Department of Dermatology, Washington University Medical School, St. Louis, Missouri 63110
Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell-cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell-cell adhesion.
Structurally related desmosomes and adherens
junctions, collectively termed adhering junctions,
are involved in anchoring the cytoskeleton to the
plasma membrane, intercellular cell type-specific adhesion, and signaling (Geiger and Ayalon, 1992 In contrast with adherens junctions that may contain
only one cadherin isoform, desmosomes always include
cadherins from two subfamilies, desmogleins (Dsg1-3)1 and
desmocollins (Dsc1-3). Alternative splicing increases Dsc diversity producing long (Dsc a) and short (Dsc b) isoforms
that differ in their intracellular domains (Garrod, 1993 Involvement of the desmosomal cadherins in cell-cell
adhesion was underscored by cell culture observations that
antibodies against the extracellular regions of Dsc interfered with the formation of the epithelial sheet (Cowin et al.,
1984 Several observations suggest that efficient desmosome
formation, and hence interactions between desmosomal cadherins, may require the function of the classic cadherins
(Wheelock and Jensen, 1992 Plasmid Construction
The plasmid BlDc1a encoding the entire sequence of the mature bovine
Dsc1a isoform was obtained by subsequent insertions in the Bluescript
vector of the XhoI-NotI and XhoI-KpnI inserts cut out from the plasmids
BDC 7.5 and BDC 6.1, respectively (Koch et al., 1991 To express Dsg and Dsc extracellular fragments Dc12M (Arg1-Asp212),
Dg12F (Glu1-Asp212), and Dg123F (Glu1-Val335), in Escherichia coli, the
QIA-expression system (Qiagen, Chatsworth, CA) was used. The corresponding sequences of the bovine Dsg1 and Dsc1a were amplified, ligated
either with single myc or flag (Sigma Chemical Co., St. Louis, MO) sequences, and then inserted in a pQE18 vector. Correct amplification and
cloning of all recombinant plasmids was checked by restriction endonuclease mapping and nucleotide sequencing. Plasmid CMVSyPg encoding a
chimeric protein consisting of the entire synaptophysin and plakoglobin
has been described (Chitaev et al., 1996 Cell Culture, DNA Transfection, and
Immunological Methods
The HT-1080 human fibrosarcoma cells were provided by Dr. G. Goldberg (Washington University, St. Louis, MO). The cells were grown in
DME (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FCS.
Transfection of HT-1080 cells, as well as the selection, growth, immunofluorescence microscopy, and immunoprecipitation, was done as described
for A-431 cells (Troyanovsky et al., 1993 Solid Phase and Reconstitution Assays
The in vitro solid phase assay was described previously (Chitaev et al.,
1996 For the reconstitution assay the Dc12M fragment was mixed with the
Dsg fragments, Dg12F, or Dg123F in 1.5 ml PBS in final concentration of
1 µg/ml. For control, Dc12M was not added. Samples were incubated 15 min, and then subsequently treated with 75 µl 9E10 anti-myc antibodies
and with 15 µg protein A-Sepharose (Pharmacia, Piscataway, NJ) suspended in PBS. The beads were then washed five times with PBS supplemented with 1% Triton X-100. The immunoprecipitates were analyzed by
immunoblotting with different primary antibodies in conjunction with an
enhanced chemiluminescence detection system (Boehringer Mannheim
Biochemicals, Indianapolis, IN).
Expression of Desmocollin 1a in Human Fibrosarcoma
HT-1080 Cells Induces Intracellular Redistribution and
Lateral Clustering of Endogenous Desmoglein 2
Although interactions between desmosomal cadherins are
likely to be involved in desmosome assembly and in cell-
cell adhesion of epithelial tissues, expression of Dsg and/or
Dsc in the adhesion-deficient L-fibroblasts did not produce a detectable increase in intercellular adhesion (Amagai et al., 1994
Since desmosomes contain two distinct cadherins, Dsg
and Dsc, we examined whether expression of the full-length bovine Dsc1a can bring about lateral clustering of
the endogenous Dsg2 in HT-1080 cells. Double immunofluorescence analysis using polyclonal anti-Dsg2 and monoclonal anti-Dsc1 2.10 antibodies revealed a redistribution of endogenous Dsg2 in HT-1080 cells stably transfected
with Dsc1a (HTDc cells). In these cells both desmosomal
cadherins Dsg2 and Dsc1a were colocalized in large clusters of cell-cell contact regions (Fig. 2, b and b To determine the contribution of the intracellular and
extracellular domains of Dsc to the assembly of the clusters of desmosomal cadherins, we transfected HT-1080 cells
with several mutants of Dsc1a (Fig. 3 A). Two deletion
mutants were constructed and stably expressed in HT-1080 cells. The first mutant, Dsc
Desmosomal Cadherins, Dsg and Dsc, Are Involved in
Ca2+-dependent Heterophilic Interactions
The ability of the Dsc1a to interact with Dsg2 in HTDc
cells was tested in coimmunoprecipitation experiments
(Fig. 3 B). The total NP-40 lysates of the control HT-1080
cells, and cells stably expressing Dsc1a or its mutants, were
immunoprecipitated with anti-Dsc antibodies. The presence of Dsg in the immunoprecipitates was monitored by
immunoblot analysis. These experiments revealed that Dsc1a
was able to coprecipitate Dsg2 from the HTDc cell lysate (Fig. 3 B). Substitution of the homologous sequence from
bovine Dsg1 for the extracellular region of Dsc (the
Dsc[1-170Dg] mutant) completely abolished Dsg/Dsc interactions in the coimmunoprecipitation assay, whereas
deletions of the intracellular domain of Dsc, Dsc To address the question of whether Dsg2 directly interacts with Dsc1a or these interactions are mediated by an
additional protein(s), the control and HTDc cells were
metabolically labeled before immunoprecipitation. A prominent 120-kD band reacting with anti-Dsc antibodies was
present in the immunoprecipitates obtained from HTDc
cells using mAb 2.10 (Fig. 3 C). Only Dsg2 consistently coimmunoprecipitated with Dsc1a as a protein with a molecular mass of 160 kD that reacted with both anti-Dsg
3.10 and G129 antibodies (Fig. 3 C). An association between Dsg and Dsc was also detected in similar immunoprecipitation experiments using anti-Dsg antibodies (Fig.
3). The absence of the other specific bands in addition to
Dsg or Dsc in these immunoprecipitates suggests a direct
interaction between these two proteins in the HTDc cells.
A disproportionately small amount of coprecipitating protein in these experiments could be caused, in part, by low
solubility of the Dsg/Dsc complexes compared with free
forms of both cadherins. Indeed, a significant amount of
both desmosomal cadherins was found in the pellets after NP-40 extraction (not shown).
It is well documented that interaction of classic cadherins is Ca2+ dependent (Ozawa et al., 1990
Since immunoprecipitation experiments revealed that the
first two cadherin-like domains of Dsc are required for interactions with Dsg, we constructed and expressed the recombinant protein Dc12M in E. coli. This protein consists
of the cadherin-like domains 1 and 2 of Dsc followed by
the myc epitope and histidine hexamer (Fig. 5 A). To determine whether the Dsg domain(s) is involved in the interaction with Dc12M protein, we constructed the Dsg fragments Dg12F and Dg123F containing its extracellular
cadherin-like domains 1 and 2 or 1, 2, and 3, respectively.
Carboxyl termini of the Dsg-derived polypeptides were
tagged by the FLAG epitope and polyhystidine (Fig. 5 A).
SDS-PAGE and immunoblot analysis of the recombinant
proteins purified by Ni-agarose chromatography showed
that the samples contained homogenous polypeptides of
the expected molecular mass of 30 kD for Dg12F and
Dc12M, and 45 kD for Dc123M (Fig. 5 A).
To analyze direct interactions between Dc12M and Dsg
fragments we used a solid phase assay developed previously for examination of plakoglobin-cadherin binding
(Chitaev et al., 1996 As an alternative approach, the molecular interactions
between different Dsg and Dsc fragments were tested in a
reconstitution assay. Fig. 5 C shows a representative experiment in which fragments Dc12M were mixed with fragments Dg12F or Dg123F and immunoprecipitated using
anti-myc antibody. The results of these experiments are in
good agreement with those obtained in the solid phase
binding assay, demonstrating the strong interaction between Dc12M with Dg123F and Dg12F fragments.
Heterophilic Binding of Desmosomal Cadherins
Contribute to Cell-Cell Adhesion
Endogenous expression of N-cadherin interferes with
the measurement by conventional aggregation assays of
the adhesive properties of HT-1080 cells that are due to
the expression of transfected desmosomal cadherins. To
test the contribution of the Dsg/Dsc interaction to cell-cell
adhesion, HT-1080 cells expressing Dsc
In addition, to show that interactions between desmosomal cadherins were present at the intracellular interface,
we investigated whether the amount of the Dsc/Dsg complex is proportionate to cell density in culture. Intercellular contacts in HT-1080 cells at ~75% confluence are
more extensive than in those at low density. Thus, HT-1080 cells expressing Dc A number of studies have shown that expression of Dsg
and/or Dsc in adhesion-deficient L-fibroblasts fails to produce detectable levels of intercellular adhesion (Amagai
et al., 1994 Here we have shown that expression of the full-length
Dsc1a in HT-1080 cells results in a redistribution of endogenous Dsg2. Both desmosomal proteins, Dsg2 and Dsc1a,
were efficiently incorporated into the cell-cell contacts of
HTDc cells, where they become associated with adherens
junction proteins, such as N-cadherin and Another important feature of Dsc/Dsg complexes is revealed by the fact that MPg coimmunoprecipitated with
anti-Dsc antibodies from the coculture of Dsc The low level of Dsg/Dsc interactions in sparse cultures
of HTDc cells is consistent with the data of Kowalczyk et al.
(1996); Schmidt et
al., 1994
; Klymkowsky and Parr, 1995
; Peifer, 1995
; Cowin
and Burke, 1996
; Gumbiner, 1996
). Classic and desmosomal cadherins are featured in both adherens and desmosome junctions. It is widely accepted that classic cadherins
mediate homophilic calcium-dependent cell-cell adhesion
(Nose et al., 1990
; Grunwald, 1993
; Shapiro et al., 1995
; Brieher et al., 1996
; Nagar et al., 1996
). An exception to
this rule, heterophilic binding of the chicken B-cadherin to
LCAM, has been documented (Murphy-Erdosh et al.,
1995
). On the intracellular face of the plasma membrane,
cadherins are integrated into plaques consisting of junctional-specific proteins. These proteins function in the formation of anchoring sites for microfilaments and intermediate filaments (in adherens junctions and desmosomes,
respectively) and are critical for adhesion and signaling
properties of cadherins (Nagafuchi and Takeichi, 1988
;
Ozawa et al., 1989
; Green and Jones, 1990
; Geiger and Ayalon, 1992
; Schmidt et al., 1994
).
; Koch
and Franke, 1994
). Recent experiments with chimeric proteins consisting of the gap junction protein connexin32 and
the intracellular regions of desmosome cadherins indicate
that Dsg and Dsc have different functions. The CoDsc chimera containing the intracellular portion of Dsc1a nucleated the formation of the intracellular desmosomal plaques. The cytoplasmic domain of Dsg1, in a similar construct, displayed a dominant negative effect on desmosome formation (Troyanovsky et al., 1993
, 1994a
,b
).
). Also, auto anti-Dsg antibodies caused a blistering
skin disease (Stanley, 1995
). Perturbation in epidermal
cell-cell interactions was found in transgenic animals producing a dominant negative form of Dsg (Allen et al., 1996
). Structural similarity between the extracellular repeats of
Dsg and Dsc with those of the classic cadherins involved in
homophilic adhesion provides additional support for the
adhesive functions of the desmosomal cadherins. However, in contrast with classic cadherins, desmosomal cadherins, alone or in combination, failed to support cell-cell
adhesion upon expression in nonadhesive fibroblast-like cells (Amagai et al., 1994
; Chidgey et al., 1996
; Kowalczyk
et al., 1996
). This suggests that the functional properties of
classic and desmosomal cadherins are distinct despite their
overall structural homology. Moreover, the molecular mechanism of coassociation of the different desmosomal cadherins in the actual desmosome is not well understood.
While direct interactions between desmosomal cadherins
were not documented, it seems likely that they are essential for desmosome assembly.
; Lewis et al., 1994
; Amagai
et al., 1995
). To investigate this possibility, we expressed
bovine Dsc1a in HT-1080 cells that feature extensive adherens junctions and produce endogenous Dsg2. We found
that expression of Dsc1a in these cells induces the formation of stable complexes between Dsc and Dsg. In addition,
we show a direct Ca2+-dependent interaction between the
extracellular regions of two desmosomal cadherins. These
observations suggest that heterophilic interactions between
desmosomal cadherins are important for targeting these
proteins to desmosomes and for cell-cell adhesion.
Materials and Methods
). The sequence coding for the leader peptide lacking in BlDc1a was amplified from plasmid
pDGK5(B), encoding bovine Dsg1 (Koch et al., 1990
). It was inserted upstream to the Dsc1a sequence in BlDc1a between the unique HindIII and
NarI sites, resulting in plasmid BlLDc1a. The fusion protein encoded by
this plasmid contains the entire sequence of the Dsg-derived leader peptide and the entire sequence of the mature Dsc1a. In Dsc1a only the first
amino acid residue Arg is replaced with Glu to create a Dsg-specific processing site. The same pDGK5(B) plasmid was used for construction of
BlDc(1-170Dg) that contains the coding sequence for the leader peptide
and for the Glu1-Arg170 sequence of the mature Dsg1 preceding the corresponding sequence of Dsc1a. Deletion mutagenesis for constructing BlDc
(697-761) and BlDc
(597-609) was done using PCR-mediated site-directed
mutagenesis. BlMPg, containing the entire sequence of human plakoglobin tagged on 5
end by 6myc epitope, was constructed using a plasmid
HPG Ca 2.1 (Franke et al., 1989
) and a plasmid CS26MT provided by Dr.
R. Kopan (Washington University, St. Louis, MO). The HindIII-XbaI inserts of all Bluescript subclones were further subcloned into the eukaryotic expression vector pBEHpac18 (Horst et al., 1991
) containing the puromycin resistance gene and SV-40 early promoter element.
).
; Chitaev et al., 1996
). The following primary antibodies were used: (a) rabbit polyclonal antibodies against
human Dsc2 and Dsg2 (Demlehner et al., 1996); (b) monoclonal murine
U114 antibodies against human Dsc3 (Nuber et al., 1996
); (c) mAbs
Dsg2E-G129 specific for human Dsg2 (Schafer et al., 1996
); (d) mAbs 2.10 against bovine Dsc1 (this antibody cross-reacts with human Dsc1); (e) 3.10 specific for human Dsg1 and Dsg2 (Schafer et al., 1996
); (f) 5.1 against human plakoglobin and a mixture of antibodies 2.15, 2.17, and 2.19 against
desmoplakin (for references describing these antibodies, see Troyanovsky
et al., 1994a
); (g) rabbit pan-cadherin antibody and mAbs GC-4 against
N-cadherin, M2 against FLAG, and 9E10 against myc epitopes (Sigma
Chemical Co.); (h) murine mAb against
-catenin (Transduction Laboratories, Lexington, KY); (i) rabbit anti-synaptophysin antibody (DAKO,
Hamburg, Germany). For immunoprecipitation 75 µl of supernatants containing 3.10 or 2.10 antibodies was added to each sample. Lysates were
precleared by centrifugation at 100,000 g for 1 h before immunoprecipitation.
). In brief, Dsg fragments isolated as described previously (Chitaev et
al., 1996
) were diluted in loading buffer (20 mM Tris HCl, pH 7.8, 1 mM
DTT with or without 2 mM EDTA) and immobilized on a 96-well dish
and incubated with increasing amounts of Dc12M. Binding was detected
by an ELISA assay with myc 9E10 mAb. The solid phase assay was always
performed in the absence or presence of 2 mM EDTA added in each solution of the binding assay. This EDTA concentration did not change the affinity of the 9E10 antibody to the myc epitope, as shown by direct ELISA assay.
Results
; Chidgey et al., 1996
; Kowalczyk et al., 1996
).
Absence of the functional adherens junctions in L-fibroblasts could be one of several reasons contributing to the
failure to demonstrate interactions between desmosomal
cadherins. From this point of view, human fibrosarcoma
HT-1080 cells present an advantage for studying Dsc-Dsg
interactions. Supporting previous observations (Sacco et al.,
1995
), we have demonstrated (Figs. 1 and 2) that, in contrast with L-fibroblasts, HT-1080 cells exhibit prominent
intercellular contacts containing adherens junctions proteins N-cadherin and
-catenin. Surprisingly, we found that HT-1080 cells, despite their fibroblast-like phenotype, synthesize Dsg2 at essentially the same level as epithelial A-431
cells (Fig. 1). By immunofluorescence analysis (see list of
the antibodies in Materials and Methods), HT-1080 cells
were devoid of other desmosomal proteins such as plakoglobin, desmoplakin, and all known isoforms of Dscs (Fig. 1).
Immunofluorescent staining with either monoclonal or
polyclonal anti-Dsg antibodies revealed that a major portion of Dsg2 is randomly distributed on the surface of the
HT-1080 cells (Fig. 2 a). Compared with A-431 cells, only a small fraction of Dsg2 can be detected at cell-cell contact regions where it is colocalized with N-cadherin and
-catenin (Fig. 2 a
). Dsg2 was sensitive to the treatment
of HT-1080 cells with trypsin (not shown), confirming its
cell surface localization.
Fig. 1.
Immunoblot analysis of the junctional proteins
synthesized in epithelial
A-431 cells and in fibrosarcoma HT-1080 cells. Equal amounts of protein from total lysates of A-431 and HT-1080 cells (lanes A431 and
HT1080, respectively) were
separated by SDS-PAGE (8%) and analyzed by immunoblotting with N-cadherin (N-cad),
-catenin (
-cat), plakoglobin (Pg), Dsg2-specific (Dsg), rabbit
Dsc2 (Dsc), and desmoplakin (Dp) antibodies.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
Redistribution of endogenous Dsg2 in HT-1080 cells expressing bovine Dsc1a or its mutants. Double-label immunofluorescence microscopy of control HT-1080 cells (a and a) and HT-1080 cells producing Dsc1a (subclone HTDc-7; b and b
and c and c
),
Dsc
(697-761) (subclone HTc-3; d and d
), and Dsc(1-170Dg) (subclone HTDcg-9; e and e
), using anti-Dsg serum (a-e) and mAbs
against
-catenin (a
and c
) or bovine Dsc1a (b
, d
, and e
). Note that only a small portion of Dsg is located at the cell-cell junctions in
parental or in HTDcg cells. The majority of Dsg is localized to the junctions in cells expressing Dsc1a or Dsc
(697-761). Bars, 25 µm.
[View Larger Version of this Image (162K GIF file)]
) that were
also positive for
-catenin (Fig. 2, c and c
). Notably, codistribution of both desmosomal cadherins was found exclusively at cell-cell contacts. For example, Dsc-positive
polymorphic vesicles detected in the cytoplasm of the
transfected cells were devoid of Dsg (Fig. 2).
(697-761), lacked the entire sequence of the C-domain mediating interaction of
Dsc1a with plakoglobin. The second mutant, Dsc
(597-
609), had an internal deletion of the CSI region involved in
binding to desmoplakin (Troyanovsky et al., 1994b
). Both
deletions had no effect on the capacity of Dsc1a to form
lateral clusters that incorporated endogenous Dsg (Fig. 2,
d and d
). Expression of Dsc in HT-1080 cells did not induce synthesis of endogenous desmoplakin or plakoglobin as determined by either immunofluorescence microscopy
or immunoblotting (not shown). Taken together, these observations suggested that known intracellular desmosomal
proteins are not required for relocation of Dsg2 in HTDc
cells. We then constructed a chimeric protein Dc(1-
170Dg) in which the sequence (Arg1-Thr170) corresponding to the first, and approximately half of the second, extracellular cadherin-like repeats of Dsc1a was replaced with the homologous sequence of the bovine Dsg1 (Fig. 3 A).
This chimeric protein was faithfully delivered to the cell surface upon expression in HT-1080 cells; however, it failed
to trigger the clustering of endogenous Dsg (Fig. 2, e and e
).
Thus the extracellular region of Dsc1a, but not its intracellular domain, is required to effect lateral clustering of
Dsg2 in HT-1080 cells.
Fig. 3.
Schematic representation of the Dsc1a gene constructs
and coimmunoprecipitation experiments showing Dsc-Dsg interactions. (A) Four extracellular cadherin-like domains (numbered
from I to IV), the intracellular conserved sequence motif CSI (I),
and the C-domain (C) involved in binding to desmoplakin or plakoglobin, respectively (Troyanovsky et al., 1994b), of bovine
Dsc1a are indicated by open boxes. The mutants Dsc
(597-609)
and Dsc
(697-761) contain the complete deletion of the CSI or
C-domain, respectively. In the mutant Dsc(1-170Dg), the Dsg1-derived sequence (stippled) substitutes for the corresponding sequence of Dsc. The plasma membrane is denoted by the vertical
double line. (B) Western blot analysis of immunoprecipitates obtained using anti-Dsc 2.10 antibody from HT-1080 cells transfected to produce Dsc1a (lane 1), Dsc
(597-609) (lane 2), Dsc
(697-761) (lane 3), and Dsc(1-170Dg) (lane 5); control HT-1080
cells (lane 4). Blots were developed either with 2.10 (Dsc) against
Dsc1a or with 3.10 (Dsg) against Dsg antibodies. (C) Autoradiograms (35S) of immunoprecipitates obtained with anti-Dsg 3.10 (IP Dsg) or anti-Dsc 2.10 (IP Dsc) antibodies from HT-1080
(lanes 1) or HTDc (lanes 2) cells. Total cell lysate of HTDc cells
was loaded in lane t; control immunoprecipitate was obtained by
anti-myc antibody in lane c. Immunoprecipitates were separated
by SDS-PAGE (8%). Note that anti-Dsg antibody precipitates an
equal amount of Dsg (arrowhead) from both cell lines in the IP
Dsg experiment; the Dsc band is only seen in lane 2 (dot). Dsc
immunoprecipitates in IP Dsc were also analyzed by immunoblotting using Dsg (Dg)- or Dsc1a (Dc)-specific antibodies. Note
that bovine Dsc1a and endogenous Dsg are seen only in transfected cells (lane 2). Asterisks on autoradiograms indicate two
bands of unknown nature that inconsistently appeared in some of
the immunoprecipitates. Bars (left) denote the relative positions
of coelectrophoresed reference proteins (from top to bottom:
-galactosidase, 116,000; phosphorylase b, 97,400; BSA, 67,000).
[View Larger Version of this Image (71K GIF file)]
(697- 761) and Dsc
(597-609), had no effect. Therefore, the
first two cadherin-like domains of the extracellular portion
of Dsc1 are required for Dsg binding. N-cadherin and
-catenin were not detected in immunoprecipitates obtained
with anti-Dsc or -Dsg antibodies (not shown), indicating
the absence of a strong interaction between desmosomal
and classic cadherins.
; Geiger and
Ayalon, 1992
; Gumbiner, 1996
; Brieher et al., 1996
). To
test whether Dsc/Dsg interactions detected in HTDc cells
were sensitive to Ca2+, the cells were lysed in the presence
of an increasing concentration of EGTA. The data presented at Fig. 4 show that 2 mM EGTA reduced the
amount of Dsc/Dsg complexes over twofold. EGTA, however, was unable to completely abolish interactions between the two desmosomal cadherins.
Fig. 4.
The calcium-dependent coimmunoprecipitation of Dsg
and Dsc. (A) HTDc cells were immunoprecipitated using anti-
Dsc 2.10 (IP Dc) or anti-Dsg 3.10 (IP Dg) antibodies. Immunoprecipitates were analyzed by immunoblotting with antibodies used
for immunoprecipitation (upper line; Dc for IP Dc, or Dg for IP
Dg) and with antibodies against the coprecipitated component
(bottom line; Dg or Dc). Increasing the EGTA concentration in
the lysis buffer raises the amount of the immunoprecipitated
component, but decreases the amount of coimmunoprecipitated
protein. (B) Bands from the coimmunoprecipitated experiments
depicted in A were scanned and the ratios of immunoprecipitated to coimmunoprecipitated bands (Co-IP/IP; open boxes for IP DC and open circles for IP Dg) are shown in the graph. Each point represents an average ratio from four independent experiments. Vertical bars indicate the SD.
[View Larger Version of this Image (39K GIF file)]
Fig. 5.
Direct interactions of the extracellular Dsg and Dsc domains in binding assays in vitro. (A) Scheme (left) and SDS-PAGE
(right) of recombinant Dsg and Dsc fragments. The fragments
Dg12F and Dg123F contain extracellular cadherin-like repeats 1 and 2, or 1, 2, and 3 of bovine Dsg1, respectively, followed by a
FLAG epitope (filled circle). Dc12M consists of the first two cadherin-like repeats of the bovine Dsc1a placed in front of the myc
epitope (filled box). Each fragment was tagged by polyhistidine
(solid line) at the carboxyl terminus. Recombinant proteins after
purification using Ni-NTA-agarose were separated by 15% SDS-PAGE. Mr of the coelectrophoresed size markers in lane M is indicated. (B) Solid phase binding assay. For the assay, 100 µl of a
10 µg/ml solution of purified Dg12F (open boxes) or Dg123F
(open triangles) was applied to each well. Plates were incubated
with increasing amounts of Dc12M as indicated. Experiments
were performed in the presence of 0.1 mM CaCl2 (solid line) or
5 mM EDTA (dashed line). Binding was monitored using anti-myc 9E10 mAb. (C) Reconstitution assay. Dg123F (lanes 1 and
3) or Dg12F (lane 2) fragments were incubated for 30 min in the
presence (lanes 1 and 2) or absence of Dc12M that was then immunoprecipitated by myc antibody. Coimmunoprecipitation of
the Dsg-derived fragments was then monitored by immunoblotting with FLAG antibody (Flag). The parallel gel was stained
with myc antibody (Myc). H and L indicate the positions of the
heavy and light IgG chains of the 9E10 antibody used for precipitation. Position of Dc12M comigrating with the light chains;
Dg123F and Dg12F are indicated with arrows.
[View Larger Version of this Image (30K GIF file)]
). Dsg fragments were immobilized on
a 96-well dish and incubated with increasing amounts of
Dc12M. Binding was detected by subsequent ELISA assay using anti-myc antibody. In this assay the Dc12M polypeptide interacts with Dg12F very weakly, and only in the
presence of calcium ions (Fig. 5 B). Significant binding,
however, was observed between Dc12M and Dg123F proteins containing extracellular domains 1-3. Addition of
5 mM EGTA also reduced but did not completely abolish these interactions (Fig. 5 B).
(697-761) were
cocultured with HT-1080 cells lacking any form of Dsc but expressing myc-tagged plakoglobin (MPg). In the latter
cells, plakoglobin and endogenous Dsg form stable complexes that can be detected by coimmunoprecipitation
(Fig. 6 B). Thus, in this coculture system, specific coimmunoprecipitation of MPg by anti-Dsc antibodies in a Ca2+-dependent fashion will be indicative of intercellular interactions mediated by desmosomal cadherins (Fig. 6 A). The
direct complex between Dsc and MPg could not be formed
because of the absence of the plakoglobin binding site in
Dsc
(697-761). A coculture of MPg- and Dsc(1-170Dg)-
expressing cells was used as an additional negative control.
To verify that the subpopulations of the corresponding cells in both cocultures were present in an approximately
equal ratio, aliquots of the cellular lysates were subjected
to Western blot analysis with myc- and Dsc-specific antibodies before immunoprecipitation. Experiments shown
in Fig. 6 C demonstrate that Dsc
(697-761), but not
Dsc(1-170Dg), interacts with Dsg/MPg complexes present in the opposing cellular subpopulation of the coculture. In
a separate experiment (Fig. 7) the HTDc cells were cocultured with HT-1080 cells stably producing the chimeric
protein SyPg (Chitaev et al., 1996
). This protein, as we reported previously, binds to classic and desmosomal cadherins. As a result, it is incorporated into cell-cell junctions of the transfected cells. Double immunofluorescence microscopy of these cocultures using polyclonal anti-synaptophysin and monoclonal anti-Dsc antibodies showed
that Dsc1a was specifically incorporated into the junctions
arising between cells from two subpopulations of Dsc1a-positive and -negative cells.
Fig. 6.
Coimmunoprecipitation of MPg with Dsc(697-761) but not with Dsc(1-170Dg) from cocultures. (A) Schematic representation
of the interface between MPg- and Dsc
(697-761)-producing cells (II and III), or Dsc(1-170Dg)-producing cells (I). Dsg and Dsc are shown by stippled or open cylinders, respectively. MPg associated with the intracellular region of Dsg in MPg cells is denoted by the single open cylinder. Note that two different models may account for coimmunoprecipitation of MPg with Dsc
(697-761). In model II,
Dsg and Dsc form a head-to-head complex while, in model III, desmosomal cadherins form side-to-side dimers. (B) Autoradiogram showing strong interactions between endogenous Dsg2 and MPg in MPg-producing cells. Immunoprecipitates were obtained with anti- Dsg 3.10 antibodies from metabolically labeled HT-1080 cells (HT1080) or MPg-producing cells (HTMPg). Dsg and MPg are indicated
by the arrowhead or the arrow. (C) Immunoblot analysis of the total lysates (Lysates) and immunoprecipitates (IP) obtained with anti-
Dsc 2.10 antibodies of the cocultures containing Dsc
(697-761) and MPg cells (lanes 1 and 2) or Dsc(1-170Dg) and MPg cells (lane 3).
Nitrocellulose blots were developed with anti-Dsc 2.10 (Dc) or anti-Myc (M) antibodies. (Lane 2) Immunoprecipitates were obtained in the presence of 5 mM EGTA. Note that EGTA or replacement of the Arg1-Thr170 Dsc sequence in the Dsc(1-170Dg) mutant abolished the interactions with MPg. Lower band in lane 3 is a degradation product of the Dsc(1-170Dg) chimera. (D) Western blot analysis
of immunoprecipitates obtained from dense (lane 1) or sparse (lane 2) HTDc cells using mAb Dsc 2.10. Immunoprecipitated (Dc) and
coimmunoprecipitated (Dg) bands in precipitates were developed by 2.10 or 3.10 antibodies, respectively.
[View Larger Version of this Image (19K GIF file)]
Fig. 7.
Double-label immunofluorescence microscopy of
HTDc cells producing bovine Dsc1a and of HT-1080 cells producing the chimeric protein SyPg in coculture. (Sy) The distribution of the SyPg in Dsc-negative cells visualized by immunostaining with rabbit anti-synaptophysin antibody. This is compared
with the localization of the Dsc1a in HTDc cells detected by mAb
2.10 (Dc). Both proteins, although present in opposing cells, form
clusters located at the same areas of cell-cell contacts (arrows).
Bars, 25 µm.
[View Larger Version of this Image (90K GIF file)]
(697-761) were plated at a density of 12 and 75% confluence and left overnight to allow
intracellular contacts to stabilize. The lysates of low and
high density cultures containing the same amount of total
protein were immunoprecipitated with an anti-Dsc antibody, and immunoprecipitates were analyzed by immunoblotting (Fig. 6 D). A five- to sixfold reduction in the
amount of Dsg in Dsc immunoprecipitates was obtained
from cell cultures plated at low as compared with high
densities. This shows that Dsg/Dsc interaction in coculture
correlates well with the number of intercellular contacts. It
is important to note that, while plated at low density, HT-1080 cells always contain some number of cell-cell contacts that cause coimmunoprecipitation of Dsg.
Discussion
; Chidgey et al., 1996
; Kowalczyk et al., 1996
).
Although intercadherin interactions are very likely to be
involved in desmosome assembly and in cell-cell adhesion
of the epithelial tissues, no physical association between
these two desmosomal cadherins was reported. The major
reasons for failure to demonstrate interactions between desmosomal cadherins might have been (a) insolubility of
the corresponding complexes in the epithelial cells, and
(b) malfunction of Dsg and Dsc in L-fibroblasts in the absence of functional adherens junctions and classic cadherins in particular. As an alternative model for study of
the interaction between Dsc and Dsg, we chose fibrosarcoma HT-1080 cells. In contrast with L-fibroblasts, they have prominent intercellular contacts containing N-cadherin and
-catenin. HT-1080 cells produce a single desmosomal protein, Dsg2, at the same level as in epithelial
A-431 cells. In these cells, however, only a minor portion
of this cadherin is localized to cell-cell junctions.
-catenin. Coimmunoprecipitation experiments revealed that in these
cells Dsc1a is able to interact directly with Dsg2, but not
with N-cadherin. Notably, formation of Dsc/Dsg complexes was found in the absence of major desmosomal proteins, plakoglobin and desmoplakin, that were undetected
in parental HT-1080 cells and in HTDc cells. In addition,
deletions of plakoglobin or desmoplakin binding sites in
Dsc1a had no effect on its ability to interact with Dsg2,
demonstrating that known cytoplasmic interactions of
Dsc1a are not required for Dsg/Dsc association. In contrast, replacement of the extracellular domain of Dsc1a
with the corresponding sequence of Dsg completely abolished both Dsg redistribution and Dsg/Dsc complex formation in coimmunoprecipitation. Furthermore, association between Dsg and Dsc was calcium dependent, which
is characteristic of interactions involving extracellular domains of cadherin. In vitro binding experiments provided
further support for a direct interaction between the extracellular segments of two desmosomal cadherins. Using two
different binding assays, we have found that Dc12M, a Dsc
fragment containing extracellular repeats 1 and 2, binds directly to the extracellular region of Dsg. Chelation of Ca2+
ions decreased but did not completely abolish this binding.
Our data are not sufficient to conclude whether Dsg/Dsc
heterodimers are formed through head-to-head or side-to-side interactions (see hypothetical models of Dsg-Dsc
complexes in Fig. 6). Recently, Brieher et al. (1996)
showed that lateral dimers of the extracellular region of
the C-cadherin in vitro are relatively stable after the removal of Ca2+ ions. Similarly, incomplete inactivation of the
Dsg-Dsc interaction even in the presence of a high concentration of EGTA suggests that the cadherin dimers
have lateral alignment. However, in the solid phase assay,
the Dc12M fragment binds more strongly to the Dsg fragment containing extracellular domain 3. This observation may be interpreted to mean that Dsg-Dsc forms antiparallel head-to-head complexes in which the extracellular domains 1 and/or 2 of Dsc bound to domain 3 of Dsg. Additional experiments are required to determine the exact
structural features of Dsg/Dsc dimers.
(697-761)-
and MPg-expressing HT-1080 cells. This unequivocally
demonstrated that interacting Dsc and Dsg are derived
from opposing cells. The number of Dsc/Dsg complexes directly correlates with the propagation of cell-cell contacts in HTDc coculture. Furthermore, clusters incorporating
Dsc and Dsg are also assembled along the contacts with
Dsc-negative cells. These observations are consistent with
the idea that these complexes are formed only on the interface between two neighboring cells. Thus, heterophilic
interactions between desmosomal cadherins are involved
in intercellular adhesion of epithelial cells, and corresponding complexes can be functional elements in desmosome assembly. In support of this assumption we found
that the bovine Dsc1a as well as its mutants Dsc
(697-
761) and Dsc
(597-609) were efficiently incorporated into
human desmosomes upon expression in epithelial A-431
cells (Chitaev, N.A., unpublished results). In contrast, the
mutant Dsc(1-170Dg), unable to interact with Dsg in HT-1080 cells, was also unable to form desmosomes in A-431
cells. The question remains, however, how Dsg/Dsc complexes assemble in a mature desmosome. The function of
the intracellular desmosomal plaque proteins, such as plakoglobin and desmoplakin, may be necessary for segregation of the Dsg/Dsc complexes from adherens junctions and for further desmosome assembly (Hinck et al., 1994
;
Allen et al., 1996
; Bornslaeger et al., 1996
; Chitaev et al.,
1996
; Demlehner et al., 1996; Ruiz et al., 1996
; Troyanovsky et al., 1996
). The subsequent expression of these
desmosomal proteins in HTDc cells is likely to provide an
excellent system for examining the molecular mechanisms
involved in this process.
showing the absence of detectable intercadherin interactions when both Dsg and Dsc are coexpressed in
mouse fibroblasts lacking adherens junctions. The requirement of adherens junctions for intercadherin interactions
was suggested by observations that the malfunction of adherens junctions, induced either by E-cadherin antibodies
or by dominant negative mutants of the classic cadherins, delays desmosome assembly in keratinocytes after raising
the calcium concentration (Wheelock and Jensen, 1992
;
Lewis et al., 1994
; Amagai et al., 1995
). In HT-1080 cells,
Dsc/Dsg clusters were found in areas where N-cadherin-
-catenin complexes are abundant. Therefore, it is reasonable to hypothesize the existence of cross talk between adherens junctions and desmosomes, which allows Dsg/Dsc interactions only after the establishment of the cell type-
specific contacts mediated by classic cadherins.
Received for publication 7 January 1997 and in revised form 1 May 1997.
1. Abbreviations used in this paper: Dsc, desmocollin; Dsg, desmoglein; MPg, myc-tagged plakoglobin.We thank Drs. G. Goldberg and A. Eisen for valuable discussion.
This work has been supported in part by a Pfizer Pharmaceutical Career Development Award from the Dermatology Foundation and National Institutes of Health (grant 1R01 AR44016-01).
1. | Allen, E., Q.-C. Yu, and E. Fuchs. 1996. Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epithelial differentiation. J. Cell Biol. 133: 1367-1382 [Abstract]. |
2. | Amagai, M., S. Karpati, V. Klaus-Kovtun, M.C. Udey, and J.R. Stanley. 1994. Extracellular domain of pemphigus vulgaris antigen (desmoglein 3) mediates weak homophilic adhesion. J. Invest. Dermatol. 103: 609-615 [Abstract]. |
3. | Amagai, M., T. Fujimori, T. Masunaga, H. Shimizu, T. Nishikawa, N. Shimuzu, M. Takeichi, and T. Hashimoto. 1995. Delayed assembly of desmosomes in keratinocytes with disrupted classic-cadherin-mediated cell adhesion by a dominant negative mutant. J. Invest. Dermatol. 104: 27-32 [Abstract]. |
4. | Bornslaeger, E.B., C.M. Corcoran, T.S. Stappenbeck, and K.J. Green. 1996. Breaking the connections: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly. J. Cell Biol. 134: 985-1001 [Abstract]. |
5. | Brieher, W.M., A.S. Yap, and B. Gumbiner. 1996. Lateral dimerization is required for the homophilic binding activity of C-cadherin. J. Cell Biol. 135: 487-496 [Abstract]. |
6. | Chidgey, M.A., J.P. Clarke, and D.R. Garrod. 1996. Expression of full-length desmosomal glycoproteins (desmocollins) is not sufficient to confer strong adhesion on transfected L929 cells. J. Invest. Dermatol. 106: 689-695 [Abstract]. |
7. | Chitaev, N.A., R.E. Leube, R.B. Troyanovsky, L.G. Eshkind, W.W. Franke, and S.M. Troyanovsky. 1996. The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential. J. Cell Biol. 133: 359-369 [Abstract]. |
8. | Cowin, P., and B. Burke. 1996. Cytoskeleton-membrane interactions. Curr. Opin. Cell Biol. 8: 56-65 |
9. |
Cowin, P.,
D. Mattey, and
D.R. Garrod.
1984.
Identification of desmosomal
surface components (desmocollins) and inhibition of desmosome formation
by specific FAB![]() |
10. | Demlehner, M.P., S. Schafer, C. Grand, and W.W. Franke. 1995. Continual assembly of half-desmosomal structures in the absence of cell contacts and their frustrated endocytosis: a coordinated sisyphus cycle. J. Cell Biol. 131: 745-760 [Abstract]. |
11. | Franke, W.W., M.D. Goldschmidt, R. Zimbelmann, H.M. Mueller, D.L. Schiller, and P. Cowin. 1989. Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein. Proc. Natl. Acad. Sci. USA. 86: 4027-4031 [Abstract]. |
12. | Garrod, D.R.. 1993. Desmosomes and hemidesmosomes. Curr. Opin. Cell Biol. 5: 30-40 |
13. | Geiger, B., and O. Ayalon. 1992. Cadherins. Annu. Rev. Cell Biol. 8: 307-332 . |
14. | Green, K.J., and J.C.R. Jones. 1990. Interaction of intermediate filaments with the cell surface. In Cellular and Molecular Biology of Intermediate Filaments. R.D. Goldman and P.M. Steinert, editors. Plenum Publishing Corp., New York. 147-174. |
15. | Grunwald, G.B.. 1993. The structural and functional analysis of cadherin calcium-dependent cell adhesion molecules. Curr. Opin. Cell Biol. 5: 797-805 |
16. | Gumbiner, G.M.. 1996. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell. 84: 345-357 |
17. | Hinck, L., I.S. Nathke, J. Papkoff, and W.J. Nelson. 1994. Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly. J. Cell Biol. 125: 1327-1340 [Abstract]. |
18. |
Horst, M.,
N. Harth, and
A. Hasilik.
1991.
Biosynthesis of glycosylated human
lysozyme mutants.
J. Biol. Chem.
266:
13914-13919
|
19. | Klymkowsky, M.W., and B. Parr. 1995. A glimpse into the body language of cell: the intimate connection between cell adhesion and gene expression. Cell. 83: 5-8 |
20. | Koch, P.J., and W.W. Franke. 1994. Desmosomal cadherins: another growing multigene family of adhesion molecules. Curr. Opin. Cell Biol. 6: 682-687 |
21. | Koch, P.J., M.J. Walsh, M. Schmelz, M.D. Goldschmidt, R. Zimbelmann, and W.W. Franke. 1990. Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules. Eur. J. Cell Biol. 53: 1-12 |
22. | Koch, P.J., M.D. Goldschmidt, M.J. Walsh, R. Zimbelmann, M. Schmelz, and W.W. Franke. 1991. Amino acid sequence of bovine muzzle epithelial desmocollin derived from cloned cDNA: a novel subtype of desmosomal cadherins. Differentiation. 47: 29-36 |
23. | Kowalczyk, A.P., J.E. Borgwardt, and K.J. Green. 1996. Analysis of desmosomal cadherin-adhesive function and stoichiometry of desmosomal cadherin-plakoglobin complexes. J. Invest. Dermatol. 107: 293-300 [Abstract]. |
24. | Lewis, J.E., P.J. Jensen, and M.J. Wheelock. 1994. Cadherin function is required for human keratinocytes to assemble desmosomes and stratify in response to calcium. J. Invest. Dermatol. 102: 870-877 [Abstract]. |
25. | Murphy-Erdosh, C., C.K. Yoshida, N. Paradies, and L.F. Reichardt. 1995. The cadherin-binding specificities of B-cadherin and LCAM. J. Cell Biol. 129: 1379-1390 [Abstract]. |
26. | Nagar, B., M. Overduin, M. Ikura, and J.M. Rini. 1996. Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature (Lond.). 380: 360-364 |
27. | Nagafuchi, A., and M. Takeichi. 1988. Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO (Eur. Mol. Biol. Organ.) J. 7: 3679-3684 [Abstract]. |
28. | Nose, A., K. Tsuji, and M. Takeichi. 1990. Localization of specificity determining sites in cadherin cell adhesion molecules. Cell. 61: 147-155 |
29. | Nuber, U.A., S. Schafer, S. Stehr, H.-R. Rackwitz, and W.W. Franke. 1996. Patterns of desmocollin synthesis in human epithelia: immunolocalization of desmocollins 1 and 3 in special epithelia and in cultured cells. Eur. J. Cell Biol. 73: 1-13 . |
30. | Ozawa, M., H. Baribault, and R. Kemler. 1989. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO (Eur. Mol. Biol. Organ.) J. 8: 1711-1717 [Abstract]. |
31. | Ozawa, M., J. Egel, and R. Kemler. 1990. Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function. Cell. 63: 1033-1038 |
32. | Peifer, M.. 1995. Cell adhesion and signal transduction: the Armadillo connection. Trends Cell Biol. 5: 224-229 . |
33. | Ruiz, P., V. Brinkmann, B. Ledermann, M. Behrend, C. Grund, C. Thalhammer, F. Vogel, C. Birchmeier, U. Gunthert, W.W. Franke, et al . 1996. Targeted mutation of plakoglobin in mice reveals essential functions of desmosomes in the embryonic heart. J. Cell Biol. 135: 215-225 [Abstract]. |
34. |
Sacco, P.A.,
T.M. McGranahan,
M.J. Wheelock, and
K.R. Johnson.
1995.
Identification of plakoglobin domains required for association with N-cadherin
and ![]() |
35. | Shapiro, L., A.M. Fannon, P.D. Kwong, A. Thompson, M.S. Lehman, G. Grubel, J.-F. Legran, J. Als-Neilsen, D.R. Colman, and W.A. Hendrickson. 1995. Structural basis of cell-cell adhesion by cadherins. Nature (Lond.). 374: 327-337 |
36. | Schafer, S., S. Stampp, and W.W. Franke. 1996. Immunological identification and characterization of the desmosomal cadherin Dsg2 in coupled and uncoupled epithelial cells and in human tissues. Differentiation. 60: 99-108 |
37. | Schmidt, A., H.W. Heid, S. Schafer, U.A. Nuber, R. Zimbelmann, and W.W. Franke. 1994. Desmosomes and cytoskeletal architecture in epithelial differentiation. Cell type-specific plaque components and intermediate filament anchorage. Eur. J. Cell Biol. 65: 229-245 |
38. | Stanley, J.R.. 1995. Autoantibodies against adhesion molecules and structures in blistering skin diseases. J. Exp. Med. 181: 1-4 |
39. | Troyanovsky, S.M., L.G. Eshkind, R.B. Troyanovsky, R.E. Leube, and W.W. Franke. 1993. Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage. Cell. 72: 561-574 |
40. | Troyanovsky, S.M., R.B. Troyanovsky, L.G. Eshkind, V.A. Krutovskikh, R.L. Leube, and W.W. Franke. 1994a. Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage. J. Cell Biol. 127: 151-160 [Abstract]. |
41. |
Troyanovsky, S.M.,
R.B. Troyanovsky,
L.G. Eshkind,
R.E. Leube, and
W.W. Franke.
1994b.
Identification of amino acid sequence motifs in the desmosomal glycoprotein, desmocollin, that are required for plakoglobin binding
and plaque formation.
Proc. Natl. Acad. Sci. USA.
91:
10790-10794
|
42. |
Troyanovsky, R.B.,
N.A. Chitaev, and
S.M. Troyanovsky.
1996.
Cadherin binding sites of plakoglobin: localization, specificity and role in targeting to adhering junctions.
J. Cell Sci.
109:
3069-3078
|
43. | Wheelock, M.J., and P.J. Jensen. 1992. Regulation of keratinocyte intercellular junction organization and epidermal morphogenesis by E-cadherin. J. Cell Biol. 117: 415-425 [Abstract]. |