Institute of Biochemistry and Molecular Cell Biology, Vienna Biocenter, 1030 Vienna, Austria
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Abstract |
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Recent studies with patients suffering from
epidermolysis bullosa simplex associated with muscular
dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and
intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its
interaction with the uniquely long cytoplasmic tail domain of the 4 subunit of the basement membrane laminin receptor integrin
6
4 that has been implicated in
connecting the transmembrane integrin complex with
hemidesmosome-anchored cytokeratin filaments. In
vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains.
Furthermore, we show in vitro self-interaction of integrin
4 cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of
hemidesmosome-associated endogenous plectin upon
ectopic overexpression of this domain in PtK2 and/or
804G cells. The close association of plectin molecules
with hemidesmosomal structures and their apparent
random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our
data support a model in which plectin stabilizes
hemidesmosomes, via directly interlinking integrin
4
subunits and cytokeratin filaments.
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Introduction |
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INTEGRINS comprise a large family of heterodimeric receptors that mediate the adhesion of cells to extracellular matrices and other cells (Buck and Horwitz,
1987; Hynes, 1987
, 1992
; Ruoslahti and Pierschbacher,
1987
; Ginsberg et al., 1988
; Hemler, 1990
; Springer, 1990
;
Watt et al., 1993
). In addition, they are involved in transducing extracellular signals into the cell (Hynes, 1992
; Juliano and Haskill, 1993
; Giancotti and Mainiero, 1994
).
Both the
and
subunits of integrins have a large extracellular portion, a transmembrane segment, and generally
a short cytoplasmic domain. The cytoplasmic domains of
integrins interact with the cytoskeleton and possibly with
signaling molecules, but the molecular mechanisms of
these interactions are not well understood.
The 6
4 integrin is a basement membrane receptor for
laminins (Kajiji et al., 1989
; De Luca et al., 1990
; Sonnenberg et al., 1990a
,b; Lee et al., 1992
) that engages in cytoplasmic interactions distinct from those of all other known
integrins. Instead of being localized at adhesion plaques
that serve as anchoring structures of actin filament networks, this receptor is part of hemidesmosomes (Carter et al.,
1990
; Stepp et al., 1990
; Jones et al., 1991
; Sonnenberg et al.,
1991
), junctional complexes that anchor cytokeratin intermediate filament (IF)1 networks and mediate adhesion of
epithelial cells to the underlying basement membrane. The
intracellular interactions of integrin
6
4 are mediated by
the
4 subunit, the intracellular portion of which is much
larger (~1,000 amino acids) than that of all the other
known
subunits (~50 amino acids) and bears no apparent sequence homology to them (Hogervorst et al., 1990
;
Suzuki and Naitoh, 1990
; Tamura et al., 1990
). It contains
four regions with homology to fibronectin type III (FNIII)
repeats, arranged in two pairs separated by a 143-amino
acid-long connecting segment. This large cytoplasmic tail
of integrin
4 is required, and probably sufficient, for incorporation of the integrin into hemidesmosomes. Specifically, a minimal region on the integrin
4 subunit located in the linking segment between the second and third repeat has been reported to be critical to its localization in
hemidesmosomes (Niessen et al., 1997a
). The targeted inactivation of the integrin
4 gene in mice convincingly
demonstrated that hemidesmosome formation and proper
skin attachment to the basal lamina are crucially dependent on the expression of this integrin subunit (Dowling
et al., 1996
; van der Neut et al., 1996).
Cytoplasmic components of the hemidesmosome that
have been implicated in the attachment of IFs to this adhesion structure include the bullous pemphigoid antigen 1 (Stanley et al., 1981), also referred to as BP230 or
BPAG1e (Yang et al., 1996
), plectin (Wiche et al., 1984
),
the 200-kD 6A5 antigen (P200; Kurpakus and Jones,
1991
), HD1 (Owaribe et al., 1991
; Hieda et al., 1992
), and
the IF-associated protein 300 (Skalli et al., 1994
). Among these, plectin, IF-associated protein 300, and HD1 have
been shown to bind to IFs in vitro (Foisner et al., 1988
;
Skalli et al., 1994
; Fontao et al., 1997
), and the IF-binding
site of plectin has recently been mapped to the fifth repeat
domain within the carboxy-terminal globular region of the
molecule (Nikolic et al., 1996
). Also, BP230, which shares
partial sequence homology with plectin and desmoplakin
and is structurally related to these molecules (Tanaka et
al., 1991
; Green et al., 1992
; Klatte and Jones, 1994
), is
likely to bind to IFs (Yang et al., 1996
). A direct interaction of the intracellular domain of the integrin
4 subunit
with any of the cytoplasmic hemidesmosome-associated
proteins has not been demonstrated to date. There is evidence that integrin
4 interacts with HD1, based on coimmunoprecipitation of purified recombinant integrin
4
polypeptides expressed in bacteria with HD1 present in
COS-7 cell lysates, and the observation that overexpression of integrin
4 leads to a redistribution of HD1 in
transfected cells (Sánchez-Aparicio et al., 1997
; Niessen
et al., 1997a
,b), but the question whether this interaction
was of direct or indirect nature remains unsolved.
Plectin, the most versatile cytoskeletal linker protein
characterized to date, remains a strong candidate for
bridging cytokeratin filament networks to hemidesmosomes. In fact, due to the very large size (>500 kD) of
plectin molecules predicted on the basis of cDNA sequencing (Wiche et al., 1991; Liu et al., 1996
; Elliott et al.,
1997
), and their extended (~200-nm-long) multi-domain structure, as visualized by electron microscopy (Foisner
and Wiche, 1987
), plectin molecules would have the dimensions to span the entire tripartite structure characteristic of hemidesmosomes. In this way, they could extend
from the plasma membrane, the location of integrin
4, to
the filament anchorage site at the hemidesmosome inner
plate structure. A role of plectin in hemidesmosome stabilization by physical linkage of proteins constituting this
multi-component complex appears likely also in light of
recent studies showing that defects in plectin expression
lead to epidermolysis bullosa simplex (EBS)-MD, a severe
hereditary skin blistering disease combined with muscular
dystrophy (Chavanas et al., 1996
; Gache et al., 1996
;
McLean et al., 1996
; Pulkkinen et al., 1996
; Smith et al.,
1996
). Moreover, plectin-deficient mice generated by targeted gene inactivation showed severe skin blistering and
degeneration of keratinocytes, apparently caused by a significant reduction in number and mechanical stability of
epidermal hemidesmosomes (Andrä et al., 1997
).
The aim of the work presented here was to extend the
human disease and plectin gene-knockout studies to the
molecular level, and to investigate whether plectin directly
interacts with integrin 4, and if so, to define the molecular
domains of both proteins involved in this interaction. In
addition, we were interested in the consequences that
overexpression of integrin
4 mutant proteins containing
putative plectin-binding sites might have on the integrity
of cytokeratin filament networks and on the subcellular distribution of plectin. We show here that partially truncated versions of the integrin
4 cytoplasmic domain expressed in bacteria bind to recombinant carboxy- and
amino-terminal domains of plectin in two independent in
vitro binding assays, and that protein species shown to interact in vitro colocalized when ectopically coexpressed in living cells. In addition, we observed self-association
of integrin
4 mutant proteins in vitro, and show that plectin proteins can modulate this effect. Furthermore, carboxy-terminal integrin
4 mutant proteins lacking plasma
membrane anchoring sequences, but comprising plectin-binding region(s) in their tail domain, are shown to cause
bundling and collapse of IF network arrays upon overexpression in PtK2 and 804G cells, and similar fragments dislocated endogenous plectin from hemidesmosomes in
804G cells.
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Materials and Methods |
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cDNA Constructs
Integrin 4.
The integrin
4 subunit protein and cDNA sequences were
numbered according to Suzuki and Naitoh (1990; Genbank/EMBL/DDBJ
accession number X51841). Nucleotide and primer numbers refer to the nucleotide position (3') relative to the first bp of the start codon (position
127 in X51841). For cloning, a part of the cytoplasmic tail region of integrin
4 was amplified by PCR from human placenta cDNA (Quick-Clone;
CLONTECH Laboratories, Inc., Palo Alto, CA) using primers U3330 (5'-GAG CTT CAC GAG TCA GAT GTT GTC-3') and L5250 (5'-GGG
GCA GGG TGC GGT CAA GTT TGG-3'). A mixture of Klen-Taq (AB
Peptides, St. Louis, MO) and PfuI (Stratagene, Heidelberg, Germany)
polymerases was used under high fidelity conditions described by Barnes
(1994)
. The 1944 bp PCR product obtained was used as template for
nested PCR with EcoRI-tailed primers and the amplified fragments were
subcloned into the unique EcoRI site of the bacterial expression vector
pBN120 (Nikolic et al., 1996
), a derivative of pET-15b (Novagen Inc.,
Madison, WI). Clones generated encoded the following domains of the integrin
4 subunit:
4-F1,2 (amino acid residues 1,126-1,315; plasmid construct pGR1),
4-L (1,316-1,457; pGR2),
4-F3,4C'(1,486-1,752; pGR3),
4-F2L (1,219-1,457; pGR4),
4-F1,2L'(1,126-1,485; pGR5), and
4-F1,2LF3,4C (1,126-1,752; pGR6). The clone encoding
4-F3,4 (1,457-1,662;
pJP5) was generated by PCR from pGR6 (
4-F1,2LF3,4C; see Fig. 1 A for
an overview). The correctness of all PCR-generated clones was verified by
DNA sequencing. The clone encoding
4-F1,2L (1,126-1,457; pGR36) was
obtained by exchanging the SmaI/PstI fragment from pGR5 with that of
pGR4. Plasmids encoding
4-LF3,4C (1,316-1,752; pGR37) and
4-F3,4C
(1,457-1,752; pGR44) were constructed by exchanging the SmaI/HindIII
fragment of pGR2 with that of pGR6 and the XbaI/NotI fragment of
pGR6 with that of pJP5, respectively. For expression of carboxy terminally c-myc-tagged proteins in mammalian cells, EcoRI fragments were
subcloned into pAD29 (Nikolic et al., 1996
), and excised XbaI/HindIII
fragments were subcloned into the eukaryotic expression vector pRc/
CMV (Invitrogen Corp., San Diego, CA). This yielded the mammalian expression constructs encoding
4-F3,4C'myc (pGR13, derived from pGR3),
4-F1,2myc (pGR16, derived from pGR1),
4-F1,2L'myc (pGR19, derived
from pGR5), and
4-F1,2LF3,4Cmyc (pGR20, derived from pGR6). In addition, the EcoRI fragment from pGR6 was also subcloned into the eukaryotic expression vector pGR29, a modified version of pEGFP-N3 (CLONTECH Laboratories, Inc.). The resulting protein encoded by this
construct (
4-F1,2LF3,4CGFP; pGR30) carried an enhanced version of the
Aequorea victoria green fluorescent protein (GFP) at its carboxy terminus.
Correct expression of proteins encoded by mammalian vector constructs was verified by Western blot analysis of lysates of transfected 804G cells.
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Plectin.
Rat plectin cDNA constructs were generated by PCR and/or
other cloning techniques on the basis of the complete rat cDNA sequence (Genbank/EMBL/DDBJ database entry X59601). All constructs used were subcloned into the bacterial expression vector pBN120 (Nikolic et al.,
1996), which enabled expression of amino terminally his-tagged proteins,
except for the ones encoding Ple-N1 (pGR48) and Ple-R3-6T (pGR49),
which were inserted into the unique EcoRI site of pFS23, a pET23a derivative driving the expression of carboxy terminally his-tagged proteins.
Plectin constructs encoded the following segments of the protein (see Fig.
1 B for an overview): Ple-N1 (amino acids 1-1,128; construct pGR48), Ple-N2 (546-1,128; pMZ4), Ple-R1 (2,777-3,161; pJD11), Ple-R1-3 (2,777-3,851;
pJD22), Ple-R3-6T (3,346-4,687; pGR49), Ple-R3 (3,346-3,851; pJD21),
Ple-R4-6T (3,850-4,687; pJD23), Ple-R4,5 (3,780-4,367; pMZ3), Ple-R4
(3,780-4,024; pBN135), Ple-R5 (4,025-4,367; pBN132), Ple-R6T (4,262-
4,687; pMZ5), and Ple-R6 (4,277-4,620; pBN144). For expression in mammalian cells, EcoRI fragments of pGR48 (Ple-N1) and pGR49 (Ple-R3-6T)
were subcloned into the eukaryotic expression vector pGR29, resulting in
constructs encoding GFP-tagged Ple-N1GFP (pGR31) and Ple-R3-6TGFP
(pGR33). The latter plectin fragment was also expressed as a c-myc-tagged
version (Ple-R3-6Tmyc), encoded by pBN72, a pRc/CMV derived plasmid.
Cell Culture, DNA Transfection, and Immunofluorescence Microscopy
Rat kangaroo PtK2 (CCL 56; American Type Culture Collection, Rockville, MD) and rat bladder carcinoma 804G cells (Izumi et al., 1981) were
cultured in DME, supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% of heat-inactivated fetal calf serum, at 37°C and 5% CO2.
For transfection, cells were grown on glass coverslips and, while still subconfluent, transiently transfected with either 30 µg plasmid DNA per 10-cm
plate using the calcium phosphate precipitation method (Graham and
van der Eb, 1973
) or 0.6 µg plasmid DNA per 13-mm coverslip in wells of 24-well tissue culture plates (Falcon Plastics, Cockeysville, MD) using the
LipofectAMINE (GIBCO BRL, Paisley, Scotland) transfection method.
In the latter case, the transfection medium (0.6 ml/coverslip) contained
DNA and LipofectAMINE at final concentrations of 1 µg/ml and 8 µg/ml,
respectively, in serum-free medium (Opti-MEM, GIBCO BRL); it was replaced with DME/10% FCS after 4.5 h of incubation at 37°C and 5% CO2.
Cells were fixed 24-48 (PtK2) or 8-24 h (804G) after transfection using
chilled (
20°C) methanol and processed for immunofluorescence microscopy as previously described (Wiche et al., 1993
). The following immunoreagents were used: rabbit anti-human
4-antiserum (Giancotti et al.,
1992
; kindly provided by F.G. Giancotti), mouse monoclonal anti-rat
plectin antibody (mAb) 5B3 (Foisner et al., 1994
), guinea pig anti-mouse
liver cytokeratin antibody (Denk et al., 1981
; kindly provided by H. Denk), mouse monoclonal anti-vimentin antibody (clone V9; DAKOPATTS, Copenhagen, Denmark), and anti-myc monoclonal antibody
1-9E10.2 (American Type Culture Collection) as primary antibodies; and AMCA-, fluorescein (FITC)- or Texas red-conjugated AffiniPure donkey
anti-mouse IgG (H+L), anti-rabbit IgG (H+L), and anti-guinea pig IgG
(H+L; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as
secondary antibodies. Specimens were viewed in a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Oberkochen, Germany) or using the
Bio-Rad MRC600 confocal scanning laser microscope (Richmond, CA).
Photographs were taken using Ilford ASA 400 black and white film, and
digital images were processed using the NIH Image 1.59 and Adobe Photoshop 4.01 software packages.
Expression of Recombinant Proteins in Bacteria
His-tagged recombinant proteins encoded by pBN120 or pFS23 vector
constructs were expressed in Escherichia coli BL21(DE3) and purified
from inclusion bodies by solubilization in 6 M urea, 500 mM NaCl, 20 mM
Tris-HCl, pH 7.9 (binding buffer) containing 5 mM imidazole, followed by
affinity binding to His-Bind metal chelation resin, according to the manufacturer's (Novagen Inc.) protocol. Bound proteins were eluted from affinity columns using 250 mM imidazole in binding buffer and stored frozen at 20°C. Samples were dialyzed against desired buffers before use to
remove urea.
Gel Electrophoresis, Immunoblotting, and Preparation of 804G Cell Lysates
SDS-polyacrylamide gel electrophoresis (PAGE) was carried out under
reducing conditions (Laemmli, 1970). Proteins on gels were visualized by
staining with Servablue-G (Serva, Heidelberg, Germany). For immunoblotting, proteins were transferred to nitrocellulose sheets (Schleicher & Schuell, Dassel, Germany) for 4 h at 35 V in 25 mM Tris, 191 mM glycine,
0.01% SDS. After blocking with 3% BSA in TBS-T (TBS, containing
0.1% Tween-20), membranes were incubated with mAb 5B3 (1 h, room
temperature), washed with TBS-T, and incubated with alkaline phosphatase-conjugated secondary goat anti-mouse antibody (Promega,
Heidelberg, Germany). After washing, antibody binding was visualized
with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in
100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2.
Lysates of 804G cells were prepared by boiling cells in Laemmli's sample buffer (400 µl per confluent 10-cm plate) for 5 min.
Eu3+ Labeling of Recombinant Proteins and Microtiter Plate Overlay Assay
Recombinant mutant proteins were labeled with Eu3+ following the recommendations provided by the manufacturer (Wallac, Turku, Finland).
50-200 µg of recombinant integrin (4-F1,2,
4-F1,2LF3,4C,
4-F1,2L,
4-LF3,4C,
4-F3,4C,
4-F3,4) and plectin (Ple-N1, Ple-R3-6T) mutant proteins
were dialyzed against labeling buffer (50 mM sodium carbonate, pH 8.5)
overnight at 4°C, and subsequently incubated with the labeling reagent
(Eu3+-chelate of N1-(p-isothiocyanatobenzyl)-DTTA) at room temperature
for ~30 h. Unreacted labeling reagent was removed by gel filtration
through a P6 column (Bio-Rad) equilibrated with 50 mM Tris-HCl, pH
7.5, 0.9% NaCl, 0.01% NaN3. Proteins were stored at 4°C until use. Microtiter plates were coated with recombinant proteins expressed in bacteria
(100 µl of a 100 nM solution in 25 mM sodium borate buffer, pH 9.2) overnight at 4°C. Blocking was carried out with 4% BSA in TBS for 1 h and
then binding with Eu3+-labeled proteins in 100 µl overlay buffer (TBS, pH
7.5, containing 1 mM EGTA, 2 mM MgCl2, 1 mM DTT, and 0.1% Tween
20) for 90 min at room temperature. After extensive washing with overlay
buffer, the amount of bound proteins was determined by releasing complexed Eu3+ with enhancement solution and measuring the fluorescence
with a Delfia time-resolved fluorometer (Wallac). The fluorescence values
were converted to concentrations by comparison with an Eu3+ standard.
125I Labeling of Proteins and Blot Overlay Assay
The recombinant integrin 4 mutant protein
4-F1,2LF3,4C (~0.8 mg) was
dialyzed against 20 mM Tris-HCl, pH 7.5, and incubated with one Iodo-Bead (Pierce, Rockford, IL) and 0.5 mCi of Na125I (DuPont-NEN, Boston, MA) at room temperature for 15 min. Free iodine was removed by
gel filtration using a Sephadex G-25 (Pharmacia Biotech Sverige, Uppsala, Sweden) column (0.9 × 14 cm) equilibrated with 20 mM Tris-HCl,
pH 7.5, 1 mM DTT. Labeled proteins were stored at 4°C until use.
Purified recombinant integrin and plectin mutant proteins and lysates
of induced bacterial cultures expressing plectin protein fragments were
analyzed by SDS-10% PAGE. Proteins were blotted onto 0.2-µm nitrocellulose membranes (Schleicher & Schuell), overnight in 25 mM Tris, pH
8.8, and 192 mM glycine, at 25 V using a wet mini-blot apparatus (Bio-Rad). Membranes were incubated with 0.1% gelatin (Merck, Darmstadt,
Germany) in PBS for 4 h at room temperature, and rinsed once with
TSDT (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT, 1% (vol/vol)
Triton X-100). After incubation with 10 µg/ml of 125I- 4-F1,2LF3,4C in
TSDT containing 0.5% BSA for 4 h and washing with TSDT, sheets were
dried, sealed in plastic bags, and X-Omat film (Eastman Kodak, Rochester, NY) was exposed to them.
Immunoelectron Microscopy
Vascular perfusion of Wistar rats was performed with 4% paraformaldehyde in PBS, pH 7.4. Skin samples were immersed in fresh paraformaldehyde solution for 30 min and embedded in lowicryl-HM20 (Agar Scientific
Ltd., Stansted, UK) as described in detail by Villinger (1991). Small pieces
(~1-2 mm2) of fixed rat skin were rinsed in PBS and dehydrated in a series of ethanol dilutions at
20°C and ethanol finally replaced by overnight infiltration of pure lowicryl. Samples were transferred into transparent tubes (Eppendorf, Hamburg, Germany) that were filled with freshly
prepared lowicryl. Polymerization was complete after 24 h of exposure to
UV light
28°C.
Lowicryl thin sections (60-80 nm) cut with an ultramicrotome (Ultracut
S; Reichert, Vienna, Austria) were mounted on formvar-coated nickel slot
grids. Postembedding immunolabeling was performed as previously described (Polak and Varndell, 1984). Free aldehyde groups were reduced
by incubating sections in 0.1 M glycine in PBS, three times for 5 min, and
blocking was performed in normal goat serum (BioCell Res. Lab, Cardiff,
UK) for 30 min. For immunolabeling, sections were incubated for 1 h at
room temperature, or overnight at 4°C with one of the following domain-specific anti-plectin antibodies: a) anti-rod mAb 7A8 (Foisner et al.,
1996), b) anti-amino terminus rabbit serum E1A raised against a synthetic
peptide corresponding to a 12-amino acid residue-long sequence of human exon 1a, and c) anti-carboxy terminus mouse serum 135C raised
against a recombinant rat plectin mutant protein corresponding to the carboxy-terminal repeat 4 domain (see Andrä et al., 1997
), or with non-
immune serum or PBS as controls. Secondary labeling using 5 nm gold-conjugated goat-anti-mouse IgG and 1 nm gold-conjugated goat-anti- rabbit IgG (BioCell Res. Lab) was performed for 1 h at room temperature. For
visualization in the electron microscope, 1 nm or 5 nm colloidal gold particles were silver enhanced (Stierhof et al., 1991
). Thin sections were counterstained with uranyl acetate and lead citrate and were viewed at 80 kV
in a JEOL JEM-1210 electron microscope.
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Results |
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Expression and Purification of Recombinant Forms of
Integrin 4 and Plectin
Localization of plectin at hemidesmosomes (Wiche et al.,
1984) and its function as an IF (cytokeratin)-binding protein make it a likely candidate to act as a linker between
the cytokeratin filament network and the hemidesmosomal integrin
6
4. To test whether this proposed function of plectin involves its direct interaction with the
uniquely long cytoplasmic tail of the
4 subunit of integrin
6
4, we generated a series of human integrin
4 and rat
plectin truncation mutants (Fig. 1) for use in biochemical in vitro binding, as well as in vivo cell transfection assays.
The original design of our integrin 4 expression constructs was based on a report by Spinardi et al. (1993)
, who
showed that a 303-amino acid-long region of the cytoplasmic tail comprising the first of its two pairs of FNIII repeats and the following segment between the first and second pair was necessary for the recruitment of the integrin
4 subunit into hemidesmosomes. Integrin
4 subdomain-specific clones generated in the pET expression system are
shown in Fig. 1 A. Encoded proteins contained the cytoplasmic tail starting at the beginning of the first FNIII repeat (
4-F1,2LF3,4C), the 303-amino acid region described
above (
4-F1,2L'), and the rest of the tail, starting in the
third FNIII repeat, close to its beginning (
4-F3,4C').
4-F1,2L and
4-F3,4C were similar to
4-F1,2L' and
4-F3,4C',
respectively, except for excluding (
4-F1,2L) and including
(
4-F3,4C) the third FNIII repeat in its entirety.
4-F1,2
corresponded to the first FNIII repeat pair,
4-F3,4 to the
second;
4-LF3,4C lacked the first FNIII repeat pair. SDS-PAGE analysis of purified recombinant integrin
4 polypeptides expressed in E. coli confirmed that they were of the
sizes predicted on the basis of the corresponding cDNA
sequences, including the 2.6-kD his-tags (Fig. 2).
|
Plectin mutant proteins corresponding to different molecular regions of the amino- and carboxy-terminal globular domains of the molecule (Fig. 1 B) were generated by subcloning rat plectin cDNAs into the pET expression system. Two amino-terminal protein fragments used covered the amino acid sequence encoded by exons 1-24 (Ple-N1) and exons 12-24 (PleN2). The other fragments covered, partly overlapping, the complete carboxy-terminal globular domain with its repeat structure. Ple-R1 started within the hinge region after the rod domain and included the first and part of the second repeat domain; Ple-R3-6T contained the rest of plectin's carboxy terminus. Two constructs, Ple-R3 and Ple-R4-6T, split construct Ple-R3-6T into two shorter ones, one corresponding to repeat 3 and parts of the flanking repeats 2 and 4, the other to the carboxy terminus starting in repeat 4. Ple-R1-3 was a combination of Ple-R1 and Ple-R3, ranging from plectin's hinge region into repeat 4. Ple-R4,5 contained repeat 4 and 5, Ple-R4 repeat 4 alone, and Ple-R5 repeat 5 alone. Ple-R6T contained a small portion of repeat 5, repeat 6 and the carboxy-terminal tail. Ple-R6 was equivalent to Ple-R6T but lacked the tail. Plectin mutant proteins expressed from these plasmids were all of the expected sizes, as determined by SDS-PAGE (Fig. 3, A and B). The double band observed for Ple-N1 was likely due to an additional translation initiation event at an in-frame ATG codon, resulting in a ~15-kD shorter his-tagged protein version.
Different Subdomains of the Integrin 4 Cytoplasmic
Tail Region Each Bind to Amino- and Carboxy-terminal
Domains of Plectin
To detect direct interaction between the candidate integrin 4 cytoplasmic tail region and various subdomains of
plectin, we first used a qualitative blot overlay assay in
which recombinant plectin fragments immobilized on nitrocellulose membranes were overlaid with 125I-labeled
purified recombinant integrin
4-F1,2LF3,4C (Fig. 3, C and
D). The plectin fragments used in these experiments covered the complete cDNA sequence (exons 1-32; Liu et al.,
1996
) with the exception of the predicted rod region (exon
31), a small part of the preceding amino-terminal globular
domain (exons 25-30), and the three additional alternative
start exons (1a, 1b, and 1c) identified recently (Elliott et al.,
1997
). Among nearly a dozen recombinant plectin polypeptides tested, the one displaying highest binding activity to
4-F1,2LF3,4C was Ple-R3-6T, which contained most of plectin's carboxy-terminal globular domain. Plectin protein
fragments corresponding to individual repeats (Ple-R3,
Ple-R4, Ple-R5) showed no or hardly any binding activity, neither did fragments containing repeat 1 and part of the
hinge region (Ple-R1), repeats 1-3 (Ple-R1-3), or repeats 4 and 5 combined (Ple-R4,5). Aside from Ple-R3-6T, the
4-F1,2LF3,4C polypeptide bound to all other plectin mutant
proteins that contained repeat 6, such as Ple-R4-6T, Ple-R6T, and Ple-R6.
4-F1,2LF3,4C also reacted with the
amino-terminal polypeptide fragments Ple-N1 and Ple-N2,
suggesting that one or more additional interaction site(s) resided in this region of the plectin molecule.
To verify the blot overlay, and in order to assess plectin-integrin 4 interaction in a more quantitative way, we used
an alternative, nonradioactive microtiter plate-binding assay involving Eu3+-labeled proteins, which had successfully been used in the molecular mapping of plectin's IF-binding site (Nikolic et al., 1996
). The two plectin protein
fragments representing amino- and carboxy-terminal domains and exhibiting highest binding efficiency in the blot overlay assay (Ple-N1 and Ple-R3-6T, Fig. 3) were used for
this assay.
4-F1,2LF3,4C was coated onto 96-well microtiter
plates and overlaid with increasing concentrations of the
Eu3+-labeled plectin proteins (Fig. 4 A). The amounts of
proteins bound to the coated integrin fragment were determined by measuring released Eu3+ by time-resolved
fluorometry (Soini and Lövgren, 1987
). Non-specific binding to coated BSA was also determined and subtracted to
give results for specific binding to
4-F1,2LF3,4C. Binding of
both plectin proteins was similar up to 500 nM, where Ple-N1 reached saturation, contrary to Ple-R3-6T, which exhibited
approximately twofold higher binding at 1,000 nM, consistent
with the apparently stronger binding of 125I-
4-F1,2LF3,4C
to this protein in the blot overlay assay (Fig. 3).
|
To map the plectin-binding site(s) in the integrin 4 cytoplasmic domain more precisely, Ple-R3-6T and Ple-N1
were separately coated and overlaid with samples of Eu3+-labeled integrin
4 mutant proteins (Fig. 5, A and B). Consistent with the qualitative blot overlay assay (Fig. 3),
strongest binding was observed between
4-F1,2LF3,4C and
the carboxy-terminal plectin fragment Ple-R3-6T (Fig. 5 A,
first bar). The same integrin
4 fragment bound to the
amino-terminal plectin fragment Ple-N1 with just ~30%
lower efficiency (Fig. 5 B, first bar). Among a panel of
truncated versions of
4-F1,2LF3,4C tested, only those containing either the region between the second and third
FNIII repeat (
4-F1,2L and
4-LF3,4C) and/or the carboxy-terminal tail region following the last FNIII repeat (
4-
LF3,4C and
4-F3,4C) showed significant binding to either
one of the plectin fragments (Fig. 5, A and B).
4-F1,2 and
4-F3,4 hardly showed any detectable binding. These results suggested that at least two different plectin-binding domains were present in the cytoplasmic domain of the integrin
4 subunit, one between the two pairs of FNIII repeats, the other in the ultimate tail region trailing the second pair of FNIII repeats. Moreover, both binding domains
apparently comprised interaction sites for amino- as well
as carboxy-terminal domains of plectin.
|
To examine whether the different binding sites on integrin and plectin molecules have differential affinities for
each other, integrin fragments 4-F1,2L and
4-F3,4C, each
containing just one of the two putative plectin-binding regions, were coated onto microtiter plates and overlaid with
either Eu3+-labeled Ple-N1 with or without a 10-fold molar
excess of unlabeled Ple-R3-6T (Fig. 4 B, first column) or,
vice versa, with Eu3+-labeled Ple-R3-6T with or without a
10-fold molar excess of unlabeled Ple-N1 (Fig. 4 B, second
column). The results of these competition experiments,
documented as the relative percentage of inhibition of
binding in the presence of the competitor, are summarized
in Fig. 4 B. Ple-R3-6T was able to efficiently displace Ple-N1
from both,
4-F1,2L and
4-F3,4C, whereas reversely, Ple-N1
hardly could displace Ple-R3-6T from
4-F1,2L and inhibited its binding to
4-F3,4C by only ~50%. The ability of
the plectin mutant proteins to displace each other from the
more carboxy-terminal integrin
4 plectin-binding domain
(contained in
4-F3,4C) indicated that both exhibited affinities for the same binding site. Apparently, however, Ple-R3-6T bound to this site with higher affinity compared to
Ple-N1, as indicated by its higher competition efficiency. This was consistent with the more efficient binding of
4-F3,4C to immobilized Ple-R3-6T, as compared to Ple-N1 in
the microtiter plate assay (compare bars
4-F3,4C in Fig. 5,
A and B). The inability of Ple-N1 to displace Ple-R3-6T
from
4-F1,2L indicated also a significantly higher affinity
of Ple-R3-6T, compared to Ple-N1, for the second, more
amino-terminal plectin-binding site contained in this integrin
4 fragment (
4-F1,2L).
Integrin 4 Mutant Proteins Exhibit Self-association
upon Immobilization
Competition experiments similar to those shown above
(Fig. 4 B) using immobilized plectin proteins and Eu3+-
labeled integrin fragments in solution failed due to excessive self-interaction of integrin 4 mutant proteins upon
immobilization, a situation which also prevented the determination of dissociation constants. The ability of integrin
4 cytoplasmic tail domains to self-associate potentially could
have implications for certain functions of the protein, such
as integrin clustering in hemidesmosomal complex formation and/or hemidesmosomal plaque-cytoskeleton anchorage. To obtain some information on possible molecular
mechanisms and to identify the domain(s) mediating self-association, purified recombinant integrin
4 polypeptides
representing various molecular domains were resolved by
SDS-PAGE, blotted onto nitrocellulose, and overlaid with
125I-
4-F1,2LF3,4C (Fig. 6, A and B).
4-F1,2LF3,4C reacted
strongly with itself,
4-F1,2L, and
4-F3,4C, but not detectably with
4-F1,2 or
4-F3,4. This indicated that the domains
involved in the aggregation of integrin
4 were similar to
those mediating integrin
4-plectin interactions. In solution, no such aggregation of integrin
4 proteins was found
(data not shown). When
4-F1,2LF3,4C was coated and
overlaid with Eu3+-
4-F1,2LF3,4C (Fig. 6 C, filled bars) in
either the standard overlay assay buffer (150 mM NaCl),
buffer without NaCl, or buffer with increased NaCl concentration (500 mM), binding under high-salt conditions
was fivefold increased over standard condition and about
10-fold reduced at low-ionic strength. Non-specific binding to BSA (Fig. 6 C, open bars) was low and even decreased
with increasing ionic strength. This experiment indicated
that self-interaction of
4-F1,2LF3,4C was of hydrophobic
nature. In a further experiment, increasing concentrations
of Ple-R3-6T were added to a constant amount of Eu3+-
4-F1,2LF3,4C. As seen in Fig. 6 D, plectin fragments decreased the extent of integrin
4 self-association in a concentration dependent manner. Presumably by binding to
coated integrin
4 molecules, plectin fragments blocked integrin
4 self-association sites, which reside near or are
even overlapping with the plectin-binding sites, as indicated by the blot overlay assay (Fig. 6, A and B).
|
Colocalization of Integrin 4 and Plectin
Mutant Proteins Ectopically Expressed in
Transfected PtK2 Cells
To test whether integrin 4 and plectin mutant proteins
shown to interact in vitro would also interact when ectopically expressed in living cells, we transiently cotransfected
PtK2 cells using various plectin and integrin
4 cDNA vector constructs driving the expression of c-myc-tagged and
GFP-tagged proteins. Based on the in vitro binding data,
we chose to express two plectin fragments, Ple-N1GFP, containing the amino-terminal integrin
4-binding site(s)
identified above, and Ple-R3-6Tmyc (Ple-R3-6TGFP), the carboxy-terminal polypeptide exhibiting the strongest binding to integrin
4 among all the recombinant plectin mutant proteins tested. In a first series, PtK2 cells were
cotransfected with plasmids encoding the carboxy-terminal
plectin fragment and one of the plasmids encoding correspondingly tagged integrin
4 fragments
4-F1,2LF3,4CGFP,
4-F1,2LF3,4Cmyc,
4-F1,2L'myc,
4-F1,2myc, and
4-F3,4C'myc
(Fig. 7). Most transfected cells were double-transfected,
expressing both the plectin and integrin proteins. A few
cells bearing only one plasmid served as controls for single-transfected cells.
|
In cells coexpressing Ple-R3-6Tmyc and 4-F1,2LF3,4CGFP,
the bulk of both ectopically expressed proteins showed codistribution and association with dense structures surrounding the nucleus; in addition, both were associated
with small dot- or patch-like structures of unknown nature
throughout the cytosol in superimposable patterns (Fig. 7,
A and B). In rarely observed single-transfected cells expressing the plectin mutant protein alone, similar dot- and patch-like structures were not observed; such cells rather
displayed filament association of mutant proteins (e.g.,
single-transfected cell in upper right-hand corner of Fig.
7 B), or bundling and collapse of IFs, as previously reported (Nikolic et al., 1996
). As revealed by triple-staining, the cytokeratin filaments of such cotransfected cells
seemed to have completely collapsed onto the nuclei, rendering the remainder of the cytoplasmic space a cytokeratin-free zone, clearly outlined by adjacent cytokeratin-positive cells (Fig. 7 C).
4-F1,2L'myc, containing the first pair of FNIII repeats
plus the region between the second and third FNIII repeat, colocalized with the carboxy-terminal plectin protein
(Ple-R3-6TGFP) in dense aggregates as well as in more delicate filamentous structures (Fig. 7, D and E). These delicate structures were not seen in cells expressing Ple-R3-6TGFP
(see e.g., single-transfected cell in upper right-hand part of Fig. 7 E) or
4-F1,2L'myc alone (see e.g., transfected cell in
center of Fig. 10 I), suggesting that their appearance was
dependent on the coexpression of both the plectin and integrin
4 mutant proteins. This notion was supported by
the observation that in double-transfected cells, expressing comparatively low levels of
4-F1,2L'myc compared to Ple-R3-6TGFP, delicate structures were hardly observed, but integrin-specific staining was confined to dense structures
typical of collapsed filaments (see e.g., double-transfected
cell in lower right-hand part of Fig. 7, D-F).
|
The expression product 4-F3,4C'myc, similar to
4-F1,2LF3,4CGFP and
4-F1,2L'myc, colocalized with the coexpressed carboxy-terminal plectin polypeptide in dense
perinuclear structures (Fig. 7, G and H), different from the
dispersed patches observed when this integrin construct
was expressed alone (Fig. 7 G). In contrast,
4-F1,2myc,
which lacked plectin-binding activity in vitro, was diffusely localized in small dots throughout the cell, regardless of
whether or not the cell was cotransfected with the plectin
construct (Fig. 7, I and J). As expected, plectin staining
was independent of integrin
4 localization and its characteristics were similar to those of plectin only expression
(Fig. 7 J).
The plasmid encoding the amino-terminal plectin protein Ple-N1GFP was used in a second set of similar cotransfection experiments using PtK2 cells (Fig. 8). Cells,
cotransfected with the plasmid encoding 4-F1,2LF3,4Cmyc,
displayed colocalization of both ectopically expressed
polypeptides in brightly stained patches (Fig. 8, A and B).
Colocalization was also observed with
4-F1,2L'myc (Fig. 8,
C and D), but the dots and patches double-stained in this
case were smaller and distributed more uniformly throughout the cell as compared to the structures formed with
4-F1,2LF3,4Cmyc. A similar confinement of both mutant proteins within distinct dots and patches was also found in
cells coexpressing
4-F3,4C'myc and Ple-N1GFP (Fig. 8, E and
F). In a single-transfected cell the weakly expressed
4-F3,4C'myc was found diffusely distributed within the nuclear
compartment and in form of dots and patches in the cytosol (Fig. 8 E). In contrast,
4-F1,2myc, which lacks both putative plectin-binding sites, remained diffusely distributed
throughout the cell (Fig. 8 G), without showing the dotty
staining pattern of the plectin protein (Fig. 8 H).
|
Ultrastructural Localization of Plectin at Hemidesmosomes Using Domain-specific Antibodies
To examine whether plectin molecules found at hemidesmosomal junctions in tissues are located within distances
short enough to enable their interaction with plasma membrane-anchored integrin 4, immunogold electron microscopy of acrylic resin-embedded rat skin samples was performed. Embedding in lowicryl HM20 was found to
provide excellent conditions for thin sectioning of rat skin specimens (below 100 nm), and enabled adequate hemidesmosomal fine structure resolution, comparable to other
protocols (Rappersberger et al., 1990
; Shimizu et al.,
1992
). Considering that individual plectin molecules have
been visualized as structures 200 nm in length, and probably can extend over even longer distances (Foisner and
Wiche, 1987
; Foisner et al., 1995; Svitkina et al., 1996
), we
used antibodies raised against epitopes of plectin residing in three different domains of the molecule, the amino terminus (serum E1A), the rod (mAb 7A8), and the carboxy
terminus (serum 135C). All three of these domain-specific
immunoreagents were found to be reactive with epitopes
at the periphery as well as in the cytoplasmic interior of
keratinocytes. Labeling was most intense near basolateral
plasma membrane domains, in particular at the locations
of hemidesmosomes (Fig. 9, A-C) and desmosomes (not
shown; see also Eger et al., 1997
), and along cytoplasmic cytokeratin filament bundles. In the area of hemidesmosomes, label was found at the plaque, at the inner plate,
and in association with cytokeratin filaments (Fig. 9, A-C).
|
None of the domain-specific anti-plectin antibodies showed noticeable preference in labeling of any hemidesmosomal substructures. Furthermore, based on the resolution of the indirect immunogold labeling used (20-30 nm), it was unlikely that plectin molecules in hemidesmosomal basolateral regions were oriented in the same way and arranged in equidistal positions. These data were consistent with a model, in which randomly oriented plectin molecules can span over the entire distance between plasma membrane-associated hemidesmosomal integrin receptor complexes and hemidesmosomal inner plate-anchored cytokeratin filament bundles (Fig. 9 D).
Overexpression of Integrin 4 Cytoplasmic Tail
Domain Leads to Intermediate Filament Collapse in
PtK2 and 804G Cells
To assess whether the cytoplasmic domains of integrin 4
involved in plectin interaction would have any influence
on cytoskeleton integrity, specifically IF-anchorage at
plasma membrane sites, we monitored vimentin and cytokeratin network arrays in transfected PtK2 cells using
double and triple immunofluorescence microscopy. Upon
expression of
4-F1,2LF3,4CGFP, cytokeratin arrays were
found aggregated and partially codistributed with the integrin mutant protein in perinuclear areas (Fig. 10, A and B).
In fact, this phenotype was very similar to that observed after cotransfection of PtK2 cells using integrin
4 and
plectin vector constructs in combination (compare to Fig.
7 C). Triple immunofluorescence microscopy revealed
that both the vimentin and cytokeratin network arrays had
undergone collapse in such cells (Fig. 10, C-E). Similar observations were made when PtK2 cells were transfected
using the integrin
4 tail construct
4-F3,4C'myc (Fig. 7, F-H).
In contrast, ectopic expression of
4-F1,2L'myc, which lacked
the carboxy-terminal part contained in
4-F1,2LF3,4CGFP/myc
and
4-F3,4C'myc, did not lead to such a drastic collapse of
IF networks (Fig. 10, I-K). Instead, a partial association of
the expressed protein with the IF network, specifically
with a dense perinuclear ring-like structure, was observed.
This ring-like structure, which was also present in untransfected cells, appeared more pronounced in transfected cells; and cytoplasmic IFs, especially the cytokeratin network, appeared diminished (Fig. 10, J and K). Because
both,
4-F1,2LF3,4CGFP and
4-F3,4C'myc, but not
4-F1,2L'myc
contained the carboxy-terminal region shown to exhibit
plectin-binding activity in vitro, we conclude that this tail
domain was sufficient to cause the collapse of both IF systems in PtK2 cells.
To confirm these studies, similar transfection experiments were carried out using rat bladder carcinoma 804G
cells, a cell line that forms hemidesmosome-like structures
when grown on uncoated glass coverslips or plastic tissue
culture dishes (Riddelle et al., 1991). Similar to PtK2 cells,
transfection of 804G cells with the plasmid encoding
4-F1,2LF3,4Cmyc led to bundling of cytokeratin filaments and
their partial collapse in perinuclear areas; the ectopically
expressed protein was again visualized within dense, patchy
structures, and partly colocalized with the collapsed cytokeratin filaments (Fig. 11, A and B). Complete network
collapse was observed in cells expressing
4-F3,4C'myc; in
this case, the mutant protein was found colocalized with cytokeratins in dense, ring-like structures around the nucleus (Fig. 11, C and D). In cells transfected with plasmids
encoding
4-F1,2L'myc (Fig. 11, E and F), the expressed protein was found more or less diffusely distributed throughout the cell, with an apparent association with dense, perinuclear cytokeratin structures.
4-F1,2myc (Fig. 11, G and
H) was diffusely distributed throughout the cytoplasm with no apparent effect on the cytokeratin network array.
Thus, the phenotypes of transfected 804G cells were fully
consistent with those of PtK2 cells.
|
Expression of Integrin 4 Cytoplasmic Tail
Domain Prevents Hemidesmosome Association of
Plectin in 804G Cells
Because some of the integrin 4 mutant proteins overexpressed in 804G and PtK2 cells had a dramatic impact on
cytoplasmic IF network organization, and were able to
bind to recombinant plectin fragments, we wanted to test
whether overexpression of these polypeptides in such cells
would also affect the distribution of endogenous cytoplasmic plectin structures. As revealed by double immunofluorescence microscopy of untransfected 804G cells using antiserum raised against the 17 most carboxy-terminal amino
acids of the integrin
4 subunit (Giancotti et al., 1992
), and
mAb 5B3 reacting with plectin's central rod domain (Foisner et al., 1994
), plectin and integrin
4 showed colocalization in a pattern previously described as Swiss cheese-like
(Jones et al., 1991
) and characteristic of the hemidesmosome-like structures abundantly expressed by these cells
(Fig. 12, A and B). Both antigens were predominantly located at the basal membrane of these cells, as confirmed
by confocal laser scanning microscopy of vertical sections
(Fig. 12, A and B, insets). This hemidesmosome-associated
5B3-immunoreactive plectin species represented the major form of the protein in 804G cells, as confirmed by
Western blot analysis of 804G cell lysates. Using mAb5B3,
a single major immunoreactive band of apparent high molecular weight (>300,000) comigrating with plectin isolated from rat glioma C6 cells, the original source of the
immunogen (Foisner et al., 1994
), was observed (Fig. 12 C).
|
Upon transient expression of integrin 4 mutant proteins containing one or both of the putative plectin-binding regions, the distribution of endogenous plectin was significantly altered. In cells expressing mutant proteins that
caused the collapse of cytokeratin network arrays, such as
4-F1,2LF3,4Cmyc and
4-F3,4C'myc, the punctuate plectin-staining pattern typical of hemidesmosomes was greatly
diminished (Fig. 13, A-D). Instead, remaining plectin-specific staining appeared rather diffuse, with the possible exception of peripheral areas, where a few positively stained
filamentous structures became apparent. Similarly, upon
expression of
4-F1,2L'myc, the punctuate plectin-specific
signal associated with hemidesmosomes was hardly detectable anymore (Fig. 13 F). In fact, such cells seemed almost
completely depleted of plectin. Expression of the integrin
mutant protein corresponding to the first and second FNIII repeat (
4-F1,2myc), which lacked a plectin-binding
site and was diffusely distributed over the entire cell (Fig.
13 G), left the punctuate plectin-staining pattern intact, indicating no alteration of plectin's localization at hemidesmosomes (Fig. 13 H).
|
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Discussion |
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The uniquely long cytoplasmic domain of the 4 subunit
protein has been implicated in connecting the
6
4 integrin complex with cytoskeletal elements (Schwarz et al.,
1990
; Spinardi et al., 1993
; Borradori and Sonnenberg,
1996
; Green and Jones, 1996
), likely via cytoplasmic components of hemidesmosomes that are capable of cytokeratin filament interaction. Plectin, as a versatile IF-binding and cytoskeletal linker protein that has been shown to occur at hemidesmosomes (Wiche et al., 1984
), was a strong
candidate for mediating this interaction. In this report, we
demonstrate a direct interaction between recombinant integrin
4 and plectin proteins using two independent in
vitro binding assays and in vivo cotransfection experiments. Furthermore, we show that integrin
4 polypeptides containing a ~250-amino acid-long carboxy-terminal
sequence led to bundling and collapse of IF network arrays,
possibly through interference with IF-membrane association, and affected the subcellular distribution of cellular
plectin when ectopically expressed in a nonmembrane-
associated state.
The integrin 4 cDNA constructs used in our study were
designed such that they encoded the entire or truncated
forms of the cytoplasmic tail domain starting with the first
FNIII repeat. One of the objectives pursued in this way
was to express, in cells, integrin
4 proteins that would not
associate with the cell membrane but would remain soluble in the cytoplasm. In this form they would also resemble
fragments of ~70 kD resulting from proteolytic processing of cellular integrin
4, which has been investigated by Giancotti et al. (1992)
. As suggested by these authors, formation of these cytoplasmic, nonmembrane-attached
4 subunit fragments is likely to be functional and related to the
remodeling of adhesive and cytoskeletal structures occurring in the skin when basal cells detach from the basement
membrane at the onset of differentiation and movement
to upper cell layers. In addition, we used plectin cDNA
constructs that encoded parts of the amino- and carboxy-terminal globular domains, both of which have been
shown to harbor functional sites, including a highly conserved amino-terminal actin-binding domain (Elliott et al.,
1997
), and an IF-binding site within the carboxy-terminal
repeat 5 domain (Nikolic et al., 1996
).
We used affinity blotting, a commonly used method to
detect direct interaction between molecules (for review
see Phizicky and Fields, 1995), to demonstrate that plectin's carboxy-terminal repeat 6 domain is essential for
binding to integrin
4. However, our data also indicated
that other synergy sites contained in domains flanking repeat 6 facilitated binding, because larger fragments extending beyond repeat 6 showed stronger binding. Additional integrin
4-binding sites were found to be contained
in the amino-terminal globular domain of plectin. These
binding data were confirmed by a microtiter plate-binding
assay using Eu3+-labeled proteins as soluble reagents.
Moreover, at least two separate plectin-binding sites were
found within the cytoplasmic domain of integrin
4, one
within the 142-amino acid-long segment connecting the
two pairs of FNIII repeats, the other within the ultimate
tail domain of the molecule. Both showed affinities for the
same amino- as well as carboxy-terminal parts of plectin, as indicated by competition assays. Cotransfection of PtK2
cells with plasmids encoding mutant integrin
4 and plectin polypeptides fully confirmed these in vitro results. In
fact, the in vivo expression experiments can be considered
essentially as a third binding assay, as interacting proteins
were found codistributed in patterns distinctly different
from those observed when only one of the mutant proteins was ectopically expressed in cells. Thus, in vitro and in
vivo binding data both suggested that plectin-integrin
4
binding was complex, involving multiple interfaces of the
proteins.
Sequences in the linker segment between the first and
second FNIII repeat pair have been shown to be important for integrin 4 functions. Spinardi et al. (1993)
showed
that a 303-amino acid-long region of the integrin
4 cytoplasmic tail domain starting with the first FNIII repeat was
essential for association of the molecule with hemidesmosomes. The necessary sequences were later narrowed down to the second FNIII repeat and a stretch of 27 amino
acids (1,329-1,355) of the linker segment (Niessen et al.,
1997a
). Furthermore, the regions responsible for hemidesmosome localization have also been implicated in regulating the distribution of HD1 upon overexpression in COS-7
cells (Niessen et al., 1997a
). We show here for the first time that this linker segment apparently is also involved in
integrin
4 self-interaction, aside from harboring one of
the two plectin-binding regions identified in our study.
Self-association of integrin
4 may allow for efficient, cooperative clustering of these molecules in the dense
hemidesmosomal plaque. Interestingly, our competition binding experiments indicated that plectin fragments containing integrin-binding domains attenuated the self-association of integrin
4, suggesting a potential role of plectin
in the regulation of this process. Whether integrin
4 and
plectin molecules compete for binding sites located close
enough to sterically hinder binding of the second ligand, or
even compete for the same sites, remains to be established. Further functional diversity of the segment between the FNIII repeat pairs is further underlined by the
complexity of the gene locus encoding it and the identification of variants generated by alternative splicing of exons 33 and 35 within this locus (Hogervorst et al., 1990
;
Tamura et al., 1990
; Pulkkinen et al., 1997
).
The integrin 4 tail segment following the second FNIII
repeat pair apparently is another subdomain of integrin
4
involved in multiple functions. Similar to the segment between the first and second FNIII repeat pair, this region
exhibited binding to the integrin
4 cytoplasmic domain itself, as well as plectin amino- and carboxy-terminal domains. In addition, fragments containing this segment
were highly efficient in causing IF collapse upon overexpression in PtK2 and 804G cells. In none of the previous studies we are aware of were specific functions assigned to,
or binding partners identified in this region of integrin
4.
A possible reason for this may be the predominant usage
of carboxy terminally truncated mutants of integrin
4, because functions of carboxy-terminal fragments may easily
escape detection, particularly when they are partially redundant, such as in the case of integrin
4 self-interaction and binding to plectin.
The role of the FNIII repeats remains unclear. We have
shown that the pairs of FNIII repeats present in the integrin 4 cytoplasmic tail do not interact directly with plectin.
However, they may be important for providing a structural
framework allowing other parts to function efficiently. Supporting this point, integrin
4-LF3,4C, which lacked the
first FNIII repeat pair, showed reduced binding to plectin
in vitro, compared to
4-F1,2LF3,4C. Alternatively, some of
the repeats may directly take part in interaction with other
components. In a recent study, the second FNIII repeat, in
addition to parts of the linker segment between the two
FNIII pairs, has been shown to be crucial for the targeting
of recombinant integrin
4 mutants to hemidesmosomes
and interaction with HD1 (Niessen et al., 1997a
). Additionally, the FNIII repeats may also interact with elements of
the signal transduction pathway(s) coupled to the
6
4 integrin (Mainiero et al., 1995
).
In view of plectin's putative major role as a cytoskeletal
linker and stabilizer molecule, one may expect that overexpression of a plectin-binding protein would exert some
influence on plectin's cellular function(s) by competitive
binding to plectin, possibly leading to blocking of, or interference with, some of the endogenous binding partners.
This could result in dominant negative phenotypes, detectable in structures, the integrity of which is dependent on a
particular plectin linkage function. In line with this notion, we found that overexpression of integrin 4 mutant polypeptides containing the second putative plectin-binding site located in the carboxy-terminal region (
4-F1,2LF3,4Cmyc/GFP
and
4-F3,4C'myc) led to the complete collapse of vimentin
and cytokeratin IF network systems in PtK2 and 804G cells.
Because ectopically expressed
4-F1,2L'myc, which lacked this
domain but still contained the more amino-terminal plectin-binding region, was much less effective, we conclude the carboxy-terminal tail segment of integrin
4, starting with
the second FNIII repeat pair, is the dominant effector of
this phenotype.
Niessen et al. (1997a,b), who expressed membrane-
associated integrin
4 molecules comprising the complete
or parts of the cytoplasmic domain in COS-7 cells, contrary to our results, did not observe any cytoskeletal phenotype. There are a number of possible explanations for
this difference. First, integrin mutant effector molecules
need to be freely available (soluble) in the cytoplasm, because in a membrane-bound state they may be too distant from target plectin molecules. Second, the membrane-bound mutant proteins do in fact interact with plectin, but
keep the IF network bound to the membrane, and thus, in
a noncollapsed state. Third, though unlikely, COS-7 cells
are phenotypically different from PtK2 and 804G cells. We
favor the second explanation, which allows for a mechanism, in which cytosolic integrin
4 mutant proteins are in
competition with their wild-type counterparts as well as other membrane-associated plectin-binding proteins, such
as fodrin and
-spectrin (Herrmann and Wiche, 1987
).
Prohibiting the attachment of IFs to the plasma membrane, this situation could eventually lead to a collapse of
IFs toward the perinuclear region. This interpretation is
supported by the observation that endogenous 804G cell
plectin, the localization of which was restricted largely to
basal hemidesmosome-like structures in untransfected cells, upon expression of the plectin-binding sites containing integrin
4 polypeptides (
4-F1,2LF3,4Cmyc and
4-F3,4Cmyc),
showed a diffuse cytoplasmic localization. Downregulation of plectin expression, in addition to relocalization of
the membrane-bound species, may have been responsible
for the even more drastic phenotype in the case of
4-F1,2Lmyc, where endogenous plectin was hardly detectable
any more (Fig. 13 F). Similar phenomena involving IF collapse and/or relocalization of endogenous proteins have
been observed after overexpression of wild-type or mutant
versions of IF-associated proteins, including desmoplakin
(Stappenbeck and Green, 1992
), plectin (Wiche et al., 1993
),
and various IF subunit proteins themselves (Albers and Fuchs, 1987
; for review also see Fuchs and Weber, 1994
),
and after microinjection of antibodies to a whole range of
antigens, including IF-associated and other proteins, into
cultured cells.
In all, the plectin phenotypes of cells, transfected with
various truncated versions of the integrin 4 cytoplasmic
domain, were fully compatible with the data-characterizing plectin as a direct interaction partner of hemidesmosomal integrin
4. Notably, mutant integrin
4 proteins
leading to filament collapse also caused relocalization of
plectin from hemidesmosomes in 804G cells, suggesting
that the collapse may in fact be indirect, mediated through
plectin. Furthermore, overexpression of integrin
4 polypeptides, containing the different plectin-binding sites identified individually, resulted in distinct phenotypes, consistent in two different cell lines. Hence, our findings are in
favor of a hypothesis put forward by Giancotti et al. (1992)
,
that proteolytic processing of the
4 subunit of the basement membrane receptor integrin
6
4, resulting in a number of defined cleavage products is related to the remodeling of adhesive and cytoskeletal structures. Additional
support for this proposal is provided by our unpublished
observation that transfected 804G cells overexpressing integrin
4 polypeptides, representing such proteolytic fragments, frequently had a rounder morphology, were apparently in the process of detachment from the substrate, and
could be found in a second cell layer in dense cultures.
Recent studies with patients suffering from EBS-MD
linked this disease to defects in plectin expression (McLean
et al., 1996). Furthermore, in plectin (
/
) mice with a
similar phenotype, the hemidesmosomes in epidermal keratinocytes were found to be ultrastructurally intact and
numerous filaments emanated from their cytoplasmic face,
indicating that their ability to serve as filament anchorage sites was at least to a certain extent preserved (Andrä et
al., 1997
). In contrast, BPAG1-deficient mice clearly lacked
the hemidesmosomal inner plate, and consequently did
not show keratin filament anchorage at the basal cell surface membrane (Guo et al., 1995
). Thus, plectin is probably dispensable for the formation of hemidesmosomes.
However, there is mounting evidence that this protein is
important for the integrity and stability of hemidesmosomal junctions. First, the expression of integrin
4 was
found to be significantly reduced in plectin-deficient mice
(Andrä et al., 1997
), as has been reported for mice deficient in another binding partner of integrin
4, the integrin
subunit
6 (Georges-Labouesse et al., 1996
), and second,
hemidesmosome structures of plectin-deficient mice seemed to be fragile, as a peculiar, bipartite distribution of BPAG1 was observed in a discontinuous pattern both at the bottom and the top of blisters in the skin of these animals; this
suggested a mechanism of cell breakage in which rupture
can occur irregularly on both sides (apical and basal) of
the hemidesmosome inner plate, or within the plate itself.
Based on a) the results reported in this study, in particular, plectin's ability to interact via carboxy- as well as
amino-terminal domains with the cytoplasmic tail of integrin 4 and its close association with hemidesmosomes as
demonstrated by immunoelectron microscopy, b) our previous identification of a highly conserved IF (cytokeratin)-
binding site in the carboxy-terminal globular domain of
plectin molecules (Nikolic et al., 1996
), and c) the skin
phenotype observed in plectin (
/
) mice (Andrä et al., 1997
) and EBS-MD patients, we propose that plectin molecules act as stabilizers of hemidesmosomes according to
the schematic model depicted in Fig. 9 D. In this model,
plectin molecules form clamps bridging hemidesmosomal
components with the IF networks by directly interacting
with both plasma membrane-associated integrin
4 and cytoplasmic cytokeratin filaments. Binding sites for integrin
4 in both the amino- and carboxy-terminal globular domains of plectin would allow parallel as well as antiparallel
plectin oligomeres to form interactions on both ends (see
Foisner and Wiche, 1987
, and Wiche et al., 1991
, for discussions of putative dimeric and tetrameric plectin molecule structures). Multiple mutual binding sites on both integrin
4 and plectin may afford stronger resistance towards the great mechanical stress hemidesmosomes
must be exposed to, yet retain the flexibility needed for
such dynamic structures as they have shown to be (Kitajima et al., 1992
; Riddelle et al., 1992
).
In conclusion, we have identified the 4 subunit of the
hemidesmosome-associated laminin receptor complex integrin
6
4 as a novel interaction partner of plectin and we
have mapped interacting domains on both molecules. In a
more global picture our results lend further support to the
proposed role of plectin as a versatile and essential cytoskeletal linker protein that may contribute to the cytoarchitectural organization and mechanical reinforcement of
cells by cross-linking IFs, interlinking them with other cytoskeletal network systems, and participating in their anchorage at the plasma membrane at specialized structures.
![]() |
Footnotes |
---|
Received for publication 25 August 1997 and in revised form 5 February 1998.
J.M. de Pereda and S. Reipert were recipients of postdoctoral fellowships from the European Community Human Capital and Mobility Program, and the Wellcome Trust, U.K., respectively. This work was supported by grants from the Austrian Science Research Fund.We wish to thank K. Andrä, J. Dubendorff, B. Nikolic, and M. Zachlederova, all from this laboratory, for providing some of the plectin cDNA constructs and antibodies used in this study. B. Nikolic performed the Western blot analysis shown in Fig. 12 C. We also wish to thank F.G. Giancotti (New York University School of Medicine, New York, NY) for kindly exchanging antibodies with us.
![]() |
Abbreviations used in this paper |
---|
EBS-MD, epidermolysis bullosa simplex combined with muscular dystrophy; FNIII, fibronectin type III repeat; GFP, green fluorescent protein; IF, intermediate filament.
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