Integrin cytoplasmic domains connect these
receptors to the cytoskeleton. Furthermore, integrin-cytoskeletal
interactions involve ligand binding (occupancy) to the integrin
extracellular domain and clustering of the integrin. To construct
mimics of the cytoplasmic face of an occupied and clustered integrin,
we fused the cytoplasmic domains of integrin
subunits to an
N-terminal sequence containing four heptad repeat sequences. The heptad
repeats form coiled coil dimers in which the cytoplasmic domains are
parallel dimerized and held in an appropriate vertical stagger. In
these mimics we found 1) that both conformation and protein binding properties are altered by insertion of Gly spacers C-terminal to the
heptad repeat sequences; 2) that the cytoskeletal proteins talin and
filamin are among the polypeptides that bind to the integrin
1A
tail. Filamin, but not talin binding, is enhanced by the insertion of
Gly spacers; 3) binding of both cytoskeletal proteins to
1A is
direct and specific, since it occurs with purified talin and filamin
and is inhibited in a point mutant (
1A(Y788A)) or in splice variants
(
1B,
1C) known to disrupt cytoskeletal associations of
1
integrins; 4) that the muscle-specific splice variant,
1D, binds
talin more tightly than
1A and is therefore predicted to form more
stable cytoskeletal associations; and 5) that the
7 cytoplasmic
domain binds filamin better than
1A. The structural specificity of
these associations suggests that these mimics offer a useful approach
for the analysis of the interactions and structure of the integrin
cytoplasmic face.
 |
INTRODUCTION |
Integrins, a large family of heterodimeric adhesion receptors,
physically link the extracellular matrix to cytoskeletal elements within a cell (1-3). The structure of integrin cytoplasmic domains plays a key role in these linkages (4-6). Thus, deletion or mutations in the cytoplasmic domain block the co-localization of integrins with
cytoskeletal elements such as vinculin and talin (7, 8). Furthermore,
isolated
cytoplasmic domains, joined to other transmembrane proteins, target them to focal adhesions, sites of
integrin-cytoskeleton linkages (9, 10). Thus, integrin
cytoplasmic
domains link these receptors to the cytoskeleton.
Integrin
cytoplasmic domains limit certain cytoskeletal
interactions of their
partners. In intact integrins, focal adhesion targeting usually requires binding of a ligand to the extracellular domain (10), a conformational change in the extracellular domain of the
integrin (11) and an intact
tail (8). Ligand-independent focal
adhesion targeting is induced by deletion of the
cytoplasmic domain
(8, 12), suggesting that the
tail blocks cytoskeletal interaction
with the
tail. Ligand binding appears to remove this block,
permitting the
tail to target to focal adhesions. Consequently,
isolated
cytoplasmic domains resemble ligand-occupied integrins in
their interactions with the cytoskeleton.
The majority of integrin ligands are multivalent and cause receptor
clustering after binding (13). Simple antibody-mediated clustering can
initiate biochemical signals (14) and local accumulation of tensin and
pp125FAK near the clustered integrins (15). Nevertheless,
accumulation of the full complement of cytoskeletal proteins (15) and
functional cytoskeletal interaction (16, 17) requires ligand occupancy. Consequently, cytoskeletal interactions of integrins involve both receptor clustering and ligand occupancy.
We previously proposed a strategy for the chemical synthesis of
structural models of the cytoplasmic domain of multisubunit transmembrane receptors (18). The cytoplasmic domains of integrin
IIb
3 were covalently linked via a helical coiled coil. In the present study we have extended and tested this approach by preparation of recombinant mimics of the cytoplasmic face of occupied and clustered
integrins. By using recombinant proteins, we avoided limitations of
polypeptide length and modest yield encountered in the initial
synthetic approaches. Occupancy was mimicked by use of isolated
cytoplasmic domains; clustering was mimicked by use of covalent
homodimers of these domains. Helical coiled coil architecture provided
the desired parallel topology and vertical stagger of the tails. We now
report that the binding of cytoplasmic proteins to mimics of the
1A
tail is sensitive to conformational changes induced by the insertion of
Gly residues C-terminal to the helical coiled coil moieties. The
cytoskeletal proteins talin and filamin are among the polypeptides that
bind to the
1A tail. Filamin, but not talin binding, is enhanced by
the insertion of a Gly spacer. Binding of both proteins is direct and
specific, since it occurs with the purified proteins and is inhibited
in a point mutant (
1A(Y788A)) or in splice variants (
1B (19) and
1C (20)) known to disrupt cytoskeletal associations of
1
integrins (21-23). The muscle-specific splice variant
1D (24-26) binds talin better than
1A, and the
7 cytoplasmic domain binds filamin more tightly than
1A. Thus, these constructs can be used to
analyze integrin class and splice variant-specific interactions with
cytoskeletal proteins.
 |
MATERIALS AND METHODS |
Antibodies and cDNAs--
Antibodies for immunoblot analysis
were either obtained commercially (goat serum against filamin (Sigma),
rabbit serum against
-actinin (Sigma), monoclonal antibodies against
talin (clone 8d4) (Sigma), vinculin (clone hVIN-1) (Sigma), paxillin
(clone Z035) (Zymed), filamin (MAB1680) (Chemicon),
-actinin
(BM75.2) (Sigma), actin (clone C4) (Boehringer Mannheim)) or kindly
provided by Drs. J. Brugge (monoclonal antibodies against
pp60src (clone 327)) and T. Parsons (polyclonal rabbit serum
against pp125FAK (BC3). A rabbit antiserum against
pp72syk was obtained by immunization with the peptide
ESDRGPWANEREAQRE (amide).
A human
1C cDNA was the gift of Dr. J. Meredith (The Scripps
Research Institute, La Jolla) (21).
1A cDNA with the point mutation, Y788A, was kindly provided by Dr. A. F. Horwitz
(University of Illinois, Champaign, Il) (22). A cDNA for the
cytoplasmic domain of human integrin
1D (24) obtained by reverse
transcription-PCR1 of heart
muscle total RNA (kindly provided by Dr. R. Ross (University of
California, Los Angeles, CA)) was provided by Dr. J. Loftus (The
Scripps Research Institute, La Jolla, CA). The cDNA of human integrin
7 has been described (27). A cDNA coding for the human
1B subunit cytoplasmic domain was synthesized in PCR reactions using
a human
1A vector with a partially overlapping reverse oligonucleotide containing the published human
1B sequence (19).
Recombinant Cytoplasmic Domain Models--
Based on an initial
synthetic approach for the synthesis of models of the cytoplasmic
domains of integrins (18), oligonucleotides were synthesized and used
in PCR to create a cDNA encoding the four-heptad repeat protein
sequence, KLEALEGRLDALEGKLEALEGKLDALEG (G1-([heptad]4).
Variants containing 1-3 additional Gly residues (G2-4-([heptad]4) at the C terminus were synthesized by
modification of the antisense oligonucleotide. These cDNAs were
ligated into an NdeI-HindIII-restricted modified
pET15b vector (Novagen). Integrin cytoplasmic domains were joined to
the helix as HindIII-BamHI fragments. The final
constructs coded for the N-terminal sequence GSSHHHHHHSSGLVPRGSHMCG[heptad]4 linked to the cytoplasmic
domains of integrins. Different integrin cytoplasmic domain cDNAs
were cloned via PCR from appropriate cDNAs using forward
oligonucleotides, introducing a 5' HindIII site, and reverse
oligonucleotide, creating a 3' BamHI site directly after the
stop codon. PCR products were first ligated into the pCRTM
vector using the TA cloning® kit (Invitrogen). After
sequencing, HindIII/BamHI inserts were ligated
into the modified pET15b vector. Recombinant expression in
BL21(DE3)pLysS cells (Novagen) and purification of the recombinant products were performed according to the pET system manual (Novagen) with an additional final purification step on a reverse phase C18 high
performance liquid chromatography column (Vydac). Products were
analyzed by electrospray ionization mass spectrometry on an API-III
quadrupole spectrometer (Sciex; Toronto, Ontario, Canada).
Ultraviolet Circular Dichroism Spectroscopy--
Far UV CD
spectra were recorded on an AVIV 60DS spectropolarimeter with peptides
dissolved in 50 mM boric acid, pH 7.0. Data were corrected
for the spectrum obtained with buffer only and related to protein
concentrations determined from identical samples by quantitative amino
acid analysis. From these values, the predicted percentage of helical
secondary structure was calculated as described (18).
Cells and Cell Lysates--
Human platelets were obtained by
centrifugation of freshly drawn blood samples at 1000 rpm for 20 min
and sedimentation of the resulting platelet-rich plasma at 2600 rpm for
15 min. They were washed twice with 0.12 M NaCl, 0.0129 M trisodium citrate, 0.03 M glucose, pH 6.5, and once in Hepes-saline (3.8 mM Hepes, 137 mM
NaCl, 2.7 mM KCl, 5.6 mM D-glucose,
3.3 mM Na2HPO4, pH 7.3-7.4). Human
Jurkat and HT1080 cells and mouse C2C12 cells were obtained from the
American Type Culture Collection (Rockville, MD) and cultured in
RPMI1680 (Jurkat) or Dulbecco's modified Eagle's medium with 10%
fetal calf serum. For differentiation to myotubes, C2C12 myoblasts were
kept confluent in Dulbecco's modified Eagle's medium with 5% horse
serum for 6 days. Cultured cells were washed twice in
phosphate-buffered saline and biotinylated with 1 mM N-hydroxysuccinimidobiotin-biotin (Pierce) in
phosphate-buffered saline for 30 min at room temperature. Platelet
proteins were biotinylated in Hepes-saline. After two additional washes
with Tris-buffered saline, cells were lysed on ice with buffer A (1 mM Na3VO4, 50 mM NaF,
40 mM sodium pyrophosphate, 10 mM Pipes, 50 mM NaCl, 150 mM sucrose, pH 6.8) containing 1%
Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA and
protease inhibitors (20 µg/ml) aprotinin, 5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. In addition, 0.1 mM E-64 (Boehringer Mannheim), a calpain inhibitor, was in
the platelet lysate. Lysates were sonicated 5 times on ice for 10 s at a setting of 3 using an Astrason Ultrasonic Processor (Heart
Systems; Farmingdale, NY). After 30 min, lysates were clarified
by centrifugation at 12,000 g for 30 min.
Affinity Chromatography Experiments with Integrin Cytoplasmic
Domain Mimics--
500 µg of purified recombinant cytoplasmic domain
proteins were dissolved in a mixture of 5 ml of 20 mM
Pipes, 50 mM NaCl, pH 6.8, and 1 ml of 0.1 M
sodium acetate, pH 3.5, and bound overnight to 80 µl of
Ni2+-saturated His-bind resin (Novagen). In control
experiments (data not shown) we found that these conditions resulted in
saturation of the resin with recombinant protein. Resins were washed
twice with 20 mM Pipes, 50 mM NaCl, pH 6.8, and
stored at 4 °C in an equal volume of this Pipes buffer containing
0.1% NaN3 as a preservative. 50 µl of such a suspension
was added to 4.5 ml of cell lysates, which had been diluted 10-fold
with buffer A containing 0.05% Triton X-100, 3 mM
MgCl2, and protease-inhibitors. After incubation overnight
at 4 °C, resins were washed five times with this buffer and finally
heated in 50 µl of reducing sample buffer for SDS-PAGE. Samples were
separated on 4-20% SDS-polyacrylamide gels (NOVEX) and either stained
with Coomassie Blue or transferred to Immobilon P membranes
(Millipore). Membranes were blocked with Tris-buffered saline, 5%
nonfat milk powder and stained with streptavidin peroxidase or specific
antibodies. Bound peroxidase was detected with an enhanced
chemiluminescence kit (Amersham Corp.). Equal loading of
Ni2+ resins with recombinant proteins was verified by
Coomassie Blue staining of SDS-PAGE profiles of proteins eluted from
the resins with SDS sample buffer.
Binding to Purified Talin and Filamin--
Human uterus filamin
was kindly provided by Dr. John Hartwig (Massachusetts General
Hospital, Boston, MA) as a 1.5 mg/ml solution in 0.6 M KCl,
0.5 mM ATP, 0.5 mM dithiothreitol, 10 mM imidazole, pH 7.5. For binding assays performed as
described above, this solution was diluted 1/12 with buffer A, 0.05%
Triton X-100, 3 mM MgCl2, 2 mg/ml bovine serum
albumin, and protease inhibitors (see above) (omitting the 50 mM NaCl (see above)), and resins with bound model proteins
were added. Washing was performed in this buffer without bovine serum
albumin and with additional 50 mM KCl.
Talin was provided by Dr. Keith Burridge (University of North
Carolina) and was purified from human platelets essentially as
described in (28) with an additional purification step using chromatography on phosphocellulose and stored at 1 mg/ml in 10 mM Tris acetate, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 0.05%
-mercaptoethanol, 100 mM
NaCl, 50% glycerol. This solution was diluted to either 87 or 17 µg/ml talin with buffer A, 0.05% Triton X-100, 3 mM
MgCl2, 2 mg/ml bovine serum albumin, and protease
inhibitors (see above, including 0.1 mM E-64) and processed
as indicated in the binding assays with cell lysates. For
densitometric analysis, scans of Coomassie-stained gels were
processed using the program NIH-Image (NIH, Bethesda,
MD).
 |
RESULTS |
Recombinant Integrin Cytoplasmic Domains--
We developed a
strategy for recombinant production of model proteins consisting of an
N-terminal His tag sequence followed by a thrombin cleavage site, a
Cys-Gly linker, and four heptad repeats connected to the integrin
cytoplasmic domain (Fig. 1). These
polypeptides are predicted to form parallel coiled coil dimers under
physiological conditions (18). A cystine bridge ensures a parallel
orientation and a correct stagger of the coiled coil sequences within
the dimer. All synthesized proteins were >90% homogenous as judged by
reverse phase C18 high pressure liquid chromatography (Fig.
2A) and had a monomer mass
that varied by less than 0.1% from that predicted for the desired
sequence as judged by electrospray ionization mass spectrometry (Table
I). The formation of covalent dimers in
aqueous solution was observed by mass spectrometry (Fig. 2B)
and by SDS-PAGE (Fig. 2C), thus confirming the parallel
orientation of the helices.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Amino acid sequences of recombinant models of
integrin cytoplasmic domains. Above, the N-terminal and
heptad repeat structures common to all constructs are depicted. In the
example shown, these are connected to the G1- 1A cytoplasmic domain.
Below is a list of the integrin-specific sequences of all
constructs used in this study. All integrin peptides correspond to
published human integrin sequences (as cited in the text). To preserve
the HindIII site, the Arg residue at the N terminus of the
cytoplasmic domain of the human integrin 7 chain was replaced by
Lys. Arrows indicate the positions of hydrophobic residues
corresponding to the positions a and d in heptad
repeats of the type [abcdefg] (31). The position of the Gly
insertions in the G2, G3, and G4 constructs is also indicated.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Characterization of purified recombinant
G4- 1A model protein. A, analytical reverse phase C18 high
pressure liquid chromatography analysis in 0.1% trifluoracetic acid.
B, ion spray mass spectrum of an oxidized G4- 1A
preparation. C, SDS-PAGE analysis on a 20% SDS acrylamide
gel. In the right lane, 20 mM dithiothreitol (DTT) has been added to reduce the protein before heating
and electrophoresis. Positions of marker proteins (kDa) are indicated on the left.
|
|
The beginning of the integrin cytoplasmic domain sequence could provide
the a and d residues of a fifth heptad repeat
(Fig. 1). Consequently, direct linkage to the coiled coil sequence
could induce helical structure in the tail. To address this
possibility, we synthesized protein models containing Gly residues
inserted between the coiled coil and the cytoplasmic domain sequence
(Fig. 1). We compared the CD spectra of
1 integrin constructs
containing either no (G1-
1A) or three (G4-
1A) additional Gly
residues inserted between the coiled coil and the cytoplasmic domain.
Insertion of glycines sharply reduced the minima at 208 and 222 nm.
Consequently, predicted
-helical content in the protein model was
reduced from 65 to 36% (Fig. 3). 29% of
the amino acids in the construct are in the four heptad repeats;
therefore, 36% helical content is consistent with most of the helical
structure being limited to these repeats. Thus, the Gly insertion
appears to eliminate
-helical structure induced in the cytoplasmic
domain by the direct linkage to the coiled coil sequence.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Circular dichroism spectroscopy of G1- 1A
and G4- 1A model proteins. Polypeptides were dissolved at 110 µg/ml in 50 mM sodium borate, pH 7.0, and analyzed in a
AVIV 60DS spectropolarimeter. After CD spectroscopy, protein
concentrations were determined by quantitative amino acid analysis to
calculate mean molar ellipticity per residue ( ). , G1- 1A; ,
G4- 1A.
|
|
Cytoplasmic Domain Conformation Affects Cytoskeletal Protein
Binding--
To assess functional consequences of the structural
changes induced by the Gly insertions, we produced protein models of
the
1A cytoplasmic domain with one, two, and three additional Gly residues inserted after the heptad repeat motif (G2-, G3-, G4-
1A) and compared these with the G1-
1A construct. As an additional control, we produced a variant of the G4-
1A peptide, with a Tyr to
Ala substitution in the membrane-proximal NPXY-motif
(G4-
1A(Y788A)) (Fig. 1). This mutation interferes with focal
adhesion targeting (22) and activation (29) of integrins. The purified
proteins were bound via their N-terminal His tag to a Ni2+
resin and used in affinity chromatography experiments with lysates of
human platelets. Marked changes in protein binding properties were
observed as a consequence of the Gly insertions (Fig.
4): In Coomassie Blue-stained gels,
polypeptides migrating at 45, 56, 58, 140, and 240 kDa bound only to
the mimics with Gly insertions. Moreover, comparison to the cell lysate
(Ly in Fig. 4A) showed that this binding was
selective. Immunoblotting identified the 240-kDa and 45-kDa proteins as
filamin and actin, respectively (Fig. 5).
The enriched 56-, 58-, and 140-kDa polypeptides have not been
identified. They failed to react with antibodies specific for
pp60src (Fig. 5), paxillin, pp125FAK,
-actinin, and
vinculin (data not shown) in immunoblotting experiments. Talin bound to
both the G1- and G4-
1A protein. In addition, we used biotinylated
extracts to enhance sensitivity of protein detection. In such extracts,
we confirmed talin binding to the G1-
1A protein and enhanced filamin
binding with the Gly insertions (Fig. 4B). Thus, the
structural changes (Fig. 3) induced by the insertion of Gly between the
coiled coil motif and the integrin cytoplasmic domain sequence alter
interactions of these proteins with cellular components.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 4.
Gly insertions change the protein binding
properties of the model proteins. Ni2+ resin was
loaded with G1- 1A (G1), G2- 1A (G2), G3- 1A (G3), or G4- 1A
(G4). As a control, the resin was loaded with a construct containing a
1 mutation known to disrupt cytoskeletal associations of integrins
(G4- 1A(Y788A) (YA)). Unloaded Ni2+ resin
(Co) was also used. Depicted are reduced SDS-PAGE (4-20%) analyses of proteins bound from biotinylated platelet lysates and of
the lysate itself (Ly). Panels A and C
represent Coomassie Blue-stained gels; panel B depicts
biotinylated proteins as detected by streptavidin peroxidase and
chemiluminescence. Note equal loading with the model proteins shown in
panel C.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 5.
Western blot analysis of samples from the
experiments shown in Fig. 4 with antibodies specific for filamin,
talin, actin, and pp60src. Ly, lysate;
Co, unloaded Ni2+ resin; YA,
G4- 1A(Y788A).
|
|
To establish specificity of the binding of cellular proteins to
G4-
1A, we examined the effect of structural changes known to inhibit
cytoskeletal associations of the intact integrin. Introduction of the
Y788A mutation into the G4-
1A construct (YA) suppressed the interaction with both talin and filamin but not the binding of the
other polypeptides (Fig. 4). Selective suppression of the binding of
talin and filamin was also observed with G4-
1B and G4-
1C
constructs (data not shown, but see Fig. 8). Thus, alterations of the
1A tail, which block cytoskeletal interactions, also reduce binding
to talin and filamin.
The
1A cytoplasmic domain is dimerized in these model proteins. To
assess the role of
1A dimerization in cytoskeletal protein binding,
we constructed a heterodimer containing a single G4-
1A subunit. The
other subunit (G4-
T) was identical to G4-
1A through the four
glycines. C-terminal to the glycines, we placed a KLGFFKR sequence,
representing the conserved membrane proximal region of integrin
cytoplasmic domains (4). The structure of the heterodimer was confirmed
by mass spectroscopy (Table I). The G4-
T
1A heterodimer bound
filamin and talin to the same extent as the G4
1A dimer (Fig.
6). Specificity of binding in this
experiment was confirmed by the failure of an
IIb cytoplasmic domain
construct to bind either cytoskeletal protein. Thus, the dimerization
of the
1A cytoplasmic domain is not essential for talin and filamin binding.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 6.
Role of 1A dimerization in talin and
filamin binding. N12+ resin was loaded
with the heterodimer G4- T 1A or homodimers of
G4- 1A or G4- IIb. Bound proteins from
platelet extracts were separated on 4-20% SDS-polyacrylamide gels
under reducing conditions, transferred to nitrocellulose membranes, and
stained with antibodies specific for talin or filamin. In addition,
Coomassie Blue-stained gels were examined to assess loading of the
resin. Note the migration position of the G4- T construct ( T) and
the similar quantities of G4- 1A or G4- IIb subunit in
each lane.
|
|
Differential Binding of Talin and Filamin to Integrin
1D and
7 Cytoplasmic Domains--
To extend our affinity chromatography
results, we prepared similar G1 and G4 model proteins of the splice
variant,
1D, and the
7 integrin subunits (Fig. 1). When incubated
with platelet lysates, the
1D constructs bound more talin, and
7
constructs bound more filamin compared with
1A (Fig.
7A). In addition, these differences in binding were consistently observed when lysates of a
human T-cell leukemia cell line (Jurkat), a human fibrosarcoma cell
line (HT 1080), or differentiated myotubes derived from a mouse
myoblast cell line (C2C12) were used for affinity chromatography (Fig.
7B). Moreover, stronger binding of the
1D constructs to talin and of the
7 constructs to filamin was observed both with the
G1 (data not shown) as well as the G4 variants of the model proteins,
indicating that structural changes induced by Gly insertions do not
alter the relative strengths of these interactions.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 7.
Differential binding of talin and filamin to
cytoplasmic domains. A, Ni2+ resin was
loaded with G4- 1A ( 1A), G4- 1A(Y788A) (YA), G4- 1D ( 1D), or G4- 7 ( 7). Bound proteins from biotinylated platelet extracts were separated on 4-20% SDS-polyacrylamide gels under reducing conditions and stained with antibodies specific for filamin or
talin or reacted with streptavidin-peroxidase (biotin). B, Western blot analysis with antibodies against talin and filamin of
proteins bound from lysates of human Jurkat or HT1080 cells or from
mouse C2C12 myotubes to Ni2+ resin loaded with G4- 1A
( 1A), G4- 1D ( 1D), and G4- 7 ( 7).
|
|
Interactions with Purified Talin and Filamin--
To learn whether
the observed interactions with talin and filamin in the cell extracts
are direct, we used purified preparations of these proteins. The
relative binding of purified filamin and talin to the model proteins
was similar to that observed with cell lysates (Fig.
8). Specifically,
1D constructs bound
more talin, and
7 constructs bound more filamin than
1A (Fig. 8). In addition, binding of both cytoskeletal proteins to the
G4-
1A(Y788A) construct and to the G4-
1B and G4-
1C variants was
markedly reduced compared with G4-
1A (Fig. 8). Moreover, G4
constructs of
1A,
1D, and
7 integrin cytoplasmic domains bound
more purified filamin than the corresponding G1 constructs. However,
the G1-
7 model protein still bound more filamin than G4-
1A or
G4-
1D (data not shown). A densitometric analysis of the Coomassie
Blue-stained proteins indicated that the
1D construct bound 9 times
more talin, and the
7 construct bound 8.4 times more filamin than
the
1A model protein (Fig. 8). Thus, filamin and talin bind
differentially to the different integrin
subunit cytoplasmic domain
constructs.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 8.
Binding of purified talin and purified
filamin to G4 model proteins. Ni2+ resins either
unloaded (Co) or loaded with G4- 1A ( 1A),
G4- 1A(Y788A) (YA), G4- 1D ( 1D), G4- 1B ( 1B),
G4- 1C ( 1C), and G4- 7 ( 7) were incubated with purified talin
or purified filamin. Bound proteins were detected in Coomassie
Blue-stained gels (upper panels) or on immunoblots developed
with antibodies against talin or filamin (lower panels).
Lanes marked S are samples of the purified talin or filamin preparations. The concentrations of purified proteins in
these experiments were 87 µg/ml talin (upper panel), 17 µg/ml talin (lower panel), and 125 µg/ml filamin
(upper and lower panels). Numbers
below the upper panels are relative densitometric units obtained from a densitometric analysis of scans of the Coomassie Blue-stained gels.
|
|
 |
DISCUSSION |
To study integrin-cytoskeletal interactions, we designed
structural mimics of the cytoplasmic face of occupied and clustered integrins. We joined integrin cytoplasmic domains to an N-terminal parallel coiled coil dimer, and we found that the introduction of Gly
spacers between the coiled coil and the
1A integrin tail leads to a
change in the conformation of the protein. Concomitantly, the
polypeptide binding properties are altered, resulting in an increase in
the strength of the interaction with the cytoskeletal protein, filamin.
Talin and filamin binding to these structural mimics are direct,
specific, and biologically relevant. These interactions are observed
with purified talin and filamin and are abrogated in splice variants
and a mutant that disrupt cytoskeletal interactions of the intact
integrin. The
1D integrin tail binds talin, and the
7 integrin
tail binds filamin much more tightly than the
1A cytoplasmic domain.
Thus, we provide insight into integrin class and splice
variant-specific interactions with cytoskeletal proteins and their
dependence on the conformation of integrin
cytoplasmic domains.
The model proteins described here have the topology of clustered
integrin cytoplasmic domains. The tropomyosin-derived heptad repeats
employed (30) are known to form parallel dimers (31, 32). Moreover,
they were dimeric and disulfide-bonded (Fig. 2C) via their N
termini. Thus, they must have been parallel, and the integrin tails
must have been in the desired vertical stagger. The conformation of
these constructs was sensitive to Gly insertions between the coiled
coil and the integrin tails. The coiled coil structure formed by the
heptad repeats should not be disrupted by these Gly insertions, and the
estimated 36% residual helix in the G4-
1A construct is consistent
with maintenance of the coiled coil. Consequently, the Gly insertions
may have disrupted induced helical structure in the
1A tail in the
G1-
1A construct.
These model proteins permitted direct detection of talin and filamin
binding to integrin
1A subunit cytoplasmic domains. Binding of both
talin and filamin was specific since it was sensitive to alterations in
the
tail sequence known to block cytoskeletal associations in
vivo. Previously, equilibrium gel filtration was required to
demonstrate low affinity
1A integrin-talin interactions (33).
Furthermore, affinity chromatography or yeast two-hybrid screens with
linear
1A cytoplasmic domain peptides failed to isolate either
filamin or talin (34-37). The recombinant model proteins present the
tails as vertically oriented dimers. Furthermore, they are immobilized
via an N-terminal His tag. The coiled coil also serves to introduce an
additional ~42-Å space (38) between the immobilizing surface and the
tail. However, we found that a monomeric
tail, presented on a
similar model protein, also bound talin and filamin. The vertical
orientation of the
tails and the reduction of potential steric
hindrance may also contribute to the efficiency of these model proteins
in affinity chromatography.
The differential binding of
cytoplasmic domains to talin and
filamin suggests functionally different linkages of integrins with the
cytoskeleton. Talin and non-muscle filamin are both reported to be
actin-binding proteins (3), but they seem to link to different
components of the cytoskeleton. Non-muscle filamin cross-links actin
filaments and promotes their high angle branching (39). It is thus part
of the cortical actin network. The preferential binding of the integrin
7 tail to filamin could therefore affect the cell surface
distribution of
7 integrins and contribute their role in lymphocyte
homing (40, 41). In contrast, talin concentrates at sites in which
integrins are clustered, and actin filaments insert end-on into
submembraneous focal complexes (2, 3). Such sites include
tension-bearing structures such as myotendinous junctions, intercalated
disks, and costameres in striated muscle (42). The markedly increased
binding of the muscle-specific splice variant,
1D, to talin could
provide a molecular mechanism for increased stability of
matrix-cytoskeletal linkages at these sites.
The model proteins described here provide new approaches to the
structure and function of integrin cytoplasmic domains. We found that
talin binding was increased in the
1D splice variant, yet the major
differences in amino acid sequence between
1D and
1A lie outside
of the previously identified talin binding site (43) in
1A.
Furthermore, although filamin had not been noted to bind
1A, it was
reported to bind to the
2 cytoplasmic domain in affinity
chromatography experiments (44). In the previous work, the filamin
binding site was localized to a relatively conserved membrane-proximal
REYRRFEKEK sequence. In our experiments, filamin and talin binding were
sensitive to changes at membrane-distal sites in the
1B and
1C
splice variants and the
1A(Y788A) mutant. Taken together, these data
suggest that the folded structure of the
cytoplasmic domain is
important in its interactions with cytoskeletal proteins. The G4-
1A
and G1-
1A constructs contain identical
1A sequences. Their marked
differences in cytoskeletal protein binding represents direct evidence
of the functional importance of the three-dimensional structures of
integrin
cytoplasmic domains. These model proteins may also provide
powerful tools for the elucidation of such structures.
We gratefully acknowledge the contributions
of Michael Williams, Liane Höfferer, and Virginia Keivens in the
development of the recombinant model proteins. Ulrike Schwarz assisted
in the recombinant protein production and in affinity chromatography experiments. Michael Fitzgerald helped to perform the circular dichroism spectroscopy. We are also very grateful to the many colleagues who provided reagents for this study, particularly to Keith
Burridge and John Hartwig for the purified talin and filamin,
respectively.