(Received for publication, November 21, 1994; and in revised form, March 22, 1995)
From the
Cadherins are calcium-dependent, cell surface glycoproteins
involved in cell-cell adhesion. To function in cell-cell adhesion, the
transmembrane cadherin molecule must be associated with the
cytoskeleton via cytoplasmic proteins known as catenins. Three
catenins, -catenin,
-catenin, and
-catenin (also known
as plakoglobin), have been identified. The domain of the cadherin
molecule important for its interaction with the catenins has been
mapped to the COOH-terminal 70 amino acids, but less is known about
regions of the catenins that allow them to associate with one another
or with the cadherin molecule. In this study we have transfected
carboxyl-terminal deletions of plakoglobin into the human fibrosarcoma
HT-1080 and used immunofluorescence localization and
co-immunoprecipitation to map the regions of plakoglobin that allow it
to associate with N-cadherin and with
-catenin. Plakoglobin is an armadillo family member containing 13 weakly similar internal
repeats. These data show that the
-catenin-binding region maps
within the first repeat and the N-cadherin-binding region maps within
repeats 7 and 8.
Plakoglobin is one of the plaque proteins found in both desmosomes and adherens junctions(1) . In addition to membrane-associated plakoglobin, a soluble form has been characterized(2) . Interestingly, only minor differences have been found in the plakoglobin associated with these various cellular compartments(2, 3) .
Desmosomes have two
transmembrane components, desmoglein and desmocollin, which are members
of the broadly defined cadherin family of molecules. Plakoglobin has
been shown to interact with the cytoplasmic tail of desmoglein (4, 5) and desmocollin(6) . The transmembrane
component of the adherens junction is a member of the classic cadherin
family; these proteins mediate homotypic cell-cell
adhesion(7, 8, 9, 10) . In the
cytoplasm, classic cadherins associate with three proteins known as
-,
-, and
-catenin(11, 12, 13) ;
-catenin is
identical to plakoglobin(14, 15) . The cytoplasmic
components of the desmosome and the adherens junction are important in
mediating interactions with the appropriate cytoskeletal elements of
the junction, i.e. intermediate filaments and actin,
respectively(16, 17, 18) .
The cDNA
sequences of plakoglobin from a number of species have revealed close
relationships to vertebrate -catenins and to Drosophila
armadillo, a segment polarity
gene(19, 20, 21, 22, 23) .
The central region of plakoglobin is composed of 13 weakly similar
repeats of approximately 42 amino acids. Developmental studies of armadillo mutants have suggested that the repeats in Armadillo
contribute to its function in an additive manner and that the protein
is modular(24) . This raises the possibility that plakoglobin
likewise functions as a modular protein.
The fact that plakoglobin
shares significant regions of homology with -catenin (15, 19, 22) is of particular interest in
light of recent studies showing that cells express two types of
cadherin-catenin complexes: a complex that contains predominantly
-catenin and one that contains predominantly
plakoglobin(25, 26) . The consensus is that
plakoglobin (or
-catenin) binds directly to cadherin and that
-catenin is associated with the cadherin through its association
with plakoglobin (or
-catenin). Pulse-chase experiments in
Madin-Darby canine kidney cells demonstrated that cadherin-catenin
complexes are assembled by first loading plakoglobin (or
-catenin)
onto the newly synthesized cadherin and that
-catenin associates
with the complex as it arrives at the plasma membrane(25) .
Further evidence that
-catenin associates with cadherin in the
absence of
-catenin was provided by studies in PC-9 cells, which
do not express
-catenin but form complexes of
-catenin and
cadherin(27) . These cells do not strongly adhere to one
another, but when transfected with
-catenin, they adhere normally
and form complexes that include
-catenin(28) .
-Catenin is thought to provide a link between
cadherin-
-catenin and the cytoskeleton.
To examine further the
interactions of plakoglobin with cadherin and -catenin we have
identified the regions of plakoglobin that are important in its
interaction with the cadherin complex in living cells. We first
identified a cell line, the human fibrosarcoma HT-1080(29) ,
that forms a cadherin-catenin complex but produces only minuscule
amounts of plakoglobin. We then transfected HT-1080 cells with
full-length and truncated forms of plakoglobin and examined the
transfectants for the association of plakoglobin with cadherin and/or
-catenin. With this approach we have been able to identify
distinct regions of plakoglobin that interact with cadherin and
-catenin, supporting the notion that plakoglobin functions as a
modular protein.
Figure 1:
A map showing functional domains of
plakoglobin. Full-length human plakoglobin (745 amino acids) is shown
along with the sites of the truncations used in this study. The 13
Armadillo repeats are shown as boxes. The PstI site
is located just amino-terminal to the repetitive region, the SphI site is located in the third repeat, and the SmaI site is near the start of the 11th repeat. All of the
deletions described in this report are indicated with the number of
amino acids remaining in the COOH-terminally truncated plakoglobin. The
regions of plakoglobin that allow the truncated protein to retain its
association with N-cadherin and -catenin are
indicated.
Figure 2:
HT-1080 cells express N-cadherin,
-catenin, and
-catenin but very little plakoglobin. A Nonidet
P-40 extract of HT-1080 cells was resolved on a 7% SDS-polyacrylamide
gel, transblotted to nitrocellulose, and probed with: lane1, 13A9 anti-N-cadherin (N-cad); lane2, 1G5 anti-
-catenin (
-cat); lane3, 12F7 anti-
-catenin (
-cat); lane4, 15F11 anti-plakoglobin (pg). The lines at the left indicate the molecular mass standards at 205,
116, 97, 68, and 45 kDa.
Figure 3:
Transfected plakoglobin is incorporated
into the cadherin complex in HT-1080 cells. Extracts of HT-1080 cells
transfected with full-length plakoglobin (lanes1, 3, and 5) or mock transfected (lanes2, 4, and 6) were immunoprecipitated with
anti--catenin (
-cat, lanes1 and 2), anti-plakoglobin (pg, lanes3 and 4), or anti-N-cadherin (N-cad, lanes5 and 6). The proteins were resolved on a 7% SDS-polyacrylamide gel,
transblotted to nitrocellulose, and probed with anti-plakoglobin.
Plakoglobin was abundant in all three immunoprecipitation reactions in
the transfected cells but barely detectable in the mock transfected
cells. The major bands at about 50 kDa are the antibody heavy chains.
The lines at the left indicate the molecular mass
standards at 116, 97, 68, and 45 kDa.
Figure 4: Quantitative analysis of plakoglobin expression in HT-1080 cells transfected with truncated plakoglobins. Equal amounts of total protein from Nonidet P-40 extracts of transfected HT-1080 cells were resolved on a 10% SDS-polyacrylamide gel, transblotted to nitrocellulose, and probed with anti-plakoglobin (15F11). Lane1, mock transfected cells; lane2, transfected cells expressing full-length plakoglobin; lane3, transfected cells expressing the 572-amino acid construct; lane4, transfected cells expressing the 233-amino acid construct; lane5, transfected cells expressing the 114-amino acid construct. The lines at the left indicate the molecular mass standards at 97, 68, 45, and 29 kDa.
Immunofluorescence localization with anti-plakoglobin antibodies was performed on each cell line. The 572-amino acid construct (Fig. 5B) was found along the cell-cell borders, indistinguishable from the pattern seen with full-length plakoglobin (Fig. 5A). The distributions of the 233-amino acid and 114-amino acid constructs (Fig. 5, C and D, respectively) were distinctly different from those of full-length plakoglobin or the 572-amino acid construct. The two smaller fragments were found diffusely localized in the cytoplasm and not along cell-cell borders.
Figure 5: The 572-amino acid deletion of plakoglobin localizes to sites of cell-cell contact in HT-1080 cells, but the 233-amino acid and 114-amino acid deletions do not. HT-1080 cells transfected with full-length or deleted plakoglobin were grown on glass coverslips and processed for immunofluorescence with anti-plakoglobin 15F11. In cells transfected with the 572-amino acid construct (B), plakoglobin localization looked similar to that seen in cells transfected with full-length plakoglobin (A). Cells transfected with the 233-amino acid construct (C) or with the 114-amino acid construct (D) showed bright, diffuse cytoplasmic staining.
As shown earlier, full-length plakoglobin co-immunoprecipitated with
-catenin and with N-cadherin (Fig. 3). Similar experiments
were done in order to determine whether or not the truncated
plakoglobins associated with these two proteins. It was found that the
572-amino acid construct co-immunoprecipitated with
-catenin as
well as with N-cadherin (Fig. 6). Thus, the 572-amino acid
truncation retained the sequences required for associating with each of
these partners.
Figure 6:
Mapping of the N-cadherin association
domain on plakoglobin. Extracts of HT-1080 cells transfected with
truncations of plakoglobin retaining the first 572 amino acids (AA, lanes 1 and 2), 472 amino acids (lanes 3 and 4), 375 amino acids (lanes 5 and 6), or 233 amino acids (lanes7 and 8) of plakoglobin were immunoprecipitated with anti-N-cadherin (N-cad, lanes 1, 3, 5, and 7) or
anti--catenin (
-cat, lanes 2, 4, 6, and 8). For these experiments the immunoprecipitating reagents
were purified IgG from ascites fluid conjugated to Sepharose. The
proteins were resolved on a 12% SDS-polyacrylamide gel, transblotted to
nitrocellulose, and probed with monoclonal anti-plakoglobin (15F11). Arrows point out the truncated plakoglobin molecules. The lines at the left indicate the molecular mass
standards at 68, 45, and 29 kDa.
When transfectants expressing the 233-amino acid
construct were examined, the truncated plakoglobin was found associated
with -catenin but not with N-cadherin. Fig. 6, lanes 7 and 8, shows the relevant immunoprecipitates
immunoblotted with anti-plakoglobin antibody 15F11. The 233-amino acid
construct migrated at about 30 kDa in the same portion of the SDS gel
as the antibody light chains. The truncated plakoglobin was apparent in lane 8 when the immunoprecipitating antibody was
anti-
-catenin. The 233-amino acid construct was not seen when the
immunoprecipitating antibody was anti-N-cadherin (lane 7).
Thus, the 233-amino acid construct was not able to stably bind
N-cadherin, showing that the ability to bind N-cadherin is provided, at
least in part, by amino acids 233-572 of plakoglobin. For these
and the following experiments we used 1G5 anti-
-catenin ascites
fluid conjugated to Sepharose as the immunoprecipitating reagent, which
resulted in an increased background due to binding of the labeled
secondary antibody.
In order to more closely map the sequences
necessary for interaction with N-cadherin, we prepared exonuclease III
deletions resulting in proteins that retained 233-572 amino acids
of plakoglobin. Immunoprecipitations with antibodies against N-cadherin
demonstrated that, as the plakoglobin molecule became shorter, the
association with N-cadherin became less evident. Fig. 6compares
the levels of truncated plakoglobins associated with N-cadherin to the
levels associated with -catenin. Comparison of lanes 1,
3, and 5 of Fig. 6revealed a gradual decrease in
the ability of the constructs to associate with N-cadherin. The
truncation retaining 375 amino acids was just able to stably interact
with N-cadherin. Data obtained from clones expressing a 421-amino acid
construct supported this gradual decline in association with N-cadherin
(not shown). Immunofluorescence localization showed cell border
staining in cells expressing the 472-amino acid construct, with some
cytoplasmic signal. Cell border staining was not evident in cells
expressing the 375-amino acid construct.
To address the question of
whether or not the borders of the Armadillo repeats delineated
functional domains, we used PCR to generate truncations terminating
precisely at the ends of repeats 5, 7, and 8 (amino acids 334, 414, and
458, respectively). Table 1presents the immunofluorescence and
immunoprecipitation data generated by all of our constructs. The
ability of these shortened molecules to compete with endogenous
-catenin for binding to N-cadherin suggests that the amino acids
from 376 to 414 (comprising repeats 7 and 8) are particularly important
for interaction with N-cadherin.
When cells expressing the 114-amino
acid construct were extracted and immunoprecipitated, neither the
anti-N-cadherin (not shown) nor the anti--catenin
immunoprecipitate included the 114-amino acid construct (Fig. 7), although the protein was present in the cell extract (lane 6, indicated by an arrow). Thus at least one
region of plakoglobin required for interaction with
-catenin
resides between amino acids 114 and 233. Fig. 7also shows that
when plakoglobin was truncated at 161 amino acids it readily associated
with
-catenin, but, when truncated at 133 amino acids, the
association was barely detected. These data suggest that the 47
residues from amino acids 115 to 161 play a major role in association
with
-catenin with the 19 amino acids from 115 to 133 allowing
weak association. All of the deletions used in these experiments as
well as the regions of plakoglobin that likely interact with
-catenin and N-cadherin are presented in Table 1and shown
diagrammatically in Fig. 1.
Figure 7:
Mapping of the -catenin association
domain on plakoglobin. Extracts of HT-1080 cells transfected with
truncations retaining 161 amino acids (AA, lane1), 133 amino acids (lane3), or 114
amino acids (lane5) of plakoglobin were
immunoprecipitated (IP) with anti-
-catenin. Cell extract (ext) was run for comparison. The proteins were resolved on a
12% SDS-polyacrylamide gel, transblotted to nitrocellulose, and probed
with anti-plakoglobin. The 161-amino acid construct
co-immunoprecipitated with
-catenin while the 133-amino acid
construct barely associated with
-catenin and the 114-amino acid
construct no longer associated with
-catenin.
To function in cell-cell adhesion, the transmembrane cadherin
molecule must be associated with the cytoskeleton via the cytoplasmic
catenins. The COOH-terminal 70 amino acids of classical cadherins have
been shown to be necessary for association with the
catenins(12, 35, 36) . Assembly of the
cadherin-catenin complex has been
studied(13, 26, 37, 38, 39) ,
but less is known of the domains of the catenins that allow them to
associate with the cadherin molecule or with one another. In this study
we show that distinct regions of plakoglobin allow its association with
N-cadherin and with -catenin.
The localization of N-cadherin,
-catenin, and
-catenin to cell-cell borders implies that
HT-1080 cells interact with one another via their cadherins. However,
the cells form an adhesion complex that contains only minute amounts of
plakoglobin. Since the endogenous plakoglobin does not localize to
cell-cell borders, HT-1080 cells apparently do not need plakoglobin in
the cadherin-catenin complex in order to form cell-cell contacts. In
AtT20 cells (25) and in Madin-Darby canine kidney
cells(26) , data have been presented showing that
cadherin-catenin complexes contain either
-catenin or plakoglobin
but not both. The sequence similarity between
-catenin and
plakoglobin suggests they may be able to substitute for one another and
perhaps perform similar functions in the cadherin-catenin complex. The
two molecules are not interchangeable, however, since plakoglobin, but
not
-catenin, associates with desmoglein and desmocollin. Perhaps
because HT-1080 cells do not form desmosomes,
-catenin alone is
sufficient to provide the necessary functions for cell-cell adhesion.
Interestingly, we could not detect obvious morphological or behavioral
differences in cells transfected with plakoglobin. Thus, complexes
containing plakoglobin appear not to confer a distinct phenotype in
HT-1080 cells.
As mentioned earlier, plakoglobin is a member of the armadillo family of proteins(40) . The effects of
carboxyl-terminal truncations of Armadillo on the development of Drosophila larvae were studied by Peifer and
Wieschaus(24) . When the severity of each mutation was scored
using time of embryonic lethality as a parameter, the severity
correlated with the extent of the truncation suggesting a modular
structure of the Armadillo protein. It was proposed that modules
contributed in an additive fashion to the function of Armadillo. Our
experiments with plakoglobin parallel those done with Armadillo; in
both studies, the effects of carboxyl-terminal truncations were
examined. Our data support a modular structure for plakoglobin since,
as the extent of the deletion increased, association with N-cadherin
was lost first and then association with -catenin. If our data and
those of Peifer and Wieschaus (24) are taken together, one
interpretation is that, as the truncations of Armadillo became longer,
the shortened protein first lost the ability to interact with cadherin
and then lost the ability to productively associate with
-catenin.
The developmental data would then suggest that preventing the
association of an armadillo family member with both
-catenin and cadherin has more serious consequences than
preventing the association with only the cadherin. This in turn
suggests that cadherin-less complexes containing
-catenin and
Armadillo may have functions during development.
It is also interesting to note that some of the mutants studied by Peifer and Wieschaus (24) resulted in Armadillo being truncated at positions corresponding to the carboxyl-terminal region of plakoglobin. Although embryos with these mutations survived a relatively long time and synthesized enough of the truncated Armadillo for it to be readily detected in immunoblots, the mutations were lethal. This underscores the importance of the carboxyl terminus of armadillo family members, although its function is unknown.
The diagram shown in Fig. 1assigns functions to some regions of plakoglobin. A domain
of plakoglobin needed for association with -catenin was mapped to
amino acids 115-161, roughly corresponding to the first repeat.
Since the truncated plakoglobins are probably competing with
-catenin's other partners (such as
-catenin), these
data suggest that sequences near repeat 1 play a major role in the
recognition of
-catenin.
In contrast to the small region
identified in the -catenin studies, our data suggest a gradual
decrease in the ability of plakoglobin and N-cadherin to associate as
plakoglobin is shortened from the end of repeat 8 (amino acid 458) to
the end of repeat 6 (amino acid 375). Such a large region of
association suggests multiple protein-protein interactions are required
for plakoglobin to interact with N-cadherin. Immunofluorescence showed
that constructs retaining at least 458 amino acids localized to cell
borders, which suggests they can successfully compete with
-catenin for binding to N-cadherin. However, we have preliminary
data, based on washing immunoprecipitations more stringently, that
suggests COOH-terminally truncated plakoglobins are not as stably
associated with N-cadherin as are full-length plakoglobin or
-catenin. Studies are in progress to more completely characterize
the stability of the interaction of truncated plakoglobins with
N-cadherin.
As mentioned above, there are regions of plakoglobin
whose function was not revealed in our assays. A number of proteins
have been localized to the plaques of adherens junctions (for a review
see (41) ). Perhaps -catenin and plakoglobin have roles in
recruiting other junctional components. It would not be surprising if
plakoglobin and
-catenin have different affinities for these other
molecules, a feature that could play a role in signaling events.
Conditions must be developed to preserve the interactions of these
other adherens junction components in order to study their interactions
with the cadherin-catenin complex; such studies are ongoing in our
laboratory.