D.).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.
§
Both authors made equal contributions.
Supported by an Established Investigator Award from the
American Heart Association and Genentech. To whom correspondence should be addressed: Dept. of Pharmacology (M/C 868), University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612. Tel.:
312-413-5928; Fax: 312-996-1225; E-mail: sclam@uic.edu.
2
S. Patil, A. Jedsadayanmata, J. D. Wencel-Drake, W. Wang, I. Knezevic, and S. C.-T. Lam, unpublished observations.
| |
ABBREVIATIONS |
The abbreviations used are:
CHO cells, Chinese
hamster ovary cells;
mAb, monoclonal antibody;
FITC, fluorescein
isothiocyanate;
PCR, polymerase chain reaction;
DMEM, Dulbecco's
TOP
ABSTRACT
INTRODUCTION
