JBC Papers in Press. Published on July 21, 2000 as ... · The LIM-only protein DRAL/FHL2 binds to...

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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 Wixler 3 , Dirk Geerts 1 , Emmanuel Laplantine, Daniel Westhoff, Neil Smyth, Monique Aumailley 2 , Arnoud Sonnenberg 1 and Mats Paulsson. Institute for Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany and 1 The 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 2 Corresponding 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] 3 Present 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://www.jbc.org/ Downloaded from

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

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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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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.

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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)

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(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

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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.

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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.

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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

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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

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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

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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,

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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

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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.

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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

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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,

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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-

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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

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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

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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

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(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

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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.

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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

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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

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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

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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

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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

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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.

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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).

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FOOTNOTES

1) V. Wixler and K. von der Mark, unpublished observations.

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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

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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.

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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

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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

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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.

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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.

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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

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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.

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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:

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