* Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; Department of
Dermatology, University of Geneva, CH-1211 Geneva, Switzerland; and § Department of Pathology, University Hospital
Vrije Universiteit, 1081 HV Amsterdam, The Netherlands
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Abstract |
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Hemidesmosomes (HDs) are stable anchoring
structures that mediate the link between the intermediate
filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the 4 integrin cytoplasmic domain in the formation of HDs in
transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient
deficient in
4 expression. We found that the expression
of wild-type
4 restored the ability of the
4-deficient
cells to form HDs and that distinct domains in the NH2-
and COOH-terminal regions of the
4 cytoplasmic domain are required for the localization of HD1/plectin and
the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the
4 cytoplasmic domain was dispensable for HD formation, although
it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate
a direct interaction between
4 and BP180 which involves
sequences within the COOH-terminal part of the CS and
the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with
cDNAs for
6 and
4 and a mutant BP180 which lacks
the collagenous extracellular domain confirmed the interaction of
4 with BP180. Nevertheless,
4 mutants which
contained the BP180-binding region, but lacked
sequences required for the localization of HD1/plectin,
failed to localize BP180 in HDs. Additional yeast two-
hybrid assays indicated that the 85 COOH-terminal residues of
4 can interact with the first NH2-terminal pair of
FNIII repeats and the CS, suggesting that the cytoplasmic
domain of
4 is folded back upon itself. Unfolding of the
cytoplasmic domain may be part of a mechanism by
which the interaction of
4 with other hemidesmosomal components, e.g., BP180, is regulated.
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Introduction |
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HEMIDESMOSOMES (HDs)1 are specialized multiprotein complexes that mediate the adhesion of basal
epithelial cells to the basement membrane in
stratified and certain complex epithelia, and provide a link
between the keratin intermediate filaments (IFs) and the extracellular matrix (Staehelin, 1974; Burgeson and Christiano, 1997
). One of the transmembrane components in
these complexes is the
6
4 integrin which is thought to
play a key role in promoting the formation of HDs and in
cell adhesion (for review see Borradori and Sonnenberg,
1996
; Green and Jones, 1996
). Support for this supposition
is derived from several observations. Mutations in the
genes for the
4 or the
6 integrin subunits cause junctional epidermolysis bullosa associated with pyloric atresia
(PA-JEB), an inherited skin blistering disorder characterized by defective dermo-epidermal adhesion and the formation of only rudimentary HDs (Vidal et al., 1995
;
Brown et al., 1996
; Niessen et al., 1996
; Pulkkinen et al.,
1997
; Ruzzi et al., 1997
). Similarly, targeted disruption of
the genes for
6 or
4 results in widespread subepidermal blistering in neonatal mice, which are unable to form HDs
(Dowling et al., 1996
; Georges-Labouesse et al., 1996
; Van
der Neut et al., 1996
).
6
4 is a receptor for various laminin isoforms, including laminin-5 (Niessen et al., 1994
; Rousselle and Aumailley, 1994
), a major component of the epidermal basement
membrane (Carter et al., 1991
; Rousselle et al., 1991
).
6
4-mediated adhesion and signaling are likely to be regulated by the cytoplasmic domain of
4 (Spinardi et al., 1993
,
1995
; Giancotti, 1996
). This domain consists of ~1,000
amino acid residues and contains two pairs of type III fibronectin (FNIII) repeats which are separated by a connecting segment (CS) (Hogervorst et al., 1990
; Suzuki and Naitoh, 1990
; Tamura et al., 1990
). Recent studies have
identified sequences within the second FNIII repeat and
the CS that appear to be critical for the localization of
6
4 in HDs (Spinardi et al., 1993
; Mainiero et al., 1995
;
Niessen et al., 1997a
). Furthermore, the
4 cytoplasmic
domain appears to form a complex with the hemidesmosomal plaque component HD1/plectin and to regulate the
subcellular localization of HD1/plectin (Niessen et al., 1997b
)
and of the bullous pemphigoid antigen 180 (BP180) (Borradori et al., 1997
). BP180, a collagenous protein, is the
other known transmembrane hemidesmosomal constituent that is also likely to function as a cell-matrix receptor
(Giudice et al., 1992
; Li et al., 1993
; Jonkman et al., 1995
;
McGrath et al., 1995
). BP180 has been suggested to interact with the
6 integrin subunit, and this interaction may
contribute to the stabilization of HDs (Hopkinson et al.,
1995
).
The cytoplasmic hemidesmosomal proteins include, in
addition to HD1/plectin (Wiche et al., 1991; Hieda et al.,
1992
), the bullous pemphigoid antigen 230 (BP230) (Stanley et al., 1988
; Sawamura et al., 1991
), IFAP300 (Yang
et al., 1985
; Skalli et al., 1994
), and the P200 protein (Kurpakus and Jones, 1991
). IFAP300 and HD1/plectin appear
to be related and may even be identical (Herrmann and
Wiche, 1987
; Baker et al., 1997
). HD1/plectin and BP230
mediate the attachment of keratin IFs to the basal plasma membrane, since in patients with epidermolysis bullosa
simplex with muscular dystrophy, who lack HD1/plectin,
and in null mutant mice lacking either HD1/plectin or
BP230 the attachment of IFs to HDs is strongly reduced
(Guo et al., 1995
; Gache et al., 1996
; McLean et al., 1996
;
Smith et al., 1996
; Andrä et al., 1997
).
Recent cell transfection studies have defined regions in
the 4 cytoplasmic domain that are important for the localization of
6
4 within HDs, based on the ability of
4
mutants to become incorporated into HDs or to disrupt
them (Spinardi et al., 1993
; Mainiero et al., 1995
; Niessen
et al., 1997a
). However, the value of these studies is limited because the cells used express
4 endogenously and
form HDs, so that mutants could associate with preexisting HDs. Thus, it was not demonstrated whether the mutants are able to initiate HD formation. The availability of
human
4-deficient keratinocytes thus provides a unique
opportunity to investigate the role
4 and of specific domains in
4 in the formation of HDs and the recruitment
of other hemidesmosomal components.
In this study, we have used immortalized keratinocytes
derived from a patient with PA-JEB, who completely
lacked expression of the 4 integrin subunit (Niessen et al.,
1996
). We report experiments aimed at defining: (a) the
ability of these immortalized PA-JEB keratinocytes to assemble HD-like structures; (b) the feasibility to reverse
their phenotype by reexpressing wild-type
4; (c) the potential of
4 mutants, lacking distinct regions of the
4 cytoplasmic domain or carrying mutations in the tyrosine activation motif (TAM), to induce the formation of HD-like
structures by recruiting the hemidesmosomal components
HD1/plectin, BP180, and BP230 to sites of cell-substrate
contact; and (d) the interaction between the cytoplasmic
domain of
4 and BP180.
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Materials and Methods |
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Generation of Immortalized Cell Lines
HPV 16 immortalized normal human foreskin keratinocytes (NHK) have
been described previously (Steenbergen et al., 1996). NHK morphologically resemble the parental cells, but they are slightly larger and flatter
(Steenbergen et al., 1996
). Also, the cells are less stratified even when they
reach confluence.
Primary keratinocytes obtained from a patient with PA-JEB who completely lacked expression of the 4 integrin subunit (Niessen et al., 1996
)
were immortalized by transfection with full-length HPV 16 DNA (p1432;
Münger et al., 1989
). This resulted in the generation of a clonal culture
that was expanded for further characterization. The cells were relatively
large with a polygonal shape and morphologically resembled the parental
cells. They grew with a doubling time of ~36 h and showed normal stratification and differentiation as detected by electron microscopy upon culture postconfluent in HAMF12/DME (1:3) medium (data not shown). Ultrastructural analysis of the PA-JEB keratinocytes showed that in the
absence of
4 only a few rudimentary HD-like structures are formed in
some cells, as in PA-JEB patients (Vidal et al., 1995
; Niessen et al., 1996
; data not shown).
Cells and Antibodies
The two keratinocyte cell lines were grown in keratinocyte serum-free medium (SFM) (GIBCO-BRL, Paisley, UK) supplemented with 50 µg/ml bovine pituitary extract, 5 ng/ml epidermal growth factor, 100 U/ml penicillin, and 100 U/ml streptomycin. Alternatively, the cells were cultured in HAMF12/DME (1:3) medium containing 10% (vol/vol) FCS, 100 U/ml penicillin, 100 U/ml streptomycin, L-glutamine, 0.4 µg/ml hydrocortisone (Sigma Chemical Co., St. Louis, MO) and 1 µM isoproterenol (Sigma Chemical Co.). The African monkey kidney cell line COS-7 was cultured in DME (GIBCO-BRL) supplemented with 10% (vol/vol) FCS, 100 U/ml penicillin, and 100 U/ml streptomycin. The cells were grown at 37°C in a humidified, 5% CO2 atmosphere.
The following antibodies against human integrin subunits were used:
the mouse mAbs P1E6 and P1H5, anti-2 (Wayner and Carter, 1987
); the
mAb J143, anti-
3 (Kantor et al., 1987
); the mAb Sam-1, anti-
5 (Keizer
et al., 1987
) and the NKI-M9, anti-
v (Von dem Borne et al., 1989
) were
obtained from C.G. Figdor (University of Nijmegen, Nijmegen, The Netherlands); the mouse mAb J8H, the rat mAb GoH3 and a rabbit polyclonal
antiserum, anti-
6 have been described (Sonnenberg et al., 1987
; Hogervorst et al., 1993
; Delwel et al., 1994
); the mouse mAb 113C, anti-
4, was
prepared by A.M. Martínez de Velasco in our laboratory (unpublished results); the mouse mAb 4.3E1 against
4 (Hessle et al., 1984
) was provided by E. Engvall (The Burnham Institute, La Jolla, CA); the mouse mAbs
450-10D and 450-9D, and the rat mAb 439-9B, anti-
4 (Kennel et al.,
1989
, 1990
), were kindly provided by S.J. Kennel (Oak Ridge National
Laboratory, Oak Ridge, TN); a rabbit antiserum (67p120) to recombinant
human
4 cytoplasmic domain was prepared as previously described
(Niessen et al., 1994
); the rat mAb AIIB2, anti-
1 (Werb et al., 1989
),
was a gift from C.H. Damsky (University of California, San Francisco,
CA); an the mAb TS2/16 against
1 was obtained from the American
Type Culture Collection (Rockville, MD). A rabbit antiserum to rat IgG
has been described previously (Sonnenberg et al., 1986
). The mouse mAb
VIIF9 against vinculin (Glukhova et al., 1990
) was a generous gift from
M.A. Glukhova (École Normale Supérieure, Paris, France). A rabbit antiserum directed against the COOH-terminal domain of BP230 (Tanaka
et al., 1990
) was kindly provided by J.R. Stanley (University of Pennsylvania, Philadelphia, PA). The mouse mAbs 1D1 and 233 against the intra- and extracellular portion of BP180, respectively (Nishizawa et al.,
1993
), and the mAb 121 directed against HD1 (Hieda et al., 1992
) were
kindly donated by K. Owaribe (Nagoya University, Nagoya, Japan). A
rabbit antiserum against the cytoplasmic domain of BP180 was generously provided by L. Bruckner-Tuderman (University of Münster, Münster, Germany). The mouse mAb anti-FLAGTM M2 against the FLAGTM
peptide (DYKDDDDK) was purchased (IBI, Eastman Kodak Co., New
Haven, CT). Species-specific FITC-conjugated goat anti-mouse IgG
(Zymed Laboratories, San Franscisco, CA), Texas red-conjugated goat
anti-rat IgG (Rockland, Gilbertsville, PA), and Texas red-conjugated donkey anti-rabbit IgG (Amersham Int., Buckinghamshire, UK) were purchased, as were species-specific horseradish peroxidase-conjugated antibodies (Amersham Int.).
cDNA Constructs
The full-length 4A and
4B cDNA constructs, and the cDNA constructs
encoding
4 with COOH-terminal truncations or internal deletions of the
cytoplasmic domain have been described previously (Niessen et al.,
1997a
,b). The cDNA plasmid encoding
4A with combined phenylalanine
substitutions of the tyrosine activation motif (Mainiero et al., 1995
) was
kindly provided by F.G. Giancotti (New York University School of Medicine, New York). The construct was assembled into pcDNA3 (Stratagene,
La Jolla, CA). The pRc-CMV expression construct encoding full-length
6A cDNA, as well as the pCI-Neo construct encoding the cytoplasmic
domain of BP180 (clone B, BP180
521-1497) have been described previously
(Borradori et al., 1997
). Correctness of all constructs was verified by sequencing. The molecular weights of the different expressed
4 proteins
correspond to that predicted based on the DNA sequences, as assessed by
Western blot analysis of transiently transfected COS-7 cells (Niessen et
al., 1997a
; data not shown).
DNA Transfections
For transfection, the keratinocytes were first grown in keratinocyte-SFM medium to 40-60% confluency in six-well tissue culture plates (Falcon; Becton Dickinson, Lincoln Park, NJ). The cells were transfected using the cationic lipid Lipofectin® (GIBCO-BRL). The DNA/Lipofectin® mixture was prepared using serum-free medium (OPTI-MEM®, GIBCO-BRL). The final concentration of plasmid DNA and Lipofectin® in serum-free transfection medium was 2.5 µg/ml and 10 µg/ml, respectively. 1 ml of transfection medium was added to each monolayer that had been previously washed with serum-free medium and cells were incubated with the transfection medium for 9-10 h at 37°C with 5% CO2. The transfection medium was then replaced with keratinocyte-SFM medium for 12 h and subsequently with HAMF12/DME (1:3) medium for an additional 24 h, after which gene expression was assessed.
COS-7 cells (1.2 × 106 cells/60 cm2) were transiently transfected using
the DEAE-dextran method (Cullen, 1987) with 2 µg of DNA per construct and assayed for gene expression after 48 h.
Immunofluorescence Microscopy
Cells grown on glass coverslips in six-well tissue culture plates in
HAMF12/DME (1:3) medium for 24 h were fixed with 1% formaldehyde in PBS for 10 min and permeabilized with 0.5% Triton X-100 for 5 min at
room temperature. After rinsing with PBS and blocking with 2% (wt/vol)
BSA in PBS for 30 min at 37°C, the cells were incubated with primary antibody for 30 min at 37°C and then washed three times with PBS. The cells
were subsequently incubated with FITC-labeled anti-mouse IgG, Texas
red-labeled anti-rabbit IgG, Texas red-labeled anti-rat IgG, or rabbit anti-
rat IgG followed by Texas red-labeled donkey anti-rabbit IgG for 30 min
at 37°C, respectively. For double labeling studies, cells were incubated with the respective antibodies as described previously (Niessen et al.,
1997a). The coverslips were subsequently washed, mounted in Vectashield (Vector Labs, Inc., Burlingame, CA), and then viewed under a Bio-Rad MRC-600 confocal scanning laser microscope (Richmond, CA).
Immunoprecipitation Studies and Immunoblotting
Keratinocytes cultured in keratinocyte-SFM medium were detached using
20 mM EDTA in PBS and washed three times with PBS. Cells were surface-labeled with 125I (Amersham Int.) by the lactoperoxidase/hydrogen
peroxide method (Sonnenberg et al., 1987; Niessen et al., 1996
). Thereafter,
the cells were washed three times with PBS and lysed on ice with NP-40 lysis buffer (1% Nonidet P-40, 25mM Tris-HCl, pH 7.5, 4 mM EDTA, 100 mM NaCl, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml soybean trypsin inhibitor). The lysates were then used for immunoprecipitation, as
described previously (Sonnenberg et al., 1993
; Niessen et al., 1996
). Immune complexes were released from the beads by boiling for 5 min in nonreducing SDS sample buffer and resolved on a 5% SDS-PAGE gel.
Alternatively, keratinocytes were washed twice with PBS and incubated with DME without methionine and cysteine (ICN Biomedicals Inc., Costa Mesa, CA) for 1 h at 37°C. Cells were then labeled with 100 µCi/ml [35S]methionine/cysteine (Amersham Int.) for 4 h, washed, and then lysed with NP-40 lysis buffer and used for immunoprecipitation analysis as described above.
Transfected COS-7 cells were washed twice with PBS and scraped in 1 ml CHAPS lysis buffer (1% CHAPS, 25 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM PMSF, 10 µg/ml leupeptin and 10 µg/ml soybean trypsin inhibitor). The lysates were clarified by centrifugation and incubated with antibodies previously bound to GammaBind plus Sepharose CL4B beads (Pharmacia LKB Biotech., Uppsala, Sweden). Immune complexes were washed three times with lysis buffer and two times with PBS. Immunoprecipitates were released from the beads by boiling for 5 min in nonreducing SDS sample buffer, resolved on an 8% SDS-PAGE gel, and blotted to polyvinylene difluoride membranes (Immobilon-P; Millipore Corp., Waters Chromatography, Bedford, MA). The immunoblots were blocked for 1 h in TBSTB (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween-20, 2% [wt/vol] baby milk powder) and probed with primary antibodies in TBSTB for 1 h at room temperature. After extensive washing in TBSTB (with only 0.2% [wt/vol] baby milk powder), blots were incubated for 1 h with secondary horseradish peroxidase-conjugated antibodies diluted 1:5,000 in TBSTB. The blots were then washed again and developed using enhanced chemiluminescence (Amersham Int.).
Yeast Two-hybrid Assay
All yeast galactose metabolism regulatory gene 4 (GAL4) expression
plasmids containing parts of the 4 or BP180 cytoplasmic domains that
were used for the yeast two-hybrid assay are listed in Figs. 10 and 12.
Numbers in superscript correspond to the
4 amino acid residues (numbered according to Niessen et al., 1997a
) that are encoded within the
GAL4 activation domain (AD) or binding domain (BD) fusion proteins.
The sequences encoding
4 were amplified by PCR from the full-length
4A and
4B cDNA constructs used for the transfection studies described
above, using
4-specific sense and antisense primers containing restriction
site tags. PCR products were cut with the appropriate restriction enzymes,
correctly sized DNA fragments were isolated from agarose gels using the
Easy-PureTM kit (Biozym, Landgraaf, The Netherlands), and ligated into
the yeast GAL4(AD) expression vector pACT2 (Harper, 1993; Clontech, Palo Alto, CA) cut with conforming restriction enzymes. This resulted in
the in-frame fusion of each
4 coding sequence to the 3' end of the GAL4
(768-881) transcriptional AD. For the experiments described in Fig. 12,
several
4 sequences were recloned into the yeast GAL4 (BD) expression
vector pAS2-1 (Durfee et al., 1993
; Clontech) using restriction sites in the
polylinkers of the vectors. This resulted in the in-frame fusion of
4-
encoding sequences to the 3' end of the GAL4 (1-147) DNA-BD.
|
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A cDNA clone containing nucleotides 1-1,398 encoding the entire cytoplasmic domain, i.e., the first 466 amino acid residues, of human BP180
(Hopkinson et al., 1992) was isolated from a
gt11 human keratinocyte library by PCR using BP180-specific sense and antisense primers containing
restriction site tags, purified and cut with the appropriate restriction enzymes as described above, and then cloned into pAS2-1. This BP180 construct, pAS2-BP180(0), caused autonomous activation of the reporter
genes in the yeast host strain, presumably because of the presence of multiple Gly and Cys residues in the BP180 protein sequence immediately preceding the putative transmembrane region. A subclone containing only the
first 1,201 nucleotides of the BP180 sequence and lacking the nucleotides
encoding the Gly and Cys repeats was isolated from pAS2-BP180(0) using
the StuI site (at position 1,201 in the BP180 sequence) and recloned into
pAS2-1, resulting in pAS2-BP180(C) which encodes the first 400 amino acids of the BP180 protein. This construct did not cause autonomous transactivation of the reporter genes in the yeast host strain. The BP180 and
4
coding sequences within the yeast expression constructs were confirmed by
sequence analysis using the T7Sequencing kit (Pharmacia Biotech.).
Yeast strain PJ69-4A (gift of P. James, University of Wisconsin, Madison, WI), which contains the genetic markers trp1-901, leu2-3, his3-200,
gal4, gal80
, LYS2::GAL1-HIS3, and GAL2-ADE2 (James et al., 1996
),
was used as the host strain for the two-hybrid assay. It contains two tightly
regulated and selectable GAL4-driven reporter genes, His and Ade, and
is therefore suited for sensitive detection of protein interactions. Strain
PJ69-4A was grown and transformed with plasmid DNA essentially as
described (Gietz et al., 1995
; James et al., 1996
). PJ69-4A yeast cells
were cotransformed with a pACT2 (-derived) plasmid as well as a pAS2-1
(-derived) plasmid, and aliquots of the same transformation were spread
on plates containing SC-LT medium, yeast synthetic complete medium (SC) lacking only the vector markers Leu (for pACT2 and derivatives) and Trp (for pAS2-1 and derivatives), and on plates containing SC-LTHA
medium, lacking Leu and Trp as well as the interaction markers His and
Ade. Plates were scored after 4 and 9 d of growth, and the number of colonies on the SC-LT plate compared with that on the SC-LTHA plate. As
a positive control for GAL4-driven activation of the Ade and His reporter
genes of PJ69-4A, two combinations of vectors were used that enable
growth on SC-LTHA plates. One combination was pCL1, full-length
GAL4 (which is able to activate the two reporter genes on its own) in a
pACT2-like vector (Fields and Song, 1989
; Clontech) together with the
empty pAS2-1 vector. In the other combination, two vectors, pTD1-1, SV-40
large T antigen in pACT2 (Li and Fields, 1993
; Clontech), together with
pVA3-1, a p53 subclone in pAS2-1 (Iwabuchi et al., 1993
; Clontech), were used, that express proteins that are known to interact and thereby cause
expression of the reporter genes.
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Results |
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Altered Distribution of Hemidesmosomal Components in Immortalized PA-JEB Keratinocytes
We investigated the distribution of hemidesmosomal proteins in preconfluent monolayers of the immortalized
NHK and PA-JEB cell lines by confocal immunofluorescence microscopy. In NHK, the 6
4 integrin and HD1/
plectin were concentrated at cell-substrate contact sites in
structures appearing as dots and large patches (Fig. 1, A,
B, and E). This staining pattern is characteristic for HD-like structures (Marchisio et al., 1991
, 1993
) or stable anchoring contacts (Carter et al., 1990
) of cultured keratinocytes. In addition, the localization pattern of BP230 and
BP180 was similar to that of
6
4 and HD1/plectin, although the staining was less extensive in most cells (Fig. 1,
C and D). In the PA-JEB cells, no
4 staining was found
using mAbs directed against either the extra- or the intracellular domain of
4 (Fig. 1 G; data not shown).
6 (Fig. 1
F) was codistributed with vinculin in short linear arrays at
the basal cell surface at the ends of actin stress fibers (data
not shown), consistent with its localization in focal contacts in the parental PA-JEB cells (Niessen et al., 1996
). In
only a few cells (less than 1%) BP230 and BP180 were
concentrated in HD-like structures at the basal cell surface
(Fig. 1, H and I). However, whereas HD1/plectin was
found in some HD-like structures in the primary PA-JEB
keratinocytes (Niessen et al., 1996
), it was completely absent from such structures in the immortalized cells (Fig. 1
J). These findings show that the formation of HD-like structures is impaired in the immortalized PA-JEB keratinocytes.
|
Coimmunoprecipitation of 6 with
1 in
PA-JEB Keratinocytes
The expression profile of integrins was determined by immunoprecipitation of 125I surface-labeled PA-JEB keratinocytes and NHK (Fig. 2). The mAb against 6 precipitated this subunit associated with
4 from NHK (Fig. 2,
left), whereas
6 and
1, but not
4, were precipitated from the PA-JEB keratinocytes (Fig. 2, right).
1 was coprecipitated together with
2,
3,
5, and
6 from the PA-JEB keratinocytes, but only with
2,
3, and
5 from the
NHK. These results demonstrate that although NHK express
6
4, PA-JEB keratinocytes express
6
1 on their
surface. In addition, no
4 was detected by flow cytometry on the surface of PA-JEB keratinocytes, whereas the expression level of
6 was substantially decreased as compared to that on NHK (data not shown). The
v subunit
was precipitated from both cell lines (Fig. 2) and was associated with both
3 and
5 subunits (data not shown).
|
Reexpressed 4 in PA-JEB Keratinocytes Is
Associated with
6 and Induces the Formation of
HD-like Structures
To restore expression of 4, the PA-JEB keratinocytes
were transiently transfected with cDNA encoding wild-type
4 (Fig. 3 A). Immunoprecipitation analysis of lysates of radiolabeled transfected PA-JEB keratinocytes is
shown in Fig. 4. A mAb against
4 precipitated
4 and
6
from transfected PA-JEB keratinocytes (Fig. 4, right), but
not from untransfected cells (Fig. 4, middle). Thus,
4 is expressed and forms a heterodimer with endogenous
6 in
the transfected cells, as in NHK (Fig. 4, left).
|
|
The subcellular distribution of the newly synthesized 4
in transfected PA-JEB keratinocytes was assessed by
confocal immunofluorescence microscopy. In transfected
cells,
4 (i.e.,
4A or
4B with a 53 amino acid insertion in
the CS) was concentrated in HD-like structures at the
basal side of the cell (Fig. 5, A-E), where it is colocalized
with
6, BP180 and BP230 (Fig. 5, F, I, and J). In addition,
HD1/plectin was no longer diffusely distributed in the cytoplasm, but colocalized with
6
4 at cell-substrate contact sites (Fig. 5, C and H). Staining for vinculin revealed the presence of focal contacts organized at the periphery
of the HD-like clusters (Fig. 5, B and G). We conclude
that expression of
4 restores the capacity of PA-JEB keratinocytes to form HD-like structures.
|
The 4 TAM Is Not Essential for the Formation of
HD-like Structures
It has been suggested that phosphorylation of the TAM,
which consists of two closely spaced tyrosine residues located at position 1,422 and 1,440 within the CS of the 4
cytoplasmic domain, is critical for the incorporation of
6
4
in HDs and HD assembly (Mainiero et al., 1995
). Therefore, we investigated whether expression of
4 with a mutated TAM affected the formation of HD-like structures.
4
with phenylalanine substitutions in the TAM was concentrated at the basal side of the cells (Fig. 6, A, C, E, and G)
together with
6, HD1/plectin, BP180, and BP230 (Fig. 6, B,
D, F, and H, respectively) in a pattern indistinguishable from
that seen upon transfection with wild-type
4 cDNA (Fig.
5). However, compared to wild-type
4, TAM-mutated
4A
appeared to have a reduced ability to induce redistribution
of BP180 to the basal side of the cell, because in ~30% of
the transfected cells the distribution of BP180 remained diffuse throughout the cell. Thus, although the
4A TAM may
influence the association of BP180 with HDs, it is largely dispensable for the assembly of these structures.
|
Identification of Sequences within the 4 Cytoplasmic
Domain Involved in the Recruitment of HD1/Plectin,
BP180, and BP230 to HD-like Structures
Previous studies have shown that the sequences within the
4 cytoplasmic domain that are responsible for inducing the
redistribution of HD1/plectin in COS-7 cells and mouse embryonic fibroblasts are also sufficient to incorporate
4 mutants into existing HDs in 804G rat bladder carcinoma cells
(Niessen et al., 1997a
; Sánchez-Aparicio et al., 1997
). To
identify the specific regions required for HD formation, we
assessed the distribution of HD1/plectin, BP180, and BP230
in PA-JEB cells transfected with mutant
4 cDNAs. We found that the NH2-terminal, but not the COOH-terminal
half of the
4 cytoplasmic domain, is involved in the recruitment of HD1/plectin to areas of cell-substrate contact in
PA-JEB cells (refer to Fig. 3 A). Transfection of PA-JEB
cells with the various cDNAs encoding COOH-terminal deletion mutants of
4 showed that the segment comprising the first pair of FNIII repeats and a stretch of 27 amino acids in the CS contains sequences that critically affect the distribution of HD1/plectin (Fig. 3 A and Fig. 7).
|
Since the COOH-terminal half of the 4 cytoplasmic
domain has previously been shown to be responsible for
the localization of BP180 in transfected COS-7 cells (Borradori et al., 1997
), we investigated whether
4 mutants
with increasing COOH-terminal truncations were able to
recruit BP180 and BP230 into newly formed HD-like
structures. In contrast to its effect on the localization of
HD1/plectin, truncation after amino acid 1,487 (
4A1,487)
already reduced the ability of the mutated
4 molecules to
localize BP180 and BP230 together with
6
4 and HD1/
plectin at the basal side of the cells (Fig. 3 A and Fig. 8).
Progressive COOH-terminal truncations up to amino acid
1355 (
41,355) resulted in a gradual increase in the percentage of
4-transfected cells in which BP180 and BP230 remained diffusely distributed throughout the cell (refer to
Fig. 3 A). Furthermore, in cells expressing
4 that was
truncated after amino acid 1,328 (
41,328), in which HD1/
plectin was no longer concentrated together with
6
4 at
the basal side of the cell, BP180 and BP230 were also diffusely distributed throughout the cell. These results suggest that sequences within the CS and the second pair of
FNIII repeats of
4 are involved in targeting BP180 and
BP230 into HD-like structures. In addition, the presence
of HD1/plectin at the basal cell surface appears to be crucial for these translocation events as well (see also below).
|
Interaction between 4 and BP180 in COS-7 Cells
To test whether 4 interacts with BP180 in mammalian
cells, we have performed immunoprecipitation and immunoblotting experiments using lysates from COS-7 cells that
were transfected with cDNAs encoding
6A and FLAG-tagged BP180 (clone B, BP180
521-1,497) together with various
4 mutants. As shown in Fig. 9,
4 is present in anti-FLAGTM M2 immunoprecipitates from lysates of cells transfected with cDNAs for
6A,
4A, and BP180 (Fig. 9,
top, lane 4). Furthermore, in agreement with our localization studies, small amounts of
41,355 mutant protein were
co-precipitated by anti-FLAGTM M2 antibodies (Fig. 9,
top, lane 6). As expected, the
41,328 mutant protein was
not detectable in these immunoprecipitates (Fig. 9, top,
lane 8), although equal amounts of mutant BP180 molecules were precipitated by the anti-FLAGTM M2 antibodies from the cells transfected with the corresponding
cDNAs (Fig. 9, bottom). Together, these data reveal that
4 and BP180 are present in immune complexes and support the results obtained with the localization studies in
PA-JEB cells.
|
Direct Interaction between the Cytoplasmic Domains of
4 and BP180
To investigate whether the cytoplasmic domains of 4 and
BP180 can directly bind to each other, and to determine
which site on
4 is involved in this interaction, a yeast two-hybrid assay (Fields and Song, 1989
) was performed (Fig.
10). Yeast strain PJ69-4A was cotransformed with the
GAL4(AD) pACT2 and the GAL4(BD) pAS2-1 vectors
or derivatives thereof. For several pACT2-
4 plasmids, cotransformation of the yeast strain together with the
pAS2-BP180 plasmid (and only then) supported growth of
colonies on SC-LTHA plates, showing that the His and
Ade reporter genes in these yeast cells were activated by a
direct interaction between the BP180- and
4-GAL4 fusion proteins. The highest plating efficiency on SC-LTHA
plates was observed with the
41,115-1,666 construct (both
the
4A and the
4B splice variant). The number and
growth rate of the colonies on SC-LTHA plates were comparable to those on SC-LT plates, indicating that both reporter genes were efficiently expressed as a result of a
strong interaction between
4 and BP180 (refer to Materials and Methods). In fact, the plating efficiency was comparable to that of the pCL1/pAS2-1 and pTD1-1/pVA3-1
positive controls.
The site of interaction was mapped using 4 with
COOH-terminal deletions. Removal of the COOH terminus together with the second part of the fourth FNIII repeat,
41,115-1,666 (again for both the
4A and
4B splice
variants), resulted in only a slight decrease in the plating
efficiency and growth rate. However, COOH-terminal deletions up to the end of the CS (
4A1,115-1,457) resulted in a
dramatic reduction in binding. Truncation up to the last 21 amino acids of the CS (
41,115-1,436) had no effect on the
binding, but there was no binding at all when 54 additional
residues were deleted (
41,115-1,382). These results suggest
that the main binding sites for BP180 on
4 reside in the
segment comprising the COOH-terminal half of the CS
and the third FNIII repeat.
To confirm this finding, fragments of the 4 cytoplasmic
domain were used. As expected, deletion of the first two
FNIII repeats (
4A1,320-1,668) did not result in decreased
binding to BP180, as compared to
4A1,115-1,666. The same
was found for binding of BP180 to
4A1,320-1,552, compared
to
41,115-1,600. Accordingly, neither the first (
41,115-1,217)
nor the second (
41,217-1,328) FNIII repeat can bind to
BP180. Efficient binding to BP180 was only observed
when both the CS and the third FNIII repeat were present
(
4A1,320-1,552). The third or fourth FNIII repeat or the
COOH terminus alone (
41,457-1,552,
41,570-1,668, and
41,667-
1,752, respectively) showed only weak binding. The results
obtained with the yeast two-hybrid assay are in good
agreement with those obtained with the cell transfection
studies (as shown above), and conclusively prove that
4
interacts directly with BP180.
A Role for HD1/Plectin in the Localization of BP180
The presence of small amounts of 41,355 protein, which
does not contain the BP180 binding site(s), in the BP180
immunoprecipitate may have been due to the presence of
HD1/plectin in these immune complexes. To determine
whether HD1/plectin play a role in the recruitment of
BP180 into HDs, we assessed the localization of BP180 in
PA-JEB cells transfected with cDNAs encoding
4 mutants that lack sequences essential for the recruitment of
HD1/plectin (
4A
1,219-1,319 and
4A
1,249-1,265; Fig. 3 B).
Neither of the
4 mutants induced a redistribution of
HD1/plectin, despite the fact that they were able to cluster with
6 at the basal side of the cells (Fig. 11 A). Remarkably, in the absence of HD1/plectin, but in the presence of
the binding sites for BP180 on
4, BP180 was not recruited
to the basal cell surface but remained diffusely distributed
(Fig. 11 B). Thus, in addition to
4, HD1/plectin is essential for the recruitment of BP180 into HD-like structures.
|
It is noteworthy that in a few cases (i.e., <25% of transfected cells) these 4 mutants recruited BP230 in an HD1/
plectin- and BP180-independent manner to the basal cell
surface (Fig. 11 C). These results together with the results
obtained with the COOH-terminal truncation mutants
suggest a BP180-independent, (in)direct interaction between
4 and BP230.
Intramolecular Interactions in the 4
Cytoplasmic Domain
The observation that the 4 internal deletion mutants
which lack the HD1/plectin binding site(s), but possess the
binding sites for BP180 failed to recruit BP180, prompted
us to investigate whether the NH2- and the COOH-terminal parts of the
4 cytoplasmic domain interact with each
other. Such an intramolecular interaction might prevent
the interaction with BP180 and proffer an explanation for
the essential role of HD1/plectin in the interaction of
4 with BP180. The potential intramolecular interaction was
tested in a yeast two-hybrid assay in which the GAL4 activation and DNA-binding domains were fused to various
parts of the
4 cytoplasmic domain (Fig. 12).
A 41,457-1,752 cDNA was cloned into the pAS2-1(BD)
vector and cotransformed to yeast in combination with an
empty pACT2 vector (to check for autonomous transactivation) or with a pACT2-derivative containing diverse
4
inserts. Strong binding was observed with
4(AD) constructs
4A1,115-1,457 and
41,115-1,436 that contained the first
two FNIII repeats together with a large part of the connecting region (Fig. 12, top). Binding was strongly, but not
completely reduced by deletion of the first and second FNIII repeat (
4A1,320-1,457). No binding could be demonstrated with either the first or the second FNIII repeat
alone (
41,115-1,217 and
41,217-1,328, respectively). To determine which part of the
4 COOH terminus binds to the
more NH2-terminal part (
41,115-1,436), the smaller COOH-terminal
4 subclones as shown in Fig. 10 were excised
from the pACT2(AD) vector and recloned in the pAS2-1(BD) vector. Subsequently, the resulting
4(BD) constructs were cotransformed to yeast in combination with
an empty pACT2 vector (to check for autonomous transactivation) or with a pACT2-derivative containing the
41,115-1,436 insert (Fig. 12 bottom). The results show that
only the COOH-terminal part of the
4 cytoplasmic domain strongly interacted with the
41,115-1,436 sequence,
whereas the third and fourth FNIII repeat had no apparent role in this intramolecular association. These observations together with the results of the cell biological studies
described in the previous paragraph suggest that the
4
protein can fold back upon itself, with the COOH-terminal 85 amino acids binding to the HD1/plectin-binding region located further NH2-terminal.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The 4 Integrin Subunit Is Critical for the Formation of
HD-like Structures
We have established an immortalized keratinocyte cell
line from a PA-JEB patient lacking 4 expression (Niessen et al., 1996
). Only a few of these cells have low numbers of disorganized HD-like structures that contain the
hemidesmosomal components BP180 and BP230, but not
6 or HD1/plectin. Whereas
6 is concentrated in focal
contacts, HD1/plectin is diffusely distributed in the cytoplasm. Our results demonstrate that transfection of these
PA-JEB keratinocytes with cDNA encoding wild-type
4
results in the reexpression of the protein that is correctly
associated with
6 and is clustered in patches at the basal
cell surface. Most strikingly, restored expression of
4 results in the formation of organized HD-like structures that
contain, in addition to the
6
4 integrin, the hemidesmosomal elements HD1/plectin, BP180 and BP230. These
HD-like structures are encircled by focal contacts in a pattern indistinguishable from that observed in cultured normal human keratinocytes (Carter et al., 1990
; Marchisio et
al., 1990). These findings demonstrate that expression of
4 affects the subcellular localization of various hemidesmosomal components, suggesting that it recruits these
components to HD-like structures.
Previous electron microscopical studies have shown that
PA-JEB patients exhibit hypoplastic HDs with poorly developed attachment plaques and subbasal dense plates
(Vidal et al., 1995; Niessen et al., 1996
). Our results provide additional support for a crucial role of
6
4 in the
formation and organization of HDs. It is noteworthy that,
in contrast to our findings in vitro, HD1/plectin, BP180, and BP230 appeared to be correctly localized at sites of
cell-substrate contact in the skin of this PA-JEB patient
(Niessen et al., 1996
). The different localization of these
proteins in vitro is puzzling, but may reflect the inability of
the immortalized PA-JEB cells to synthesize the ligand(s)
for BP180 which normally induces its correct polarization
at the basal cell surface via a ligand-receptor interaction.
Signaling via the 4 TAM Is Dispensable for the
Formation of HD-like Structures
Mutagenesis experiments have suggested that phosphorylation of the 4 TAM is required for the formation of HDs
in 804G rat bladder carcinoma cells, since a
4 molecule
with phenylalanine substitutions in the TAM was not incorporated in HDs (Mainiero et al., 1995
). However, using
the same cells we could not reproduce these results (Niessen et al., 1997a
). Consistent with and in extension to that
observation, we here show that a
4 molecule with a mutated TAM is able to initiate the assembly of well-organized HD-like structures containing HD1/plectin, BP180,
and BP230 in PA-JEB keratinocytes. Nevertheless, in
some cells expressing the
4 TAM mutant, BP180 remained diffusely distributed. This observation is in agreement with recently published results suggesting that mutations in the
4 TAM affect the localization of BP180 in
COS-7 cells (Borradori et al., 1997
). Together, our findings do not support the supposition that the TAM is critical for the formation of HDs.
Multiple Interactions between 4, HD1/Plectin,
and BP180
Sequences within 4, encompassing the second FNIII repeat and a stretch of 27 amino acids of the CS, have previously been shown to be required for the localization of
6
4 in HDs of 804G cells that endogenously express
4
(Niessen et al., 1997a
). These sequences are also important
for the localization of HD1/plectin into junctional complexes containing
6
4 (Niessen et al., 1997a
; Sánchez-Aparicio et al., 1997
). Our present study indicates that this
same region is critical for initiating the formation of HD-like structures in PA-JEB keratinocytes. In contrast to the
abrupt loss of HD1/plectin recruitment when
4 is truncated after amino acid 1328 (
41,328), increasing COOH-terminal truncations of
4 appeared to gradually decrease
the capacity of these mutants to redistribute BP180 and BP230 in PA-JEB cells.
We show here that the effect of 4 on the subcellular localization of BP180 in both transfected PA-JEB keratinocytes (this study) and COS-7 cells (Borradori et al.,
1997
) is due to its direct binding to BP180. Using the yeast
two-hybrid system, we have identified major binding sites
for BP180 in a segment comprising the COOH-terminal
half of the CS and the third FNIII repeat. The interaction
with BP180 of either the CS or the third FNIII repeat alone was only weak. In accordance with these results,
4
mutants with truncations in this region exhibited a reduced capacity to recruit BP180, as assessed by immunofluorescence analysis. Surprisingly,
4 mutants that lacked
the sequences critical for localization of HD1/plectin (i.e.,
the first pair of FNIII repeats and 27 amino acids of the
CS) were no longer able to recruit BP180, even though they contained the BP180 binding site(s). These findings
suggest that the recruitment of BP180 into HDs requires
the interaction of BP180 with both
4 and HD1/plectin.
Three observations support this hypothesis. First, preliminary studies using the yeast two-hybrid system demonstrate a direct interaction between HD1/plectin and BP180
(Aho, S., and J. Uitto. 1997. J. Invest. Dermatol. 108:546a). Second, BP180 is not localized in HD-like structures at the
basal cell side in keratinocytes derived from an epidermolysis bullosa simplex with muscular dystrophy patient lacking HD1/plectin (Gache et al., 1996
). Finally, our immunoprecipitation analysis of transfected COS-7 cells showed
the presence of the mutant
41,355 protein, containing the
HD1/plectin-binding region but lacking the binding sites
for BP180, in the BP180 immunoprecipitate. In contrast,
coimmunoprecipitation of a
41,328 mutant which is no
longer able to recruit HD1/plectin, was not observed. Although the presence of HD1/plectin in these immune complexes could not be assessed due to the lack of a suitable antibody which efficiently detects monkey HD1/plectin on
immunoblots, these observations provide indirect evidence for an association of BP180 with both
4 and HD1/
plectin.
The finding that the efficient localization of BP180 into
HDs seemed to depend on its interaction with at least two
different hemidesmosomal components is not without precedent. The linkage of keratin filaments to the hemidesmosomal plaque involves the contribution of the two IF-binding proteins, HD1/plectin and BP230 (Foisner et al., 1988;
Yang et al., 1996
). Deletion of one of these molecules is not
compensated by the remaining components and disturbs
the anchorage of the keratin filaments to the hemidesmosomal plaque (Guo et al., 1995
; Gache et al., 1996
; McLean
et al., 1996
; Smith et al., 1996
; Andrä et al., 1997
). Thus, it is
likely that proper assembly of HDs requires multiple interactions between the various components.
Recently, it was suggested that the incorporation of
BP180 into HDs is facilitated by a direct interaction with
the 6 integrin subunit (Hopkinson et al., 1995
). Although
it is not excluded that such an interaction may contribute
to the stabilization of HDs, our results do not support this
contention and they further show that the localization of
BP180 clearly depends on distinct cytoplasmic regions of
4 rather than
6. Even when
6 and the various
4 mutants were clustered at the basal cell surface, BP180 remained diffusely distributed. In addition, we recently
found that chimeric proteins consisting of the extracellular
and transmembrane domains of the interleukin 2 receptor
and the cytoplasmic domain of
4 recruit BP180 to the
basal cell surface in a laminin-5- and
6-independent
manner (Nievers et al., 1998
).
The translocation of BP180 to the preexisting complex
consisting of 4 and HD1/plectin at the basal cell surface
may constitute an intermediate step in the nucleation of
HD-like structures and precede the localization of BP230
(this study; Gagnoux-Palacios et al., 1997
). Indeed, restored synthesis of BP180 in keratinocytes, derived from a
patient suffering from generalized atrophic benign epidermolysis bullosa associated with a deficiency for BP180, affected the subcellular localization of BP230. BP230 was no
longer diffusely distributed in the cytoplasm, but was
found together with BP180 at the basal surface of the
transfected cells (Borradori et al., 1998
). However, it is
possible that additional pathways exists regulating the distribution of BP230, as inferred from the observation that
expression of
4 with a mutated TAM, appeared to impair
the recruitment of BP180 but not of BP230, into HD-like structures. Furthermore, a BP180-independent redistribution of BP230 was observed in ~10-20% of the cells transfected with the
4 internal deletion mutants. The partial
redistribution which is seen of BP180 and BP230 upon
transfection of some
4 mutants may also be due to compensatory mechanisms present in some cells but absent in
others. This would mean that other proteins critical for
hemidesmosome assembly are yet to be detected.
Are the 4 Binding Sites for BP180 Masked by an
Intramolecular Interaction?
As has been described for vinculin (Johnson and Craig,
1995; Gilmore and Burridge, 1996
) and the ERM protein
family (Bretscher et al., 1997
), conformational activation
of proteins appears to be a mechanism by which the assembly of cell surface structures can be regulated. The results of our transfection studies and the observation by
others that BP180 is not localized at the basal cell surface
in HD1/plectin-deficient keratinocytes (Gache et al., 1996
)
raise the intriguing possibility that HD1/plectin contributes to the formation of HDs not only by providing an interaction site for BP180 (as discussed above), but also by
affecting the capacity of
4 to bind to BP180. What are the
mechanisms by which HD1/plectin might regulate the
binding of
4 with BP180? The results of the two-hybrid
assay suggest the possibility of an intramolecular interaction in the
4 cytoplasmic domain, since a segment of 85 COOH-terminal residues can bind directly to a more NH2-terminally located region. Strikingly, this region encompasses the sequences essential for the recruitment of HD1/
plectin; i.e., the first pair of FNIII repeats and part of the
CS (this study; Niessen et al., 1997a
). Hence, it is possible
that the intramolecular interaction is disrupted by a high-affinity interaction of HD1/plectin with
4 and that binding of BP180 to
4 is thus facilitated. Alternatively, the
intramolecular interaction between the NH2 and COOH
regions of the
4 cytoplasmic domain is regulated by phosphorylation. Binding of HD1/plectin may then help to
maintain an unfolded structure of the
4 molecule, thereby
rendering the binding site for BP180 accessible for interaction. Strong phosphorylation of the COOH-terminal segment of
4 has indeed been found in various epithelial cell lines (Falcioni et al., 1989
; Kennel et al., 1989
). Tyrosine
phosphorylation of the TAM (Mainiero et al., 1995
, 1996)
may play only a secondary role in this process as mutation
of the tyrosine residues in this motif had only a minor effect on the localization of BP180 (this study; Borradori et
al., 1997
).
In conclusion, the results of our study indicate that immortalized PA-JEB keratinocytes lacking 4 expression
are not capable of forming organized HD-like structures.
Transfection of these cells with
4 cDNA resulted in restored synthesis of this protein, which was correctly associated with
6 and induced the recruitment of various
hemidesmosomal components to the basal side of the cell
into junctional complexes typical for HD-like structures. Mutation of the TAM within the
4 cytoplasmic domain
did not prevent the assembly of these structures. Distinct
domains within the cytoplasmic tail of
4 were shown to
regulate the localization of HD1/plectin and both BP180
and BP230 into HDs. A direct interaction between the cytoplasmic domains of
4 and BP180 is involved in this process. The observed interaction of the 85 COOH-terminal residues with a more NH2-terminally located region suggests that the cytoplasmic domain of
4 can undergo an intramolecular interaction. Finally, we propose that the localization of BP180 may be regulated by the complex
formation of
4 and HD1/plectin that changes the
4 conformation such that it can directly interact with BP180.
![]() |
Footnotes |
---|
Address all correspondence to A. Sonnenberg, Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: (31) 20-5121942. Fax: (21) 20-5121944. E-mail: asonn{at}nki.nl
Received for publication 30 March 1998 and in revised form 27 May 1998.
R.Q.J. Schaapveld, Luca Borradori, and D. Geerts contributed equally to this work.We thank L. Oomen for excellent assistance with the confocal laser microscope and assembly of the figures, N. Ong for photographic work, and J. Calafat and H. Janssen (all four from The Netherlands Cancer Institute,
Amsterdam, The Netherlands) for electron microscopy. We are grateful
to M. Jonkman (Gronnigen University Hospital, Gronnigen, The Netherlands) for diagnosing the PA-JEB patient and providing us with skin from
this patient. We thank P.M. Howley (Harvard University School of Medicine, Boston, MA) for providing the p1432 plasmid and E.M.H. van der
Raay-Helmer (Free University of Amsterdam, Amsterdam, The Netherlands) for help with immortalizing the keratinocytes from the primary culture. We acknowledge A.M. Martínez de Velasco for generation of the
mouse anti-4 mAb 113C. We thank L. Bruckner-Tuderman, C.H. Damsky,
C.G. Figdor, M.A. Glukhova, S.J. Kennel, K. Owaribe, and J.R. Stanley
for their generous gifts of antibodies, and F.G. Giancotti for the TAM-mutated
4 construct. The authors would like to thank P. James for providing yeast strain PJ69-4A and advice. We acknowledge E. Roos, C.P. Engelfriet, and L. Fontao (all three from The Netherlands Cancer Institute except Engelfriet from Central Laboratory of the Netherlands Red
Cross Blood Transfusion Service, Amsterdam, The Netherlands) for critical reading of the manuscript.
This work was supported by grants from the Dutch Cancer Society (NKI 96-1305 and NKI 95-979), the Biomedical and Health program (BIOMED, BMH4-CT97-2062), and the Dystrophic Epidermolysis Bullosa Research Association (DEBRA Foundation, Crowthorne, UK). L. Borradori was supported by the Roche Foundation (Basel, Switzerland) and a grant from the Swiss National Foundation of Scientific Research (32-51083.97).
![]() |
Note Added in Proof |
---|
Two recent publications have appeared in which a
direct interaction between 4 and plectin (Rezniczek, G.A., J.M. de
Pereda, S. Reipert, and G. Wiche. 1998. J. Cell Biol. 141:209-225) and between
4 and BP180 (Aho, S., and J. Uitto. Biochem. Biophys. Res. Commun. 243:694-699) has been demonstrated.
![]() |
Abbreviations used in this paper |
---|
AD, activation domain; BD, binding domain; BP180, bullous pemphigoid antigen 180; BP230, bullous pemphigoid antigen 230; CS, connecting segment; FNIII, type III fibronectin repeat; GAL4, galactose metabolism regulatory gene 4; HDs, hemidesmosomes; IF, intermediate filament; NHK, normal human foreskin keratinocytes; PA-JEB, junctional epidermolysis bullosa associated with pyloric atresia; SC, synthetic complete medium; TAM, tyrosine activation motif.
![]() |
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