JBC Papers in Press. Published on July 21, 2000 as ... · The LIM-only protein DRAL/FHL2 binds to...
Transcript of JBC Papers in Press. Published on July 21, 2000 as ... · The LIM-only protein DRAL/FHL2 binds to...
The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several α and β integrin
chains and is recruited to adhesion complexes.
Viktor Wixler3, Dirk Geerts1, Emmanuel Laplantine, Daniel Westhoff, Neil Smyth, Monique
Aumailley2, Arnoud Sonnenberg1and Mats Paulsson.
Institute for Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str.
52, 50931 Cologne, Germany and 1The Netherlands Cancer Institute, Plesmanlaan 121, 1066
CX Amsterdam, The Netherlands.
Key words : Four and a half LIM domain protein / Signaling / Protein-protein interaction / yeast
two hybrid
2Corresponding author : Dr. Monique Aumailley, Institute for Biochemistry II, Joseph-
Stelzmann-Str. 52, 50931 Cologne, Germany
Tel.: +49 221 478 6991
Fax: +49 221 478 3109
email : [email protected]
3Present address : Institute for Experimental Medicine, Friedrich-Alexander University,
Glückstrasse 6, 91054 Erlangen, Germany.
Running title : DRAL/FHL2 binds to α and β integrin subunits
1
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on July 21, 2000 as Manuscript M002519200 by guest on January 25, 2019
http://ww
w.jbc.org/
Dow
nloaded from
ABSTRACT
LIM proteins contain one or more double zinc-finger structures (LIM domains)
mediating specific contacts between proteins which participate in the formation of multiprotein
complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM
domains, can associate with α3A, α3B, α7A and several β integrin subunits as shown in yeast
two-hybrid assays as well as after overexpression in human cells. The amino acid sequence
immediately following the conserved membrane-proximal region in the integrin α subunits or
the carboxy-terminal region with the conserved NXXY motif of the integrin β subunits are
critical for binding DRAL/FHL2. Further, the DRAL/FHL2 associates with itself and with other
molecules that bind to the cytoplasmic domain of integrin α subunits. Deletion analysis of
DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the
different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in
cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin
signaling pathways.
Running title : DRAL/FHL2 binds to α and β integrin subunits
2
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
INTRODUCTION
Integrins are key regulators of cell adhesion and migration, processes that play a crucial
role in cell survival and differentiation (1- 4). Upon ligand binding, integrins cluster and many
intracellular components, including adaptors, structural proteins and kinases, are recruited into
large multimolecular complexes where actin microfilaments anchor. As integrins do not possess
intrinsic enzymatic activity, proteins associated with the complexes activate signalling pathways
(1, 4-6). However, the precise molecular mechanisms of signal transmission still remain elusive.
The cytoplasmic domain of integrin β subunits is assumed to play an active role in transducing
the signals, while those of the integrin α subunits are thought to be modulators. These different
functions are probably mediated by diverse proteins that have been shown to interact, for most of
them in vitro, with the cytoplasmic domains of the α or β integrin subunits. Talin, α-actinin,
paxillin, focal adhesion kinase FAK (for review see 7), filamin (8), β3-endonexin (9), integrin-
linked kinase (ILK; 10), cytohesin-1 (11), integrin cytoplasmic domain-associated protein 1
(ICAP-1; 12, 13), receptor for activated protein kinase C (Rack-1; 14) and WD protein
interacting with integrin tails (WAIT-1; 15) were shown to interact with the cytoplasmic tail of
either specific or various β integrin subunits. Similarly, several proteins have been shown to bind
the cytoplasmic domain of integrin α subunits. Five of them, calreticulin (16), mammalian
suppressor of secretion (Mss4), Bridging Integrator protein-1 (also called box dependent Myc
interaction protein-1 or BIN1), and α integrin-binding proteins 63 (AIBP63) and 80 (AIBP80)
Running title : DRAL/FHL2 binds to α and β integrin subunits
3
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
(17) interact with several integrin α subunits and bind to the conserved sequence proximal to the
transmembrane domain. Another, the calcium- and integrin-binding protein CIB, has a more
restricted binding pattern (18). With the exception of calreticulin, which has been shown to
modulate cell adhesion, the biological significance of these interactions is still unknown.
Such proteins are likely involved in the control of multiprotein complex remodelling. The
α3β1 integrin transdominantly regulates the clustering of other integrins, inclusing α6β1 (19-
21), α5β1 and α2β1 (22), and of intracellular proteins associated with adhesion complexes. To
identify proteins involved in the transdominant control exerted by the α3β1 integrin, a yeast
two-hybrid screen was performed using the cytoplasmic domain of the α3A integrin subunit. Of
five proteins identified in the screen, four bind the cytoplasmic conserved of several integrin α
subunits, while the fifth protein interacted with the unique, more distal sequence of the integrin
α3A cytoplasmic domain (17). This fifth protein is the four and a half LIM protein 2 (FHL2) of the
LIM-only family, which was originally identified as DRAL (for down-regulated in
rhabdomyosarcoma LIM protein) by subtractive cloning (23). To follow the recommended
nomenclature (HUGO/GDB Nomenclature Committee) and to acknowledge the original
discovery we will refer to the protein as DRAL/FHL2.
LIM domains are double Zn-finger motifs, defining the expanding family of LIM
proteins involved in protein-protein interactions and transcriptional regulation (24-26). They are
comprised either exclusively of LIM domains, the LIM-only proteins, or of LIM domains
associated with homeodomains, kinase domains or other functionally active sites, the LIM-plus
proteins. Of the latter several proteins such as paxillin, zyxin, CRP1, abLIM and the paxillin
Running title : DRAL/FHL2 binds to α and β integrin subunits
4
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
analog in platelets (Hic-5) are associated with the cytoskeleton (25, 27-31) and could function
as nuclear-cytoplasmic shuttlers (32). DRAL/FHL2 is one of five known LIM-only proteins
with four and a half LIM domains (33-35). We report here that DRAL/FHL2 and specific
subdomains thereof have the capacity to interact with several β integrin subunits, a restricted
number of integrin α subunits and with integrin-binding proteins. Further DRAL/FHL2 can
localise to cell adhesion complexes.
Running title : DRAL/FHL2 binds to α and β integrin subunits
5
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
EXPERIMENTAL PROCEDURES
DNA constructs
cDNA fragments encoding the complete cytoplasmic domain of integrin α2, α3A, α5,
α6A and β1A subunits were amplified by RT-PCR using as template total RNA from the human
mammary epithelial cell line HBL100. They were inserted into EcoRI/BamHI sites of the pAS2-
1 vector (Clontech. Heidelberg, Germany) as fusion proteins with the GAL4 DNA binding
domain as described previously (17). cDNA fragments containing the partial or complete
cytoplasmic domains of the integrin α3B, α6B, α7A, α7B, β1D, β2 and β3A subunits were
amplified by PCR using the full length cDNA as a template and appropriate specific sense and
antisense primers containing restriction site tags. The constructs for the cytoplasmic domain of
the integrin α1 and β6 subunits were provided by Drs. B. Eckes and D. Petersohn (Department
of Dermatology, University of Cologne, Germany) and Dr. S. Spong (Lung Biology Center,
University of California-San Francisco, CA), respectively. All integrin α3A and β1A deletion
mutants were derived by PCR amplification using the appropriate cDNA constructs and inserted
into the pAS2-1 vector as above. DRAL/FHL2, AIBP63, AIBP80, Mss4 and BIN1 were
available from our previous study (17) as clones in the pACT2 vector (Clontech). Deletion
mutants of DRAL/FHL2 generated by PCR were cloned into BamHI/XhoI sites of the pACT2
vector. AIBP80 and DRAL/FHL2 were subcloned and inserted into EcoRI/BamHI sites of the
pAS2-1 vector.
Running title : DRAL/FHL2 binds to α and β integrin subunits
6
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
For expression in mammalian cells, the cytoplasmic domains of integrin α2 (aa 1126-
1152), α3A (aa 1015-1051), α7A (aa 1060-1115) and β1A (aa 752-798) subunits were cut
from the pAS2-1 construct using NdeI and SpeI and, after fill-in of the NdeI overhang, the
inserts were cloned in-frame into the GST-vector pEBG (a gift of Dr. A. Kalmes, University of
Würzburg, Germany), which had been cut and filled-in at the BamHI site and then digested with
SpeI. The full-length human DRAL/FHL2 was cut from pACT2 construct by NheI/AvrII and
inserted into the SpeI site of the pEBG vector. For the generation of myc-tagged full-length or
truncated DRAL/FHL2 and AIBP80, the cDNAs encoding these proteins were cut from pACT2
constructs (17) by SalI/XhoI and XmaI/XhoI, respectively, and cloned in-frame into the
pCS2+MT vector (a gift of Dr. A. Kalmes). Full-length or truncated DRAL/FHL2 was inserted
into the XhoI site, and AIBP80 was inserted after filling-in of the existing overhangs into the
StuI site. Full-length cDNA coding for the entire integrin α3A subunit in the Bluescript vector (a
generous gift of Dr. M.E. Hemler, Dana-Farber Cancer Institute, Boston, MA) was cut by XbaI
and subcloned into the XbaI site of the pcDNA3 vector (Invitrogen. Groningen, The
Netherlands).
Yeast two-hybrid library screening, mating and transformation assays.
Yeast cultures were grown under standard conditions in liquid or on solid media using
YPD or minimal SD media. The yeast strain Y190 (Clontech) was transformed sequentially with
the pAS2-1 plasmid coding for the cytoplasmic domain of the integrin α3A subunit as bait and
then with a pACT2 plasmid containing the placenta cDNA library (Clontech). Transformants
Running title : DRAL/FHL2 binds to α and β integrin subunits
7
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
were grown on SD medium lacking the amino acids leucine, tryptophan and histidine in the
presence of 25 mM 3-amino-1,2,4-triazole. On day 5 the colonies were tested for the activity of
the LacZ reporter gene in a β-Gal filter assay. To remove the bait cDNA, positive clones were
recultured on SD medium without tryptophan in the presence of 10 µg/ml cycloheximide. The
cycloheximide-resistant Y190 yeast clones were verified in a mating assay with yeast strain
Y187 expressing the pAS2-1 plasmid with either the cytoplasmic domain of the integrin α3A
subunit, the unrelated protein lamin C (Clontech), or the nonfused GAL4 DNA binding domain
as baits. Clones were scored as positive when the His+ and LacZ+ phenotype of yeast cells was
dependent on the co-expression of only the cytoplasmic domain of the integrin α3A subunit as
bait. Such clones were retested in a co-transformation assay with purified plasmid cDNA using
the same controls as in mating assays.
For direct two-hybrid binding assays, yeast Y190 cells were co-transfected with
DRAL/FHL2 or its deletion mutants fused to the GAL4-transactivation domain in the pACT2
vector and with one of the cDNA constructs coding for either integrin cytoplasmic domains,
mutants thereof or DRAL/FHL2 itself, fused to the GAL4-DNA binding domain in the pAS2-1
vector. In other experiments, yeast cells were co-transfected with DRAL/FHL2 in pAS2-1 and
with AIBP63, AIBP80, Mss4, BIN1 or DRAL/FHL2 itself in pACT2. In all cases, positive
clones were scored as described above.
Expression of proteins in mammalian cells
For transient expression, human embryonic kidney 293 (HEK293) cells (American Type
Running title : DRAL/FHL2 binds to α and β integrin subunits
8
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
Culture Collection) or mouse 3T3 fibroblasts (kindly provided by Dr. U. Rapp, University of
Wurzburg) were grown for 24 h in six-well plates (2.5x105 cells/35 mm-diameter well) prior to
transfection with plasmid DNA (2 µg/well) using Superfect Transfection mixture
(Quiagen,Hilden, Germany) according to the manufacturers instructions. When cells were co-
transfected with different DNAs, the DNA content was equalised with appropriate amounts of
empty expression vectors. For stable expression, HEK293 cells were transfected with the
pTracer-CMV vector (Invitrogen) containing the full length DRAL/FHL2 cDNA with an N-
terminal insertion of the nine amino acid hemagglutinin-tag (HA-tag). The cells were
trypsinised 48 h after transfection and selected further in medium containing 25 µg|ml zeocin.
After two weeks, single colonies were picked, cells were cultured, and the expression of HA-
tagged DRAL/FHL2 was analysed by immunoblotting using HA-tag specific monoclonal
antibody 12CA5 (a generous gift from Dr. U. Rapp, University of Würzburg, Germany).
Cell cultures and subcellular fractionation
Normal human skin fibroblasts and the epithelial cell line HaCat were provided by Dr. H.
Smola (Department of Dermatology, University of Cologne, Germany). Established lines of
human lung fibroblats (Wi26), embryonic kidney (293), fibrosarcoma (HT1080), mammary
epithelia (HBL100), mammary epithelial carcinoma (MCF-7), epidermoid carcinoma (A431),
ductal mammary carcinoma (T47D) have been previously described (36-38). CaCo2 cells were
newly purchased from the ATCC. Rat PC12 and mouse NIH 3T3 cells were provided by Dr. U.
Rapp (University of Wurzburg) and human RD-9 cells by Dr. A. Hoffmann (Department of
Running title : DRAL/FHL2 binds to α and β integrin subunits
9
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
Biochemistry, University of Cologne). All cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 2 mM glutamine, a cocktail of antibiotics and 10% heat
inactivated fetal calf serum (Seromed/Biochrom, Berlin, Germany).
Cells were fractionated as previously described (39). Cell monolayers were washed twice
with phosphate-buffered saline (PBS) pH 7.4, and scraped in 1 ml of hypotonic lysis buffer (1
mM EGTA, 1 mM EDTA, 10 mM β-glycerophosphate, 2 mM MgCl2, 10 mM KCL, 1 mM Na-
vanadate, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM pepstatin, pH 7.2). After
incubation on ice for 30 min, the cells were homogenised with a tight-fitting pestle and loaded
onto 1 ml of 1 M sucrose in lysis buffer. The nuclear fraction was collected by centrifugation
(1,600 g, 10 min). The pellet was washed once with 1 M sucrose in lysis buffer. The supernatant
was further centrifuged (150,000 g, 30 min) and the resulting pellet and supernatant was taken as
membrane and cytosolic fraction, respectively. Cytosolic proteins were precipitated by the
methanol/chloroform method (40). Pellets of all three fractions were dissolved in 100 µl of
electrophoresis sample buffer and 30 µl were used for SDS-PAGE analysis.
Immunoprecipitation and immunoblotting
Transiently transfected cells were washed twice with PBS and lysed in 25 mM Hepes pH
7.5, 137 mM NaCl, 1 mM MgCl2, 1% Brij 98 or 1% Triton X-100, 2% glycerol, 1 mM Na-
vanadate, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM pepstatin at room
temperature for 20 min. The lysates were cleared by centrifugation (10,000 g, 10 min) at 4°C and
the supernatants were incubated for 3 h at 4°C with antibodies against the myc tag (clone 9E10,
Running title : DRAL/FHL2 binds to α and β integrin subunits
10
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
Oncogene distributed by Calbiochem, Schwalbach, Germany) and protein G-conjugated agarose
(Roche, Mannheim, Germany) or with glutathion-conjugated Sepharose beads (Pharmacia
Biotech, Freiburg, Germany) when precipitating GST-tagged proteins. The complexes were
washed three time with lysis buffer, suspended in electrophoresis sample buffer and heated at
95°C for 3 min. The samples were resolved by SDS-PAGE on 10% acrylamide gels and
electrophoretically transfered onto nitrocellulose membrane. Proteins were detected with goat
polyclonal antibodies against a synthetic peptide corresponding to the cytoplasmic domain of the
integrin α3A subunit (Santa Cruz Biotechnology) or against GST (Life Technologies, Berlin,
Germany), mouse monoclonal antibodies against myc (clone 9E10), or rabbit polyclonal
antibodies against recombinant GST-DRAL/FHL2 fusion protein and partially purified by
affinity chromatography on a GST column (a generous gift of Dr. B. Schäfer, University of
Zurich, Switzerland), followed by appropriate horse radish peroxidase-coupled secondary
antibodies (DAKO, Glostrup, Denmark) and the ECL detection system (Amersham Pharmacia
Biotech)
Immunofluorescence staining of cell adhesion complexes
Cells were cultured either overnight on uncoated or for 60 minutes on fibronectin-coated
(10 µg/ml) glass coverslips in DMEM containing 10% fetal calf serum. The cells were fixed with
2% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 2 min and blocked
with 1% BSA (fraction V, Sigma, Deisenhofen, Germany). The cells were processed for
immunofluorescence staining with mouse monoclonal antibody F-VII against human vinculin (a
Running title : DRAL/FHL2 binds to α and β integrin subunits
11
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
gift from Dr. M. Glukhova, Institut Curie, Paris, France), K20 (Immunotech, Marseille, France)
or TS2/16 (41) against the human β1 integrin subunit, 9E10 against the myc-tag, or rabbit
polyclonal antiserum against recombinant GST-DRAL/FHL2, followed by Cy3-conjugated
second antibodies against mouse or rabbit immunoglobulins (Jackson Immunoresearch
Laboratories distributed by Dianova, Hamburg, Germany) together with FITC-conjugated
phalloidin (Sigma Immunochemicals). For analysis of cell foot-prints the cells were lysed by
osmotic shock with or without cross-linking with 1 mM dithiobissuccinimidylpropionate (DSP)
(Pierce Chemicals, Oud Beijerland, The Netherlands) as previously described (38) and then
processed for immunofluorescence labeling as detailed above. After mounting, the samples were
observed with an Axiophot microscope (Zeiss, Oberkochen, Germany) equipped with
epifluorescence optics.
Running title : DRAL/FHL2 binds to α and β integrin subunits
12
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
RESULTS
Identification of DRAL/FHL2 as a protein binding the cytoplasmic domain of the
integrin α3A subunit.
To identify proteins that bind to the cytoplasmic domain of the α3Α integrin subunit, a
human placenta library (> 5x106 independent clones) was used in a yeast two-hybrid screen
with the C-terminal cytoplasmic part of the α3A integrin subunit (aa 1015-1051) as bait (17).
Out of 84 His+ and LacZ+ positive clones (17), 28 contained cDNAs with an identical open
reading frame coding for the 279 amino acid residues of DRAL/FHL2. The specificity of this
interaction was confirmed in a direct two-hybrid binding assay (Table I). Transformation of
yeast cells with DRAL/FHL2 in pACT2 vector alone or together with the GAL4-DNA-binding
domain in pAS2-1 vector or together with an unrelated protein, lamin C, fused to the GAL4-
DNA-binding domain instead of α3A, did not activate the His and LacZ reporter genes (Table
I). The predicted amino acid sequence contains four double Zn-finger LIM domains at the C-
terminal end and a half LIM domain at the N-terminus. It corresponds to FHL2 (23, 42, 43).
DRAL/FHL2 interacts with the cytoplasmic domain of the integrin α3A subunit when
co-transfected into human cells
To test if DRAL/FHL2 binds the integrin α3A subunit also in mammalian cells, HEK293
cells were co-transfected with the GST-tagged α3A cytoplasmic domain (aa 1015-1051) and
with myc-tagged DRAL/FHL2. These were tested for complex formation in co-precipitation
Running title : DRAL/FHL2 binds to α and β integrin subunits
13
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
experiments. The GST-tagged integrin α3A cytoplasmic domain precipitated in immune
complexes formed in the presence of antibodies against the myc tag as shown by
immunoblotting with specifc antibodies (Fig. 1A). Reciprocally, the presence of myc-tagged
DRAL/FHL2 in complexes specifically precipitated with antibodies against the cytoplasmic
domain of the α3A integrin subunit was dependent on the expression of GST-tagged α3A
cytoplasmic domain but not on the expression of GST alone (Fig. 1A). Thus, consistent with the
two-hybrid data, the LIM-only protein DRAL/FHL2 specifically interacts with the cytoplasmic
domain of integrin α3A subunit in mammalian cells.
To analyse whether DRAL/FHL2 also binds the full-length recombinant α3A subunit,
the myc-tagged DRAL/FHL2 and the full-length integrin α3A subunit were transfected in
HEK293 cells which normally express only small amounts of these polypeptides (data not
shown, but see Fig. 7). Only upon co-expression with myc-tagged DRAL/FHL2, was the
integrin α3 chain detected in myc-containing immunocomplexes (Fig. 1B).
Mapping of the DRAL/FHL2 binding site in the cytoplasmic domain of the integrin α3A
subunit by yeast direct two-hybrid assays.
The binding of DRAL/FHL2 was tested with four overlapping deletion mutants of the
cytoplasmic domain of the integrin α3A subunit. We have previously shown (17) that
DRAL/FHL2 does not bind to the stretch of highly conserved amino acids KXGFFKR proximal
to the transmembrane domain and common to all integrin α subunits. Here we show also no
interaction with a similar construct containing three additional residues at the carboxy-terminal,
Running title : DRAL/FHL2 binds to α and β integrin subunits
14
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
which are crucial for integrin function (44, 45). Further no binding occured to a construct
containing the last 18 carboxy-terminal residues. However, there was a positive reaction with all
constructs which contain a stretch of 12 amino acid residues beyond the conserved KCGFFKR
motif irrespective of other deletions (Fig. 2).
Binding specificity of DRAL/FHL2 for α or β integrin subunits and mapping of its
binding site within the integrin β1A chain.
To investigate the specificity of DRAL/FHL2 binding, direct two-hybrid interaction tests
were performed between DRAL/FHL2 and the cytoplasmic domain of nine different α integrin
subunits; the α1 and α2 integrin chains of collagen receptors, the α5 chain of the fibronectin
receptor and the A and B variants of the α3, α6 and α7 chains of laminin receptors (Table II). In
addition to the integrin α3A subunit cytoplasmic domain, α3B and α7A subunits interacted with
DRAL/FHL2 (Table II). Similar experiments with the cytoplasmic domains of different integrin
β subunits indicated that DRAL/FHL2 interacted with all β integrin chains, including β1A, β1D,
β2, β3A, and β6 (Table II).
The cytoplasmic domains of integrin β subunits have a stretch of conserved amino acid
residues proximal to the transmembrane domain and additionally share the conserved cyto-1,
cyto-2 and cyto-3 regions, which are important for integrin function (46-48). Cyto-2 and cyto-
3 contain typical protein-binding sequences, NPXY and NXXY, respectively. Six different
deletion mutants containing one or more of the characteristic motifs of the integrin β1A
cytoplasmic domain were constructed and tested for DRAL/FHL2 binding in the yeast two-
Running title : DRAL/FHL2 binds to α and β integrin subunits
15
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
hybrid assay. The results showed that the last nine C-terminal amino acids, including the NXXY
(NPKY) sequence of the cyto-3 region, are necessary for interaction between DRAL/FHL2 and
the cytoplasmic domain of integrin β1A subunit (Fig. 3). The importance of this carboxy-
terminal sequence was supported by data obtained with deletion mutants of the integrin β2, β3A
and β6 chains in which the cyto-3 region was preserved (Table II).
DRAL/FHL2 interacts with the cytoplasmic domain of the α7A or β1A integrin subunits
in co-transfected human cells.
To analyse whether the interactions reported above occur also in mammalian cells,
HEK293 cells were transiently co-transfected with the GST-tagged cytoplasmic domain of three
additional integrin subunits, α2, α7A or β1A, and with myc-tagged DRAL/FHL2. As with the
integrin α3A subunit, the myc-tagged DRAL/FHL2 was precipitated together with GST-tagged
α7A or β1A integrin cytoplasmic domains when using glutathion-conjugated Sepharose beads
(Fig. 4). As expected from the two-hybrid data, upon co-expression of the integrin α2
cytoplasmic domain and DRAL/FHL2, the GST-tagged α2 integrin subunit was not associated
with DRAL/FHL2 in the complexes (Fig. 4). The reciprocal immunoprecipitation test with
antibodies against myc confirmed these results (not shown).
DRAL/FHL2 interacts with itself and with other integrin-binding proteins.
Several LIM domain-containing proteins have been suggested to dimerise, thus
enhancing their capacity for complex formation (49). DRAL/FHL2, which contains four and a
Running title : DRAL/FHL2 binds to α and β integrin subunits
16
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
half LIM domains, could therefore represent an adaptor protein which could interact in
multimolecular complexes with other integrin-binding proteins such as those that we have
recently identified (17). This possibility was tested in direct two-hybrid interaction experiments.
The results showed that DRAL/FHL2 is able to interact with itself and with two additional
integrin-binding proteins, AIBP80 and BIN1 (Table III). To test the interactions in vivo, we
constructed expression vectors for GST-tagged DRAL/FHL2 and for myc-tagged AIBP80.
HEK293 cells co-expressing GST-DRAL/FHL2 and myc-DRAL/FHL2 or myc-AIBP80, were
lysed and proteins were precipitated with either anti-myc antibodies or with glutathion-coupled
Sepharose beads. Immunoblot analysis of the precipitates showed that also in human cells
DRAL/FHL2 self-interacts and that AIBP80 associates with DRAL/FHL2 (Figure 5).
Molecular dissection of binding sites in DRAL/FHL2.
To identify the LIM domains of DRAL/FHL2 responsible for the diverse interactions
described in this study, cDNA sequences for single LIM modules were cloned in the pACT2
vector and were tested in direct two-hybrid binding assays with the other proteins, including
DRAL/FHL2, cloned in pAS-2 vector. Surprisingly, only two out of six proteins analysed
interacted with particular single LIM domains; the cytoplasmic domain of the integrin α7A
subunit had affinity for the LIM 2 domain, while DRAL/FHL2 itself bound the C-terminal LIM
3 and LIM 4 domains (Fig. 6A). Tests with additional DRAL/FHL2 mutants, in which one, two
or three LIM domains were deleted from either the C- or N-terminus confirmed that the LIM 2
domain is responsible for binding to the integrin α7A subunit and that LIM 3 and LIM 4
Running title : DRAL/FHL2 binds to α and β integrin subunits
17
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
modules are involved in DRAL/FHL2 self association (Fig.6A). Additionally, they revealed that
both LIM 1 and LIM 2 N-terminal domains are required for interaction with AIBP80, while
binding of the cytoplasmic domains of the integrin α3A or α3B subunits needs the last 2, 3 and 4
LIM domains (Fig. 6A). Further, deletion of any LIM domain prevented the binding of
DRAL/FHL2 to the cytoplasmic domain of the integrin β1A subunit (Fig. 6A), suggesting that
the three-dimensional structure of DRAL/FHL2 is important for this interaction. The major
binding sites of the analysed proteins are summarized in Fig. 6B.
DRAL/FHL2 is expressed by normal human skin fibroblasts and is mainly localised in
the cell nucleus and cytosol .
DRAL/FHL2 was initially described as a protein preferentially localised in nuclei after
overexpression in NIH 3T3 cells, while it was distributed uniformly over nucleus and cytoplasm
in Rh30 cells (23). To analyse the subcellular localisation of DRAL/FHL2 more precisely, we
screened several human and rodent cells or cell lines for the presence of endogenous
DRAL/FHL2. Immunoblotting with an antiserum raised against the recombinant GST fusion
protein (23) showed the presence of DRAL/FHL2 in the cell lysates of normal human
fibroblasts, Wi26 human fibroblasts, mouse NIH 3T3 and DRAL/FHL2-transfected HEK293
cells as a prominent band migrating under reducing (Fig. 7A) or non reducing (not shown)
conditions at the position expected for a ca. 31 kDa polypeptide, in agreement with previous data
(23). In cell lysates from a panel of transformed or tumour cell lines of epithelial or neuronal
origin, the DRAL/FHL2 band was fainter or barely seen and had a slightly different mobility
Running title : DRAL/FHL2 binds to α and β integrin subunits
18
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
(Fig. 7A), which could indicate different post-translational modifications. Human skin
fibroblasts expressed most DRAL/FHL2 and were chosen to study the subcellular localisation of
naturally occuring protein. After fractionation by differential centrifugation, DRAL/FHL2 was
mainly found in the nuclear and cytosolic fractions with only small amounts in the cell
membrane fraction (Fig. 7B).
Endogenous DRAL/FHL2 can be recruited to cell adhesion complexes
As integrins function at the cell surface in adhesion complexes, we used
immunofluorescence microscopy to establish whether DRAL/FHL2 is localised at the periphery
of spread cells, where it would be expected to interact with the cytoplasmic tail of integrin
subunits. Normal human skin fibroblasts were the most appropriate for the study since they
express more DRAL/FHL2 than most of the established cell lines (Fig. 7). Moreover, the
polyclonal antibody raised against GST-DRAL/FHL2 recognised a single major band in
immunoblots of normal human skin fibroblast lysates (Fig. 7). Immunofluorescence staining
with the polyclonal antiserum against DRAL/FHL2 showed that naturally expressed
DRAL/FHL2 was associated with fibril-like structures within the cell body and with clusters at
the cell periphery (Fig. 8A,D,G). Double-labeling with antibodies against DRAL/FHL2 (Fig.
8A,D,G) and with monoclonal antibodies against the integrin β1 subunit (Fig. 8B) or vinculin
(Fig. 8H) showed overlap for many, but not all the clusters (Fig. 8, superimposed photogaphs C
and I). Further, visualisation of fibrillar actin by FITC-conjugated phalloidin in cells stained
with the antiserum against DRAL/FHL2 revealed that DRAL/FHL2 is clustered at the ends of
Running title : DRAL/FHL2 binds to α and β integrin subunits
19
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
actin bundles (superimposed photogaph F). Again, not all DRAL/FHL2 positive clusters were
located at the ends of the actin fibers and, vice versa, DRAL/FHL2 positive clusters were not
always present at the termini of actin fibers. In contrast, the antiserum against DRAL/FHL2 did
not stain any of vinculin-positive peripheral clusters in RD-9 cells (not shown), which agrees
with previous results showing that DRAL/FHL2 is down-regulated in rhabdomyosarcoma cells
(23).
Cell adhesion complexes are dynamic multimolecular assemblies of kinases and adaptor
proteins which are structurally or transiently associated to integrin clusters (4, 5). To confirm the
association of DRAL/FHL2 with cell adhesion complexes, foot-prints were prepared by
submitting normal human fibroblasts to osmotic shock in absence or in presence of DSP, a
membrane permeable cross-linker. Immunofluorescence labeling showed that both integrin β1
subunit and vinculin, but not DRAL/FHL2, were present in non-crosslinked cell remnants (Fig.
9). However, after cross-linking, DRAL/FHL2 was detected in the foot-prints of normal human
fibroblasts (Fig. 9).
Finally mouse 3T3 fibroblasts were transiently transfected with myc-tagged full-length
or truncated DRAL/FHL2 representing the N- and C-terminal part of the molecule.
Immunofluorescence detection of the tagged protein with an antibody against myc showed that
full-length DRAL/FHL2 localised to focal adhesion clusters (Fig. 10A). In contrast, the
immunofluorescence staining of mouse 3T3 fibroblasts transfected with constructs representing
the amino or carboxy-terminal-half of DRAL/FHL2 was only nuclear (Fig. 10B,C), indicating
that only full-length DRAL/FHL2 can be targeted to focal adhesion clusters.
Running title : DRAL/FHL2 binds to α and β integrin subunits
20
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
DISCUSSION
In this report we show that the LIM-only protein DRAL/FHL2 is a novel integrin-
binding protein. After the identification in a yeast two-hybrid screen as interacting with the
cytoplasmic domain of the integrin α3A subunit, we provide evidence that DRAL/FHL2 has the
capacity to interact, both in yeast and in mammalian cells, with itself, with the cytoplasmic
domain of integrin α3A, α3B, α7A and several β subunits, and with integrin-binding proteins.
Further, studies with mutant forms of DRAL/FHL2 demonstrate that different LIM domains are
responsible for those interactions. Finally, we show that DRAL/FHL2, previously described as a
nuclear protein, is targeted to cell adhesion complexes. Together, these results suggest that
DRAL/FHL2 may act as an adaptor protein, regulating integrin trafficking, function or
signalling.
A molecular dissection of the DRAL/FHL2 integrin α subunit binding sites showed the
importance of twelve amino acid residues immediately following the conserved membrane-
proximal region of the integrin α3A subunit. Several cytoplasmic proteins, including calreticulin,
CIB, BIN1, Mss4, AIBP63 and AIBP80 interact with the cytoplasmic tail of integrin α subunits
and require the highly conserved KXGFFKR sequence for optimal binding (16; 17, 50).
DRAL/FHL2, therefore, is the first protein shown to bind the non-conserved region of integrin α
subunit cytoplasmic domains. Further, DRAL/FHL2 has a specificity restricted to the integrin
α3A, α3B and α7A chains. These subunits have divergent sequences after the conserved
KXGFFKR motif, and it was unexpected that DRAL/FHL2 interacts with all three. A deletion
Running title : DRAL/FHL2 binds to α and β integrin subunits
21
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
mutation analysis of DRAL/FHL2 itself solved the apparent contradiction, as it was revealed that
different LIM domains interact with the α7A peptide and the α3 variants.
In integrin β subunits the C-terminal part of the cytoplasmic domain, that includes the
cyto-3 NXXY motif, is needed for binding to DRAL/FHL2. Except for the integrin β4 and β8
subunits, the cytoplasmic domains of the β chains share the functionally important cyto-1, cyto-
2 and cyto-3 regions (46-48). Cyto-2 and -3 contain the NPXY and NXXY motif, respectively.
NPXY motifs are recognition sites for phosphotyrosin-binding proteins such as those containing
SH2 domains (51). It is believed that the integrin cyto-2 region folds in a β-turn so that the C-
terminal part of the chain, containing the NXXY motif of cyto-3, can be brought in the vicinity
of the membrane proximal conserved region (52). Mutations in the NPXY sequence, truncation
or deletion of cyto-2, or peptides representing this region impair talin, filamin and α-actinin
binding to the β1 cytoplasmic tail as well as cell adhesion, spreading and formation of focal
adhesions (47; 53-57). The NXXY motif of cyto-3 is required for ICAP-1 or β3-endonexin
binding to the cytoplasmic domain of integrin β1 or β3 subunit, respectively (12, 13, 58). Our
data show that DRAL/FHL2 binds all tested integrin β subunits. Results obtained with sequential
deletion mutants of the β1A subunit and further deletion mutants of several other β subunits
indicated that the NXXY cyto-3 motif is within the critical binding site. Thus, in contrast to
ICAP-1 and β3-endonexin, DRAL/FHL2 is a binding partner common to all integrin β subunits
that possess the NXXY motif. One hypothesis is that the function of β subunits in integrin
activation is regulated through a conformational change involving folding of the C-terminal
region containing cyto-3 over the N-terminal region (52). Such a change could be under the
Running title : DRAL/FHL2 binds to α and β integrin subunits
22
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
control of cytoplasmic factors. Binding of DRAL/FHL2 to the cyto-3 region may regulate the
conformational changes of the integrin β1 cytoplasmic domain by either inducing or inhibiting
the folding or, alternatively, by transiently competing with other proteins for binding to this
domain.
Our observation that DRAL/FHL2 binds numerous proteins is not surprising as it
contains several LIM domains and those are thougth to function as protein interaction modules.
In addition, DRAL/FHL2 has the property to bind both α and β integrin subunits through
different LIM domains. Single LIM domains are involved in binding to the integrin α7A subunit
or in DRAL/FHL2 homodimerisation, while for binding to other proteins the coordinated action
of several LIM domains was needed. The most striking result was that deletion of any LIM
domain prevented the interaction with the integrin β1A subunit, suggesting that the three-
dimensional structure of DRAL/FHL2 is required for binding. At this point we do not know if
the different interactions described here for DRAL/FHL2 can take place simultaneoulsy or
whether homodimerisation of DRAL/FHL2 influences the interactions. LIM domains fold
independently and are held together by a linker region (26, 59). Further a single LIM domain, or
even a single Zn-finger module of a LIM domain, can function as protein-binding interface, so
that single LIM domain could be functionally bipartite (27). For example, while the LIM-only
protein PINCH consists of five LIM domains, it binds ILK by only the most N-terminal one
(60). Similarly, zyxin, a LIM-plus protein with a tandem of three LIM domains binds to another
LIM-containing protein, CRP1, also by a single LIM domain (27). These data, together with the
fact that DRAL/FHL2 forms dimers, suggest that this novel integrin-binding protein has great
Running title : DRAL/FHL2 binds to α and β integrin subunits
23
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
potential for forming multimeric protein complexes. Moreover, sequence comparison of
DRAL/FHL2-interacting proteins did not reveal any homology, except for the integrin β
subunits which share the cyto-1, -2 and -3 motifs, so that due to its modular structure,
DRAL/FHL2 can potentially be involved in many different interactions. Interestingly, further
interactions of DRAL/FHL2 with the the androgen receptor (61) or with hCDC47 (62) were
recently described. The interaction with the androgen receptor requires both the N- and C-
terminal domains of DRAL/FHL2 and the interaction with hCDC47 involves the LIM 2 and 3
domains together with the first half LIM motif of FHL2/DRAL, respectively. Taken together,
this suggests that by using different sets or combinations of its LIM domains this adaptor protein
could be involved in the organisation or the regulation of very diverse multimolecular complexes
including transcriptional complexes.
Although DRAL/FHL2 is mainly localised in the cell nucleus and cytosol (23; this
report), it was recruited to cell adhesion complexes in several cell types, including normal skin
fibroblasts. There, it was clustered together with integrins and vinculin at the ends of actin stress
fibers. Only full-length DRAL/FHL2 was targeted to cell adhesion complexes in transfected
mouse 3T3 fibroblasts while truncated version of the protein were not. This agrees well with the
results observed in yeast interaction assays showing that binding to the integrin β1A subunit
requires full-length DRAL/FHL2. In this aspect, the requirement for binding is similar to that
reported for the androgen receptor (61). Several protein from the LIM-plus family, like paxillin,
zyxin and abLIM, are adaptors involved in scaffolding of focal adhesion complexes or of the
cytoskeleton (25, 27-29). The C.elegans LIM-only protein UNC-97, that also has both nuclear
Running title : DRAL/FHL2 binds to α and β integrin subunits
24
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
and extranuclear distribution, colocalises in muscle with β-integrin in focal adhesion-like
structures (63). The other LIM-only proteins PINCH and CRP1 can also be recruited to integrin
signalling complexes through interactions with ILK (60) or via zyxin and α-actinin (25),
respectively.
Our cell fractionation experiments showed that only a small proportion of DRAL/FHL2
is present in the membrane fraction of human fibroblasts and that DRAL/FHL2 is more abundant
in the cell nucleus and the cytoplasm. This suggests that DRAL/FHL2 may also have other
functions within the cell such as that recently described in the formation of transcriptional
complexes (61). That only a small amount of DRAL/FHL2 was found to be membrane-
associated, can explain why we could not precipitate the endogenous protein with antibodies
against integrin chains. Only when recombinantly over-expressed in mammalian cells, an
interaction of DRAL/FHL2 and integrin cytoplasmic domains or full-length integrin α3A (this
report) or α7A1) could be observed. Alternatively, in mammalian cells interaction of
DRAL/FHL2 with integrins may be favored by a certain conformation or activation states of the
integrins such as those induced by extracellular ligand binding and which are lost under the
experimental conditions required for immunoprecipitation. Remarkably, binding of DRAL/FHL2
to the androgen receptor is strictly agonist-dependent in mammalian cells (61). Moreover, in
fibroblasts colocalisation of DRAL/FHL2 and vinculin, a marker of focal adhesion complexes,
or integrin β1 subunit was not always seen. Further, association of naturally expressed
DRAL/FHL2 within adhesion complexes of fibroblasts is weak, at least weaker than that of
vinculin, since it was retained in cell foot-prints only after cross-linking. This finding argues for
Running title : DRAL/FHL2 binds to α and β integrin subunits
25
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
a transient or regulatory role of DRAL/FHL2, a hallmark expected of proteins involved in the
signal transduction cascade initiated in adhesion complexes. The migration velocity in in vitro
wound closure assays as well as the adhesion to several extracellular matrix proteins, including
collagens, fibronectin and laminins, were not changed after overexpression of DRAL/FHL2.
Moreover, in the RD-9 rhabdomyosarcoma cells vinculin-positive adhesion complexes were
present despite the absence of DRAL/FHL2. Thus, while DRAL/FHL2 obviously participates in
protein clusters formed by integrins in fibroblasts, its presence does not appear to be essential for
adhesion complexes. In this regard it is similar to LIM proteins like PINCH (60) or Hic-5 (31).
A function as a nuclear-cytoplasmic shuttling protein has been proposed for zyxin, a
member of the LIM-plus family (32). The LIM domain structure has been resolved for a number
of proteins and they all show a striking similarity to the DNA-interacting CCCC module of the
transcription factor GATA-1 (64-66), suggesting that they can bind DNA (33, 42). Indeed, two
recent reports describe interactions of FHL2/DRAL with the androgen receptor (61) or with
hCDC47 (62) and its involvement in transcriptional complexes. Finally, one of the other four and
a half LIM domain containing proteins, ACT (activator of CREM in testis), stimulates the
transcriptional activity of CREM and CREB (35). In conclusion, DRAL/FHL2 appears as an
excellent candidate to facilitate the processing of integrin signals at the cell membrane into the
cell program since such proteins are likely to be required for the transfer of information from cell
adhesion complexes into the nucleus.
Running title : DRAL/FHL2 binds to α and β integrin subunits
26
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
ACKNOWLEDGEMENTS
We thank Monika Pesch for expert technical assistance, Drs. Beate Eckes and Dirk
Petersohn for the integrin α1/pAS2-1 construct, Dr. Marina Glukhova for the mAb against
vinculin, Dr. Martin E. Hemler for the integrin α3 chain cDNA, Dr. Andreas Kalmes for GST-
and myc-tagged vectors, Drs. Roswitha Nischt and Hans Smola for the normal human skin
fibroblasts, Wi26 and HaCat cells, Dr. Beat W. Schäfer for the antiserum against GST-DRAL
and Dr. Suzanne Spong for the pAS2-1/β6 constructs. We are very grateful to Drs. Markus
Plomann and Patrik Maurer for helpful discussions. This work was supported by the University
of Cologne, the Centre National de la Recherche Scientifique (M.A.), grants from the Deutsche
Forschungsgemeinschaft (Kr 558/10-1, FOR265/2-1 and AU 86/5-1), the Köln Fortune
Programme (Nr.160/1998 and 30/1999) and the Dutch Cancer Society (NKI 95-979).
Running title : DRAL/FHL2 binds to α and β integrin subunits
27
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
REFERENCES
1. Clark, E. A., and Brugge, J. S. (1995) Science 268, 233-238
2. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Ann. Rev. Cell Dev. Biol. 11,
549-599
3. Dedhar, S., and Hannigan, G. E. (1996) Curr. Opin. Cell Biol. 8, 657-669
4. Yamada, K. M., and Geiger, B. (1997) Curr. Op. Cell Biol. 9, 76-85
5. Burridge, K,. and Charzanowska-Wodnicka, M. (1997) Trends Cell Biol., 7, 342-347
6. Howe, A., Aplin, A. E., Alahari, S. K., and Juliano, R. L. (1998) Curr. Opin. Cell Biol. 10,
220-231
7. LaFlamme, S. E., Homan, S. M., Bodeau, A. L., and Mastrangelo, A. M. (1997) Matrix Biol.
16, 153-163
8. Sharma, C. P., Ezzell, R. M., and Arnaout, M. A. (1995) J. Immunol. 154, 3461-3470
9. Shattil, S. J., O’Toole, T., Eigenthaler, M., Thon, V., Williams, M., Babior, B. M., and
Ginsberg, M. H. (1995) J. Cell Biol. 131, 807-816
10. Hannigan, G. E., Leung-Hagersteijn, C., Fitz-Gibbon, L., Copolino, M. G., Radeva, G.,
Filmus, J., Bell, J. C., and Dedhar, S. (1996) Nature 79, 91-96
11. Kolanus, W., Nagel, W., Schiller, B., Zeitlmann, L., Godar, S., Stockinger, H., and Seed, B.
(1996) Cell 86, 233-242
12. Chang, D. D., Wong, C., Smith, H., and Liu, J. (1997) J. Cell Biol. 138, 1149-1157
13. Zhang, X. A., and Hemler, M. E. (1999) J. Biol. Chem. 274, 11-19
Running title : DRAL/FHL2 binds to α and β integrin subunits
28
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
14. Liliental, J., and Chang, D. D. (1998) J. Biol. Chem. 273, 2379-2383
15. Rietzler, M., Bittner, M., Kolanus, W., Schuster, A., and Holzmann, B. (1998) J. Biol. Chem.
273, 27459-27466
16. Rojiani, M. V., Finlay, B. B., Gray, V., and Dedhar, S. (1991) Biochemistry 30, 9859-9866
17. Wixler, V., Laplantine, E., Geerts, D., Sonnenberg, A., Petersohn, D., Eckes, B., Paulsson,
M., and Aumailley, M. (1999) FEBS Lett. 445, 351-355
18. Naik, U. P., Patel, P. M., and Parise, L. V. (1997) J. Biol. Chem. 272, 4651-4654
19. Dogic, D., Rousselle, P., and Aumailley, M. (1998) J. Cell Sci. 111, 793-802
20. Dogic, D., Hülsmann, H., Sherman, N., Fox, J. W., Broermann, R., Paulsson. M., and
Aumailley, M. (1999) Matrix Biol. 18, 433-444
21. Hodivala-Dilke, K. M., DiPersio, C. M., Kreidberg, J. A., and Hynes, R. O. (1998) J. Cell
Biol. 142, 1357-1369
22. Laplantine, E., Vallar, L., Mann, K., Kieffer, N., and Aumailley, M. (2000) J. Cell Sci. 113,
1167-1176
23. Genini, M., Schwalbe, P., Scholl, F.A., Remppis, A., Mattei, M. G., and Schäfer, B. M.
(1997) DNA Cell. Biol. 16, 433-442
24. Sanchez-Garcia, I., and Rabbitts, T. H. (1994) Trends Genet. 10, 315-320
25. Beckerle, M. (1997) BioEssays 19, 949-957
26. Dawid, I. B., Breen, J. J.,. and Toyama, R. (1998) Trends Genet. 14, 156-162
27. Schmeichel, K. L., and Beckerle, M. C. (1994) Cell 79, 211-219
28. Pomies, P., Louis, H. A., and Beckerle, M. C. (1997) J. Cell Biol. 139, 157-168
Running title : DRAL/FHL2 binds to α and β integrin subunits
29
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
29. Roof, D. J., Hayes, A., Adamian, M., Chishti, A. H., and Li, T. (1997) J. Cell Biol. 138, 575-
588
30. Hagmann, J., Grob, M., Welman, A., van Willigen, G., and Burger, M. M. (1998) J. Cell.
Sci. 111, 2181-2188
31. Thomas, S. M., Hagel, M., and Turner, C. E. (1999) J. Cell Sci. 112, 181-190
32. Nix, D. A., and Beckerle, M. C. (1997) J. Cell Biol. 138, 1139-1147
33. Morgan, M. J,. and Madgwick, A. J. (1999a) Biochim. Biophys. Res. Comm. 255, 251-255
34. Morgan, M. J., and Madgwick, A. J. (1999b) Biochim. Biophys. Res. Comm. 255, 245-250
35. Fimia, G. M., De Cesare, D,. and Sassone-Corsi, P. (1999) Nature 398, 165-169
36. Sonnenberg, A., Linders, C. J. T., Modderman, P. W., Damsky, C. H., Aumailley, M., and
Timpl, R. (1990) J. Cell Biol. 110, 2145-2155
37. Rousselle, P,. and Aumailley, M. (1994) J .Cell Biol. 125, 205-214
38. Sondermann, H., Dogic, D., Pesch, M., and Aumailley, M. (1999) Cell Adh. Commun. 7,
43-57
39. Chen, R. H., Sarnecki, C., and Blenis, J. (1992) Mol. Cell Biol. 12, 915-927
40. Wessel, D,. and Flügge, U. (1984) Anal. Biochem. 138, 141-143
41. Hemler, M. E., Sanchez-Madrid, F., Flotte, T. J., Krensky, A. M., Burakoff, S. J., Bhan, A.
K., Springer, T. A., and Strominger, J. L. (1984) J. Immunol. 132, 3011-3018
42. Morgan, M. J., and Madgwick, A. J. (1996) Biochem. Biophys. Res. Comm. 225, 632-638
43. Chan, K. K., Tsui, S. K., Lee, S. M., Luk, S. C., Liew, C. C., Fung, K. P., Waye, M. M., and
Lee, C. Y. (1998) Gene 210, 345-350
Running title : DRAL/FHL2 binds to α and β integrin subunits
30
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
44. Kassner, P. D., Kawaguchi, S., and Hemler, M. E. (1994) J. Biol. Chem. 269, 19859-19867
45. Kawaguchi, S., Bergelson, J. M., Finberg, R. W., and Hemler, M. E. (1994) Mol. Biol. Cell
5, 977-988
46. Marcantonio, E. E., Guan, J.- L., Trevithick, J. E, and Hynes, R. O. (1990) Cell Regul. 1,
597-604
47. Reszka, A. A., Hayashi, Y., and Horwitz, A. F. (1992) J. Cell Biol. 117, 1321-1330
48. Mastrangelo, A. M., Homan, S. M., Humphries, M. J. and LaFlamme, S. E. (1999) J. Cell
Sci. 112, 217-229
49. Feuerstein, R., Wang, X., Song, D., Cooke, N. E., and Liebhaber, S. A. (1994) Proc. Natl.
Acad. Sci. USA 91, 10655-10659
50. Vallar, L., Melchior, C., Plançon, S., Drobecq, H., Lippens, G., Regnault, V., and Kieffer, N.
(1999) J. Biol. Chem. 274, 17257-17266
51. Sudol, M. (1998) Oncogene 17, 1469-1474
52. Haas, T. A., and Plow, E. F. (1997) Protein Eng. 10, 1395-1405
53. Otey, C. A., Vasquez, G. B., Burridge, K., and Erickson, B. W. (1993) J. Biol. Chem. 268,
21193-21197
54. Pfaff, M., Liu, S., Erle, D. J., and Ginsberg, M. H. (1998) J. Biol. Chem. 273, 6104-6109
55. Retta, S. F., Balzac, F., Ferraris, P., Belkin, A. M., Fässler, R., Humphries, M. J., De Leo, G.,
Silengo, L., and Tarone, G. (1998) Mol. Biol. Cell 9, 715-731
56. Sampath, R., Gallagher, P. J., and Pavalko, F. M. (1998) J. Biol. Chem. 273, 33588-33594
57. Kaapa, A., Peter, K., and Ylanne, J. (1999) Exp. Cell Res. 250, 524-534
Running title : DRAL/FHL2 binds to α and β integrin subunits
31
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
58. Eigenthaler, M., Hofferer, L., Shattil, S. J., and Ginsberg, M. H. (1997) J. Biol. Chem. 272,
7693-7698
59. Konrat, R., Krautler, B., Weiskirchen, R., and Bister, K. (1998) J. Biol. Chem. 273, 23233-
23240
60. Tu, Y., Li, F., Goicoechea, S., and Wu, C. (1999) Mol. Cell Biol. 19, 2425-2434
61. Müller, J. M., Isle, U., Metzger, E., Rempel, A., Moser, M., Pscherer, A., Breyer, T.,
Holubarsch, C., Buetttner, R., and Schüle, R. (2000) EMBO J. 19, 359-369
62. Chan, K. K., Tsui, S. K. W., Ngai, S.- M., Lee, S. M. Y., Kotaka, M., Waye, M. M. Y., Lee,
C.- Y., and Fung, K.- P. (2000) J. Cell. Biochem. 76, 499-508
63. Hobert, O., Moerman, D. G., Clark, K. A., Beckerle, M. C., and Ruvkun, G. (1999) J. Cell
Biol. 144, 45-57
64. Perez-Alvarado, G. C., Miles, C., Michelsen, J. W., Louis, H. A., Winge, D. R., Beckerle,
M. C., and Summers, M. F. (1994) Nat. Struct. Biol. 1, 388-398
65. Perez-Alvarado, G. C., Kosa, J. L., Louis, H. A., Beckerle, M. C., Winge, D. R., and
Summers, M. F. (1996) Mol. Biol. 257, 153-174
66. Kowalski, K., Czolij, R., King, G. F., Crossley, M., and Mackay, J. P. (1999) J. Biomol.
NMR. 13, 249-262
Running title : DRAL/FHL2 binds to α and β integrin subunits
32
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
FOOTNOTES
1) V. Wixler and K. von der Mark, unpublished observations.
Running title : DRAL/FHL2 binds to α and β integrin subunits
33
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
FIGURE LEGENDS
Figure 1.
Interaction of DRAL/FHL2 with the integrin α3A subunit in human cells
(A) Association of DRAL/FHL2 with the cytoplasmic domain of the integrin α3A
subunit. HEK293 cells were transiently transfected with different GST- and myc-tagged
constructs as indicated. After 48 h, cell lysates were divided into two parts. From one aliquot,
proteins were precipitated with the antibody 9E10 against the myc tag followed by protein G-
conjugated agarose for precipitation of myc-tagged proteins. While with the other aliquot,
proteins were precipitated with glutathion-conjugated Sepharose beads for GST-tagged
proteins. After SDS-PAGE and immunoblottting, the cytoplasmic domain of the integrin α3A
subunit was only detected in precipitates from cells where the integrin peptide had been co-
expressed with myc-tagged DRAL/FHL2 (left blot, lane 4). In the reverse experiment
(glutathion-precipitated complexes) myc-tagged DRAL/FHL2 was detected only when co-
expressed with GST-tagged α3A peptide (right blot, lane 4), but not with GST alone. After the
first immunodetection, the blots were stripped and redeveloped with polyclonal antibody against
recombinant DRAL/FHL2 (bottom, left blot) or against GST (bottom, right blot) to ascertain that
myc-tagged DRAL/FHL2 and GST-tagged α3A peptide were equivalently precipitated.
(B) Association of DRAL/FHL2 with the full-length integrin α3A subunit. HEK293
cells were transiently transfected with different cDNA constructs as indicated. After cell lysis,
myc-tagged DRAL/FHL2 was immunoprecipitated with antibody against myc and the
Running title : DRAL/FHL2 binds to α and β integrin subunits
34
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
precipitated proteins were visualised by immunoblotting with antibodies against the cytoplasmic
tail of the integrin α3A subunit (upper blot), or against myc (lower blot).
Figure 2.
Identification of a DRAL/FHL2-binding site in the unique amino acid sequence of the
integrin α3A subunit cytoplasmic domain.
Yeast Y190 cells were transformed with pAS2-1 plasmid expressing the indicated
deletion mutants of the integrin α3A subunit and with DRAL/FHL2 in the pACT2 vector. The
interaction was determined and scored as described in the legend to table I. Numbers in
parentheses refer to amino acid positions. The conserved membrane-proximal region is marked.
Figure 3.
Identification of the major DRAL/FHL2-binding site in the C-terminal cyto-3 region of
integrin β1A subunit.
Yeast Y190 cells were transformed with pAS2-1 plasmid expressing the indicated
deletion mutants of the integrin β1A subunit and with DRAL/FHL2 in the pACT2 vector. The
interaction was determined and scored as described in the legend to table I. Numbers in
parentheses refer to amino acid positions. The conserved cyto-1, -2, and -3 regions are
highlighted.
Figure 4.
Running title : DRAL/FHL2 binds to α and β integrin subunits
35
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
Interaction of DRAL/FHL2 with the integrin α7A and β1A subunits in human cells.
HEK293 cells were transiently transfected with cDNA constructs as indicated and lysed
48 h later. GST-tagged proteins were precipitated as described in the legend to figure 2. After
SDS-PAGE and electrophoretic transfer, the blots were incubated with antibodies against myc
(upper blot) and, after stripping, with antibodies against GST (lower blot).
Figure 5.
Association of DRAL/FHL2 with itself and with AIBP80 in human cells.
After transient transfection with the indicated cDNA constructs, lysates of HEK293 cells
were precipitated as descibed in figure 2. Co-precipitating proteins were identified by
immunoblotting with the indicated antibodies. Myc-tagged DRAL/FHL2 co-migrates with the
IgG heavy chain and could therefore not be visualised with the antibodies against myc.
Figure 6.
Identification of sites on DRAL/FHL2 that bind to integrin subunits, to AIBP80 or are
required for DRAL/FHL2 self-interaction.
(A) Yeast Y190 cells were co-transformed with one of the cDNA fragments encoding
DRAL/FHL2 deletion mutants inserted into the pACT2 vector, and with the cDNA fragments
encoding one of the ligand proteins inserted into the pAS2-1 vector. The numbers in parentheses
indicate DRAL/FHL2 amino acids encoded by the corresponding constructs. The interactions
were detected by growth on His- medium and in a β-Gal filter assay. Scoring was as described
Running title : DRAL/FHL2 binds to α and β integrin subunits
36
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
in the legend to table I.
(B) Schematic representation of principal binding sites on DRAL/FHL2 identified in (A).
Figure 7.
Expression of DRAL/FHL2 in human skin fibroblasts and established cell lines (A) and
subcellular distribution of DRAL/FHL2 in human skin fibroblasts (B).
(A) Total lysates (20 µg of proteins per lane) of the indicated cells and (B) equal samples
of nuclear (N), cytosolic (C) and membrane (M) fractions from human skin fibroblasts were
separated by SDS-PAGE, electrophoretically transfered to nitrocellulose and the blots were
developed with a polyclonal antiserum against DRAL/FHL2 fusion protein.
Figure 8.
Localisation of DRAL/FHL2 to adhesion complexes in normal human skin fibroblasts.
The cells were cultured overnight in DMEM containing 10% FCS, fixed and double
stained with rabbit polyclonal antiserum against DRAL/FHL2 together with the mouse
monoclonal antibody TS2/16 (a) against the integrin β1 subunit or F-VII (c) against vinculin,
followed by the appropriate fluorescence-labeled second antibodies or by FITC-conjugated
phalloidin (e). Specimens were examined and photographed under epifluorescence microscopy
using separate detection channels (A,B,D,E,G,H). Superimposed photographs show that the
integrin β1 subunits (C) and vinculin (I) are, but not always, colocalised with DRAL/FHL2.
Superimposition of actin and DRAL/FHL2 images (F) reveals that DRAL/FHL2 is localised at
Running title : DRAL/FHL2 binds to α and β integrin subunits
37
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
the far ends of actin stress fibers which usually terminate in cell adhesion complexes.
Figure 9.
Immunofluorescence staining of human skin fibroblast foot-prints.
Fibroblasts were subjected to osmotic shock either without (a,c,e) or with (b,d,f) previous
crosslinking of proteins with DSP and further processed for immunofluorescence staining with
the mouse monoclonal antibody K20 (a,b) against integrin β1 subunit or F-VII (c,d) against
vinculin, or the rabbit polyclonal antiserum against DRAL/FHL2 (e,f).
Figure 10.
Only full-length DRAL/FHL2 is targeted to cell adhesion complexes.
Mouse 3T3 fibroblasts were transiently transfected with constructs representing myc-
tagged full-length (A), N-terminal (B) or carboxy-terminal (C) half of DRAL/FHL2. The
truncated versions of the protein corresponded to LIM1/2-2 (amino acids 1-157) or LIM 2-4
(amino acids 97-279) as represented in figure 6A. The cells were cultured on fibronectin-coated
coverslips in DMEM containing 10% FCS for 60 minutes. After fixation, they were stained with
mouse monoclonal antibody 9E10 against the myc tag followed by Cy3-conjugated second
antibodies.
Running title : DRAL/FHL2 binds to α and β integrin subunits
38
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
Running title : DRAL/FHL2 binds to α and β integrin subunits
39
Table I
Specific interaction of the cytoplasmic domain of integrin α3A subunit with
DRAL/FHL2 in a yeast two-hybrid binding assay.
-----------------------------------------------------------------------------------------------------------
Bait Prey Reporter gene
--------------------------------------------
His expression LacZ activity
-----------------------------------------------------------------------------------------------------------
pAS2-1/α3A pACT2/DRAL/FHL2 + +++
pAS2-1/α3A pACT2 - -
pAS2-1 pACT2/DRAL/FHL2 - -
pAS2-1/Lamin C pACT2/DRAL/FHL2 - -
-----------------------------------------------------------------------------------------------------------
Yeast Y190 cells were co-transformed with the indicated combinations of bait and prey.
The activation of the first reporter gene was determined by growth on His- medium and
expression of the second reporter gene, LacZ, evaluated in a β-Gal filter assay. The interaction
was scored as negative (-) when no blue colonies were visible after 8h; the interaction was scored
as: weak (+), intermediate (++) or strong (+++) when blue colonies became visible after 8 h, 4 h
or 1 h, respectively. The ubiquitously expressed lamin C was used as a negative control.
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
Running title : DRAL/FHL2 binds to α and β integrin subunits
40
Table II
Interaction of DRAL/FHL2 with cytoplasmic domains of different integrin α and β
subunits in a yeast two-hybrid assay.
-----------------------------------------------------------------------------------------------------------
α subunit bait LacZ activity β subunit bait LacZ activity
-----------------------------------------------------------------------------------------------------------
α1 - β1A +++
α2 - β1D ++
α3A +++ β2 ++
α3B ++ β3A ++
α5 - β6 ++
α6A - β2 cyto-1 +++
α6B - β3A cyto-1 +++
α7A +++ β6-11 +++
α7B -
-----------------------------------------------------------------------------------------------------------
Interaction between the cytoplasmic domain of integrin subunits in pAS2-1 vector and
DRAL/FHL2 in pACT2 vector were determined as described in the legend to Table I. The
deletion mutants of β2 cyto-1 and β3A cyto-1 regions are based on the corresponding integrin
subunit cytoplasmic domains and contain, respectively, the last 23 and 24 C-terminal amino acid
residues, including the cyto 2 and cyto 3 regions, but lacking cyto-1. In the β6-11 construct, the
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
Running title : DRAL/FHL2 binds to α and β integrin subunits
41
last 11 C-terminal amino acid residues of the integrin β6 subunit cytoplasmic domain were
deleted, but the cyto-3 region is preserved. All other constructs represent the full length
cytoplasmic domains. Scoring was as described in the legend to Table I.
Table III
Interaction of DRAL/FHL2 with itself and with other integrin α subunit-binding proteins.
---------------------------------------------------------
Proteins β-Gal activity
---------------------------------------------------------
DRAL/FHL2 +++
AIBP80 +++
BIN1 ++
AIBP63 -
Mss4 -
--------------------------------------------------------- The DRAL/FHL2 in pAS2-1 vector was tested in direct two-hybrid binding assays with the different integrin-binding proteins, including DRAL/FHL2 itself, in pACT2 vector. Positive clones were scored as described in the legend to Table I.
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from
Aumailley, Arnoud Sonnenberg and Mats PaulssonViktor Wixler, Dirk Geerts, Emmanuel Laplantine, Daniel Westhoff, Neil Smyth, Monique
integrin chains and is recruited to adhesion complexesβ and αThe LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several
published online July 21, 2000J. Biol. Chem.
10.1074/jbc.M002519200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on January 25, 2019http://w
ww
.jbc.org/D
ownloaded from