Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

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 64 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).

64 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). 64-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 64 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 64 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.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

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-FLAG^TM M2 against the FLAG^TM 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-Pure^TM 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.

View larger version (31K): [in this window] [in a new window]   Fig. 10.   Survey of the sites of interaction between the cytoplasmic domains of 4 and BP180. Yeast strain PJ69-4A was cotransformed with pAS2-BP180 and one of each of the listed pACT2-4 constructs, or with an empty pACT2. Transformation mixtures were spread on SC-LT and SC-LTHA plates and grown for 9 d at 30°C. Plating efficiency on SC-LTHA plates is expressed relative to that on SC-LT plates of the same transformation. ++, \>50%; ±, 5-25%; and , 0% indicate relative efficiencies, respectively. Plates were scored after 4 and 9 d of growth. Plating efficiencies above 25% represent fast-growing colonies that could be scored after 4 d; plating efficiencies lower than 25% represent colonies that clearly grow more slowly and could only be scored after 9 d of growth. All efficiencies listed represent an average of multiple independent transformation experiments on at least three separate days. Cotransformation efficiencies (on SC-LT plates) for all plasmid combinations listed were always at least 104 cfu/µg, and the difference between the various 4 plasmids tested never was greater than twofold. Cotransformation of yeast PJ69-4A with empty pAS2-1 and pACT2 vectors never resulted in the growth of colonies on SC-LTHA plates, nor did cotransformation of the yeast strain with either the pAS2-BP180 plasmid and an empty pACT2 vector or any of the pACT2-4 plasmids and an empty pAS2-1 vector, showing that none of the GAL4 fusion proteins encoded by these recombinant plasmids by themselves could cause activation of the His and Ade reporter genes.

View larger version (28K): [in this window] [in a new window]   Fig. 12.   Intramolecular interactions in the 4 cytoplasmic domain. Yeast strain PJ69-4A was cotransformed with two different 4 constructs as listed. Other details are as in Fig. 10, except that the efficiencies represent an average of multiple independent transformation experiments on at least two separate days.

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.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

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 64 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 64 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.

View larger version (107K): [in this window] [in a new window]   Fig. 1.   Immunolocalization of hemidesmosomal components in the NHK (A-E) and PA-JEB cell lines (F-J) by confocal laser microscopy. Cells were grown on glass coverslips in HAMF12/DME (1:3) medium, fixed, and immunolabeled using the rat mAb GoH3 directed against 6 (A and F), the mAb 450-9D against 4 (B and G), a rabbit anti-BP230 antiserum (C and H), the mAb 233 against BP180 (D and I), and the mAb 121 against HD1 (E and J). In NHK, the hemidesmosomal components are concentrated at sites of cell- substrate contact in patches characteristic for HD-like structures. In PA-JEB keratinocytes, only BP230 (H) and BP180 (I) are found concentrated in the rare HD-like structures in fewer than 1% of the cells. 6 is colocalized with vinculin (data not shown) in dots and streaks representing focal adhesions (F), whereas HD1/plectin is found diffusely distributed throughout the cell (J). No 4 reactivity is observed in PA-JEB cells (G). Sections were focused at the cell-substrate interface. Arrowheads, positions from which the perpendicular sections, shown in the insets, were taken. Bar, 10 µm.

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 64, PA-JEB keratinocytes express 61 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).

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Immunoprecipitation of integrin complexes and BP180 from NHK and PA-JEB keratinocytes. Lysates of 125I-labeled NHK (left) and PA-JEB keratinocytes (right) were immunoprecipitated with the mAbs P1E6 (against 2, lane 1), J143 (3, lane 2), Sam-1 (5, lane 3), NKI-M9 (v, lane 4), J8H (6, lane 5), TS2/16 (1, lane 6), 450-9D (4, lane 7), 439-9B (4, lane 8, right) and 1D1 (BP180, lane 8, left and lane 9, right, respectively). The antibody against 6 precipitated this subunit associated with 4 from NHK, whereas from the PA-JEB cells 6 and 1 were precipitated, but not 4. The faint band which migrates just above 1 and seen in the lanes containing the anti-4 immunoprecipitates from PA-JEB cells, represents a nonspecific product. 1 is found in association with 2, 3, 5, and 6 in PA-JEB cells, but only with 2, 3, and 5 in NHK. Precipitation of 1 with 5 is evident after prolonged exposure (data not shown). Samples were analyzed on a SDS-polyacrylamide (5%) gel under nonreducing conditions. The positions of molecular weight standards (in kD) are indicated on the left.

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).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Expression of full-length, TAM-mutated, COOH-terminally truncated (A), and internal deletion mutant (B) 4 cDNAs in PA-JEB keratinocytes. Schematic representation of the cDNA constructs encoding wild-type and mutant forms of 4 carrying deletions of the cytoplasmic domain. Boxes, FNIII repeats in which the number of the repeat is shown; triangle, the 4B-specific insert of 53 amino acids in the CS (A), or the 17-amino acid deletion in the second FNIII repeat as described for a PA-JEB patient (Vidal et al., 1995) (B). Y > F mutations of the TAM are represented by F. The immunolocalizations of the hemidesmosomal components HD1/plectin, BP180, and BP230 were investigated upon transfection of the 4 constructs in PA-JEB cells. Colocalization of HD1/plectin, BP180, and BP230, respectively, with 64 at the basal cell surface in HD-like structures is observed in ++, 75-100%; +, 25-75%; ±, 1-25%; , 0%, of the 4-transfected cells.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Immunoprecipitation analysis of PA-JEB keratinocytes transfected with cDNA encoding wild-type 4 (PA-JEB R). Lysates of [35S]methionine/cysteine-labeled NHK and PA-JEB cells were immunoprecipitated with the mAb J8H against 6 (lane 1), the anti-4 mAb 450-9D (lane 2), and the mAb TS2/16 against 1 (lane 3). Antibodies against 6 precipitate 6 and 4 from NHK, and 6 together with 1 from PA-JEB cells. Antibodies against 4 precipitate this subunit together with 6 from NHK, but not from PA-JEB cells. In contrast, from PA-JEB cells transfected with cDNA for 4 (PA-JEB R) antibodies against 4 (lane 1) precipitate 4 together with 6. Coprecipitation of 6 is evident after prolonged exposure (lane 2). Samples were analyzed on a SDS-polyacrylamide (5%) gel under nonreducing conditions. The positions of molecular weight standards (in kD) are indicated on the left and those of the 4 and 6 integrin subunits are indicated on the right.

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 64 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.

View larger version (74K): [in this window] [in a new window]   Fig. 5.   Expression of wild-type 4 in PA-JEB keratinocytes induces the formation of HD-like structures. PA-JEB cells were transfected with cDNA encoding 4A. After 36 h, cells were fixed, permeabilized, and subjected to double labeling immunofluorescence for 4 (A-E) and 6 (F), vinculin (G), HD1/plectin (H), BP180 (I), and BP230 (J). Upon transfection, expression of 4 results in the formation of HD-like structures, in which 4 is concentrated at sites of cell-substrate contact and codistributed with 6, HD1/plectin, BP180, and BP230 (insets are the perpendicular sections). In cells expressing 4, 6 is now found in HD-like structures (F), and no longer concentrated in focal adhesions at the outer periphery of these structures, as shown by staining for vinculin (G). Bar, 10 µm.

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 64 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.

View larger version (75K): [in this window] [in a new window]   Fig. 6.   Expression of a 4 TAM mutant induces the assembly of HD-like structures in transfected PA-JEB keratinocytes. PA-JEB cells transfected with TAM-mutated 4A cDNA were double stained for 4 (A, C, E, and G) and 6 (B), HD1/plectin (D), BP180 (F), and BP230 (H). As shown in the perpendicular sections, a 4 molecule with phenylalanine substitutions at the TAM becomes localized together with 6 at the basal cell side and recruits HD1/plectin, BP180, and BP230 to sites of cell-substrate contact. The redistribution of BP180 was, however, slightly impaired. Bar, 10 µm.

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).

View larger version (78K): [in this window] [in a new window]   Fig. 7.   A segment comprising the first pair of FNIII repeats and a 27-amino acid stretch of the CS is essential for the localization of HD1/plectin at the basal cell surface. Representatives of double immunofluoresence analyses of PA-JEB cells transfected with cDNA encoding COOH-terminal deletion mutants of 4 as depicted in Fig. 3 are shown. PA-JEB cells transfected with cDNA coding for 41,355 or 41,328 were immunolabeled with antibodies against 4 (red) and HD1 (green). Although 41,355 still induces the redistribution of HD1/plectin to the basal surface of the cell (left), 41,328, lacking an additional 27 amino acids of the CS does not affect the distribution of HD1/plectin (right). Noteworthy, the distribution pattern of 64 in the absence of HD1/plectin is comparable to that of 64 together with HD1/plectin and indistinguishable by confocal microscopy. Bar, 10 µm.

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 (4A^1,487) already reduced the ability of the mutated 4 molecules to localize BP180 and BP230 together with 64 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 (4^1,355) 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 (4^1,328), in which HD1/ plectin was no longer concentrated together with 64 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).

View larger version (48K): [in this window] [in a new window]   Fig. 8.   Distribution of BP180 and BP230 in PA-JEB keratinocytes is affected by COOH-terminal truncations of 4. PA-JEB cells transfected with cDNA encoding mutant forms of 4 were double-stained for 4 (red) and BP180 (green) (A and B) or for 4 (green) and BP230 (red) (C and D). Shown are representatives of transfections with 41,436 cDNA. Truncation of the second pair of FNIII repeats already impairs the recruitment of BP180 (B) and BP230 (D) to the basal cell surface, although cells showing colocalization of the BP antigens with 4 can readily be found (A and C). Increasing COOH-terminal truncations further impair the localization of BP180 and BP230 at the basal cell side (refer to Fig. 3 A). In cells transfected with 41,328 cDNA, which also do not show basal localization of HD1/plectin, BP180 and BP230 remain diffusely distributed throughout the cell (data not shown). Bar, 10 µm.

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-FLAG^TM 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 4^1,355 mutant protein were co-precipitated by anti-FLAG^TM M2 antibodies (Fig. 9, top, lane 6). As expected, the 4^1,328 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-FLAG^TM 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.

View larger version (63K): [in this window] [in a new window]   Fig. 9.   Coimmunoprecipi-tation of 4 and BP180. Lysates of COS-7 cells cotransfected with cDNAs for 6A and wild-type 4A, 41,355, or 41,328 as well as an empty pCI-Neo vector or a pCI-Neo construct encoding the BP180 cytoplasmic domain were subjected to immunoprecipitation with a mixture (1:1:1) of three anti-4 mAbs, 4.3E1, 113C and 450-9D, respectively, or with the mAb FLAGTM M2. Samples were resolved on a SDS-polyacrylamide (8%) gel under nonreducing conditions. Shown is an immunoblot analysis developed with the rabbit polyclonal anti-serum against 4 (top) and the mAb FLAGTM M2 to detect BP180 (bottom) among the immunoprecipitated proteins. When samples immunoprecipitated with the anti-4 mAbs were subjected to immunoblotting with the mAb FLAGTM M2 (bottom) or a polyclonal anti-BP180 antiserum (data not shown), the mutant form of BP180 was not detectable in the anti-4 immunoprecipitates. The positions of molecular weight standards (in kD) are indicated on the right.

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 4^1,115-1,666 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, 4^1,115-1,666 (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 (4A^1,115-1,457) resulted in a dramatic reduction in binding. Truncation up to the last 21 amino acids of the CS (4^1,115-1,436) had no effect on the binding, but there was no binding at all when 54 additional residues were deleted (4^1,115-1,382). 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 (4A^1,320-1,668) did not result in decreased binding to BP180, as compared to 4A^1,115-1,666. The same was found for binding of BP180 to 4A^1,320-1,552, compared to 4^1,115-1,600. Accordingly, neither the first (4^1,115-1,217) nor the second (4^1,217-1,328) FNIII repeat can bind to BP180. Efficient binding to BP180 was only observed when both the CS and the third FNIII repeat were present (4A^1,320-1,552). The third or fourth FNIII repeat or the COOH terminus alone (4^1,457-1,552, 4^1,570-1,668, and 4^{1,667- 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 4^1,355 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.

View larger version (27K): [in this window] [in a new window]   Fig. 11.   Basal localization of HD1/plectin together with 4 is essential for the recruitment of BP180. PA-JEB cells transfected with cDNA encoding internal deletion mutants of 4 were double-stained for (A) 4 (red) and HD1/plectin (green), (B) 4 (red) and BP180 (green), and (C) 4 (green) and BP230 (red). Shown are representatives of transfections with 4A1,249-1,265. Deletion of 17 amino acids in the second FNIII repeat or its complete deletion prevent the recruitment of HD1/plectin (A). As a consequence, 64 clustered at the basal cell surface is no longer capable of recruiting BP180 to these HD1/plectin-lacking clusters although the binding sites for BP180 are still present (B). In a few cases (i.e., <25% of 4-transfected cells) these 4 mutants were able to recruit BP230 in an HD1/plectin- and BP180-independent manner to the basal cell surface (C). Bar, 10 µm.

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