(Received for publication, November 7, 1996, and in revised form, February 20, 1997)
From the Department of Biochemistry, University of Ulm, D-89081 Ulm, Germany
Xenopus XB/U-cadherin forms
functional complexes with mouse - and
-catenins and
p120cas when expressed in murine
L-TK
fibroblasts. These cells were stably transfected
with cDNAs encoding different cytoplasmic XB/U-cadherin mutants,
each partially deleted in the different parts of the 38 most
carboxyl-terminal amino acids. The binding of
p120cas was not affected by carboxyl-terminal
deletions, confirming its binding to a region more amino-terminal and
distinct from the catenins.
- and
-catenins associate with
truncated XB/U-cadherins if either 19 amino acid half of the cadherin
38 amino acid tail is present, indicating that the site of catenin
interaction is upstream of the deletions. However, for adhesive
function of XB/U-cadherin constructs, the most carboxyl-terminal 19 amino acids are essential; if these amino acids are deleted,
cadherin-catenin complexes unable to mediate cell-cell adhesion are
formed. Nonadhesive complexes are solubilized by mild detergent,
whereas functional complexes are stable. Provided that detergent
stability of cadherin-catenin complexes is taken as a measure of their
cytoskeletal association, our results give first evidence that
cytoskeletal stabilization occurs independent of cadherin-catenin
complex formation and requires the 19-amino acid cadherin carboxyl
terminus.
Xenopus XB/U-cadherin is a member of the classical cadherin family of calcium-dependent cell-cell adhesion molecules. As a maternal cadherin, XB/U-cadherin is inserted into all newly forming plasma membranes from maternal mRNA and protein stores (1). It contributes to interblastomere adhesion and, during gastrulation, promotes convergent-extension movements of mesodermal cells (2, 3). Dominant-negative expression of XB/U-cadherin deletion mutants in Xenopus embryos results in the malformation of neural tissues in the head region (3, 4). Sequence alignment and its tissue-specific expression pattern show that XB/U-cadherin is most closely related to mammalian P-cadherins (5).
To promote stable calcium-dependent cell adhesion, classical cadherins must be linked to cytoskeletal elements, putatively to actin microfilaments (6-8). Cytosolic catenins associate with the cadherin cytoplasmic domain, which shows the highest level of conservation on the amino acid level among classical cadherins of different types and species (9).
Previous studies showed that - and
-catenin, the latter of which
is most likely identical to plakoglobin, bind to overlapping domains of
the cadherin tail directly (10-12).
-Catenin interacts with sites
shared by
- and
-catenin linking the
- or
-catenin-cadherin complex to the cytoskeleton (13, 14). Recently, other cytoplasmic proteins have been reported to interact with cadherin-catenin complexes; Knudsen et al. (15) have shown that
-catenin
can bind directly to
-actinin, and p120cas has been found to
associate with E-cadherin and the c-src kinase (16-18). The role of
these proteins in the cadherin-catenin complex is still unknown.
Using E-cadherin as a model molecule, a number of studies set out to
identify the cadherin-binding site for - or
-catenin. When
E-cadherin mutants lacking more than 37 carboxyl-terminal amino acids
were expressed in mouse fibroblasts, cadherin-catenin complex formation
as well as E-cadherin-mediated adhesive function was completely
abolished (19). However, although a larger portion of the E-cadherin
cytoplasmic domain consisting of the carboxyl-terminal 72 amino acids
was both essential and sufficient for its adhesive function and catenin
binding, these terminal 37 amino acids were not (9, 20). More recently,
the
-catenin-binding domain of E-cadherin was assigned to a core
region of 25 amino acids located 54 to 29 amino acids from the carboxyl
terminus (21). At least partial phosphorylation of a cluster of eight
serine residues within this binding site was found to be necessary for cadherin-catenin complex formation in transfected mouse fibroblasts (22).
In this study, we focus on the role of motifs within the 38 carboxyl-terminal amino acids in catenin complex formation of XB/U-cadherin and the induction of cell adhesion. Full-length XB/U-cadherin and four deletion mutants lacking different parts of the
carboxyl-terminal 38 amino acids of XB/U-cadherin were stably expressed
in murine L-TK fibroblasts, as the most established
expression system for the analysis of cadherin molecules. Catenins do
not associate with mutant XB/U-cadherins lacking the carboxyl-terminal
32 amino acids but bearing sequences corresponding to the
-catenin-binding sites reported for murine E-cadherin (21, 22).
Complex formation and adhesive function are restored when the terminal
32 amino acids are replaced by the 19 carboxyl-terminal residues
alone.
However, mutant XB/U-cadherin molecules truncated by these
carboxyl-terminal 19 amino acids do not induce cell adhesion, although they associate with - and
-catenin at cell surfaces. Thus,
cadherin-catenin complex formation itself is not sufficient to render
transfectants of mutant cadherin adhesive. Nonadhesive complexes differ
from functional ones in that they lack resistance to detergent. We conclude that the specific 19 amino acids of the XB/U-cadherin carboxyl
terminus are required to link complexes to cytoskeletal elements. This
stabilization is a prerequisite for cell-cell adhesion.
Here, we provide evidence that complex formation with catenins and cytoskeletal stabilization of complexes depend on different domains of the cadherin cytoplasmic tail. Our results complete earlier findings in that they reveal a novel aspect in the establishment of cadherin-mediated adhesion.
Construction of the expression vectors
for the full-length cDNA of XB/U-cadherin and the cDNA of
XB/U-cadherin truncated by 38 carboxyl-terminal amino acids (XBc38)
have been described previously (23). Carboxyl-terminal deletion
construct XB
c19 was generated by SpeI-DraII
cleavage, truncated XB
c32 by EcoRI-TaqI cleavage of full-length XB/U-cadherin cDNA. In both cases,
resulting fragments were religated in the presence of a NheI
nonsense linker, causing frameshifts and producing stop codons
downstream of the DraII and TaqI sites,
respectively. The XB
c32 construct encodes for a random sequence of
12 amino acids following the cadherin sequence. The internal deletion
mutant XB
i20 was generated by EspI-DraII
restriction and religation of full-length XB/U-cadherin cDNA.
Truncated forms of XB/U-cadherin were asymmetrically inserted into the
polylinker of the mammalian expression vector pRc/CMV (Invitrogen). All
constructs were controlled by DNA sequencing with a laser sequencer
(Applied Biosystems, ABI 373 A).
L-TK cells (kindly
provided by Dr. R. Kemler, Max Planck Institute, Freiburg, Germany)
were grown, transfected, and selected as described previously (2, 23).
Cadherin-positive cells were subcloned twice by limiting dilution.
Several clonal cell lines expressing the full-length or the respective
deleted forms, as well as pooled clones, were analyzed. Results of
representative clonal cell lines are shown.
For metabolic radiolabeling of proteins, confluent monolayers of cells were preincubated with methionine-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) for 2 h. Fresh methionine-free medium supplemented with 50 µCi/ml [35S]L-methionine (1100 Ci/mmol, Amersham Corp.) was added for 16 h before the cells were harvested.
AntibodiesPreviously characterized monoclonal antibody
(mAb)1 6D5 raised against U-cadherin (24)
was a gift from Dr. P. Hausen (Max Planck Institute, Tübingen,
Germany). Peptide-specific polyclonal antibodies against - and
-catenin have been described elsewhere (25, 26) and were kindly
provided by Dr. R. Kemler (Max Planck Institute, Freiburg, Germany).
mAbs against
-catenin and p120cas were purchased from
Transduction Laboratories.
Cells were grown to confluency on glass coverslips for at least 48 h before being fixed in 3% paraformaldehyde in PBS/Ca2+ (phosphate-buffered saline: 2.7 mM KCl, 137 mM NaCl, 0.15 mM KH2PO4, 0.7 mM NaH2PO4, and 0.75 mM CaCl2). For detergent extraction, cells were incubated for 5 min at 4 °C in PBS/Ca2+ containing 0.5% Nonidet P-40 prior to fixation. Indirect immunostaining was carried out as described previously (23), except that all antibodies were diluted in PBS/Ca2+. Cy3-conjugated goat anti-mouse IgG (Dianova) were used as secondary antibodies. Cells were viewed with a laser scanning confocal microscope (Leitz).
Western Blot, Immunoprecipitation, Surface Biotinylation, and Streptavidin-Agarose PrecipitationConfluent monolayers of cells were rinsed three times with PBS/Ca2+ and harvested. Cells were lysed in PBS/Ca2+ supplemented with 1% each of Triton X-100 and Nonidet P-40, 2 mM each of aprotinin, leupeptin, and pepstatin, 1 mM N-ethylmaleimide, and 2 mM each of iodoacetamide and phenylmethylsulfonyl fluoride (all from Sigma). Protein concentrations were determined by Bradford protein quantification. SDS-PAGE, immunoblotting, and immunoprecipitation, as well as biotinylation and streptavidin-agarose precipitation of cell surface proteins, were carried out as described previously (23). Immunoblot signals were scanned and quantified.
Cell Aggregation AssayCell monolayers that had just reached confluency were used for preparing single cells. The cell aggregation assay was performed according to the method described elsewhere (27). 3-4 × 105 cells were incubated at 37 °C with 1 mM CaCl2 or with 1 mM EGTA on a rotating platform at 80 rpm. Aggregation was stopped after 90 min. Cells were fixed by the addition of an equal volume of 5% paraformaldehyde in PBS/Ca2+. Aggregates were observed by phase contrast microscopy.
Four deletion mutants of the amphibian XB/U-cadherin
sequence were constructed that lack 38 (XBc38), 32 (XB
c32), or 19 (XB
c19) carboxyl-terminal amino acids or bear an internal deletion
of 20 amino acids followed by 19 carboxyl-terminal amino acids of wild-type XB/U-cadherin (XB
i20). In the construct XB
c32, 12 random amino acids follow the truncated XB/U-cadherin-specific sequence. The carboxyl-terminal sequences of full-length XB/U-cadherin and the deletion mutants are shown in Fig.
1A.
To characterize the different mutants with respect to conserved
sequence motifs, a sequence alignment of the last 52 carboxyl-terminal amino acids of XB/U-cadherin and murine E-cadherin was done in Fig.
1B. Although 11 exchanges were found in the region
containing the -catenin-binding site, 5 of them were conservative.
An accumulation of four exchanges follows directly downstream of the
cluster of eight serine residues, which itself is conserved. Both
proteins share a stretch of amino acids, "KLADMYGG," upstream of
carboxyl-terminal glutamic or aspartic acid residues.
Cadherin protein levels did not differ by more than 18% among
L-TK cell lines expressing the different cadherin
constructs under the control of the human cytomegalovirus promoter
(Fig. 2A) analyzed by immunoblotting using
mAb 6D5 against XB/U-cadherin. Mutant forms of XB/U-cadherin showed
reduced molecular weights as expected from the respective cDNA
truncations. Because cadherin expression has been reported to induce
up-regulation of catenins in L-TK
fibroblasts (28), we
assessed the steady-state levels of
- and
-catenin proteins in
the different mutant XB/U-cadherin-expressing cell lines. 20 µg of
total protein per lane were analyzed by immunoblotting and quantified.
Steady-state levels of the XB/U-cadherin constructs,
- and
-catenin, were determined by analyzing 10 experiments. Fig. 2 shows
one of these experiments as an example. Cadherin and catenin levels did
not differ by more than 15% between experiments. Transfectants
contained similar amounts of
-catenin (Fig. 2B), although
in cells expressing full-length XB/U-cadherin or XB
i20, the amount
of
-catenin was 135 and 130% compared with the other transfectants,
respectively. The
-catenin content varied extensively among
transfected cell lines (Fig. 2C).
-Catenin protein was at
the detection limit in vector-transfected controls and cells expressing
XB
c32 or XB
c38 and did in no experiment exceed 10% of the
-catenin level reached in cells bearing intact XB/U-cadherin. In
XB
i20 transfectants, we detected the largest amount of
-catenin with an average of 133%, whereas cells expressing XB
c19 exhibited only 60% both compared with the amount of
-catenin with the
full-length XB/U-cadherin transfectants. Other proteins we studied
while we characterized our cell lines, such as integrins, fibronectin, and laminin, did not detectably alter their expression level upon transfection of XB/U-cadherin constructs (data not shown).
Complex Formation of XB/U-Cadherin and Its Mutants
Cadherin-catenin complex formation was studied by
coprecipitation experiments. Cells were metabolically labeled with
[35S]methionine for 16 h and lysed. Transfected
cadherins were immunoprecipitated from cell lysates using mAb 6D5.
Immunoisolated proteins were separated by SDS-PAGE. Fluorography of all
samples showed signals corresponding to the respective forms of
XB-cadherin (Fig. 3A, uppermost bands).
Additionally, two bands migrating at 102 and 88 kDa coprecipitated with
XB/U-cadherin-specific antibodies in cells expressing the full-length
XB/U-cadherin as well as in cells bearing XBc19 and XB
i20. Those
mutants comigrated with the 102-kDa band in a way that the two bands
could not be discriminated. With XB
c32 and XB
c38, no signals
other than those corresponding to the respective mutant forms of
XB/U-cadherin were detected. When mAb 6D5 immunoprecipitates from cell
extracts were immunoblotted with
-catenin antibodies, the 102-kDa
protein that coprecipitated with XB/U-cadherin, XB
c19, and XB
i20
(Fig. 3A) was stained (Fig. 3B). Similarly, the 88-kDa
coprecipitating protein was identified as
-catenin as determined by
immunoblotting with
-catenin antibodies (Fig. 3C).
Neither
- nor
-catenin were found in a complex with XB
c32 and
XB
c38 in these Western blots, as expected from their radioimmunoprecipitation profile (Fig. 3A). However,
immunodetection of precipitated cadherins with mAb 6D5 showed that all
mutants as well as wild-type cadherin were efficiently isolated from
cell lysates (Fig. 3D). Thus, detection of
- and
-catenin as XB/U-cadherin coprecipitates did not fail in LXB
c32
and LXB
c38 lysates because of inefficient immunoisolation of
XB/U-cadherin-containing protein complexes with mAb 6D5. In
coimmunoprecipitation studies, we did not detect
-catenin in a
complex with any of the forms of XB/U-cadherin (data not shown).
We also analyzed the p120cas-binding properties to full-length
and truncated forms of XB/U-cadherin. Interestingly, p120cas
was coimmunoprecipitated with wild-type as well as all four deletion mutants of XB/U-cadherin as determined by Western blotting of cadherin
immunoprecipitates with antibodies against p120cas (Fig.
4). Although the mAb used reportedly recognizes all four isoforms of p120cas (16), only p120cas 1A and B were
found in the complex with XB/U-cadherin.
Effect of Cadherin Truncation on Calcium-dependent Cell Aggregation
The ability of transfected XB/U-cadherin constructs
to mediate calcium-dependent cell-cell adhesion was tested
using reaggregation assays. Cells were first dissociated by a 5-min
trypsin treatment. Reaggregation in the presence of 1 mM
Ca2+ resulted in the formation of stable cell-cell contacts
within 90 min in L-TK cells expressing either full-length
XB/U-cadherin or mutated XB
i20 cDNAs (Fig. 5,
A and E). Cell adhesion ability was shown to be
calcium-dependent because no cell-cell aggregates were
observed in the presence of 1 mM EGTA. An example of this
is shown for the XB/U-cadherin-expressing cell line in Fig.
5B. In cells transfected with XB
c19, XB
c32, or
XB
c38, or with vector alone, no cell-cell contacts were established;
only single cells were observed 90 min after reaggregation was
initiated (Fig. 5, C, D, and F, and data not
shown).
Strikingly, the XB/U-cadherin mutant XBc19 formed complexes with
both
- and
-catenin but did not mediate cell-cell adhesion of
transfected L-TK
cells. This observation differs from all
earlier reports on mutant cadherins in which cadherin-catenin complex
formation consistently correlated with cell-cell adhesion.
To
further confirm our results on cadherin-catenin complex formation and
make sure that complexes were localized to their normal site of
function, we tested whether catenins were found associated with the
respective XB/U-cadherin forms at plasma membranes. Cell surface
molecules were biotinylated and isolated by streptavidin-agarose precipitation. The resulting streptavidin-agarose precipitated proteins
and the non-streptavidin-bound cytosolic fraction were separated by
SDS-PAGE. Distribution of transfected forms of XB/U-cadherin as well as
- and
-catenins was determined by Western blot analysis using
antibodies to catenins or cadherin. Fig. 6A
shows the subcellular localization of XB/U-cadherin and its deletion
mutants. In all transfected cell lines, cadherin molecules were bound
to streptavidin-agarose and were lacking in the soluble supernatant
fractions. This provided biochemical evidence that forms of
XB/U-cadherin were localized at plasma membranes of transfected cells.
- and
-catenin were detected in plasma membrane-associated
fractions of the three cadherin-catenin complex-forming transfectants
bearing full-length XB/U-cadherin, XB
c19, or XB
i20 (Fig.
6B,
-catenin; Fig. 6C,
-catenin). Because
catenins do not directly bind to membranes (7), this proved that they
were present in surface-associated protein complexes, presumably
through interactions with the respective cadherin molecules (refer to
Fig. 3). In cell lines that were transfected with vector alone or that
expressed the XB/U-cadherin mutants XB
c32 or XB
c38,
- and
-catenin were detected exclusively in supernatant fractions of the
biotinylated cells (Fig. 6, B and C). As
expected, catenins remained cytosolic in cells bearing cadherin
molecules deficient in cadherin-catenin complex formation. The fact
that catenins do not appear in the streptavidin-bound fraction of
complex-forming deficient constructs serves as an internal confirmation
that the biotinylation was indeed restricted to cell surface
molecules.
Immunofluorescence Analysis of Truncated XB/U-Cadherin Distribution and Detergent Resistance
In agreement with the biochemical data
shown in Fig. 6, immunofluorescence staining of nonpermeabilized
transfected cells with XB/U-cadherin-specific mAb 6D5 showed that all
forms of XB/U-cadherin were localized to the plasma membrane (Fig.
7). The complete XB/U-cadherin as well as XBi20 were
predominantly detected at sites of direct cell-cell contact (Fig. 7,
A and G). In contrast, XB
c19, XB
c32, and
XB
c38 were distributed over the entire cell surface in a punctate
pattern (Fig. 7, C, E, and I, respectively).
L-TK
cells transfected with vector alone showed no
staining with mAb 6D5 (data not shown).
To mediate stable cell-cell adhesion, classical cadherins require
linkage to cytoskeletal elements. Because this cytoskeletal stabilization renders cadherin proteins largely resistant to extraction with nonionic detergents (11), transfected L-TK cells
were treated with 0.5% Nonidet P-40 prior to immunostaining with mAb
6D5. A significant amount of transfected XB/U-cadherin (Fig.
7B) as well as the internally deleted mutant XB
i20 (Fig. 7H) was resistant to detergent extraction, as illustrated by
the pronounced immunofluorescence in regions of cell-cell contact. On
the other hand, the three carboxyl-terminal deletion mutants of
XB/U-cadherin, XB
c19, XB
c32, and XB
c38, were completely soluble in nonionic detergents. No immunofluorescence signal was obtained when cadherin staining was performed on these cells after Nonidet P-40 extraction (Fig. 7, D, F, and
J).
Here we report that amphibian XB/U-cadherin associates
cytoplasmically with endogenous - and
-catenin and
p120cas when transfected into murine L-TK
fibroblasts. This cadherin-catenin complex induces
calcium-dependent cell-cell adhesion, confirming the
evolutionary conservation of this adhesion mechanism. The expression of
different deletion mutants of XB/U-cadherin shows that p120cas
binds upstream of the
-catenin-binding site. Most interestingly, we
observed that XB/U-cadherin detergent insolubility and its ability to
mediate cell-cell adhesion requires the 19 most carboxyl-terminal amino
acids of XB/U-cadherin, whereas cadherin-catenin complex formation does
not. Thus, we provide evidence that two separate domains in the
cadherin cytoplasmic tail mediate catenin complex formation and
induction of adhesion, respectively.
-Catenin/plakoglobin, instead of
-catenin, is able to bind to
the cytoplasmic tail of E-cadherin (12). Here, XB/U-cadherin predominantly forms complexes with
-catenin and
-catenin, whereas a minor subpopulation of XB/U-cadherin-
-,
-catenin complexes may
exist in our L-TK
transfectants. p120cas
coprecipitated with full-length or truncated XB/U-cadherin
independently of their function in catenin binding or cell adhesion.
This indicates that the p120cas-binding epitope in
XB/U-cadherin is located upstream of the 38 carboxyl-terminal amino
acids and that the XB/U-cadherin site involved in p120cas
binding is not sterically altered by the carboxyl-terminal deletions. Because full-length XB/U-cadherin and the internal deletion mutant XB
i20 did not differ from adhesion-deficient mutants XB
c19, XB
c32, or XB
c38 with respect to their
-catenin- or
p120cas-binding properties, it is not likely that these
XB/U-cadherin-binding proteins contribute to the differences in
formation and function between XB/U-cadherin-catenin complexes.
The most truncated of our deletion mutants, XBc38, lacks cadherin
adhesive function as well as cadherin-catenin complex formation. The
interpretation of earlier results from equivalent E-cadherin constructs
had been that the catenin-binding domain of E-cadherin reached into the
carboxyl-terminal 37 amino acids (9, 19). Our mutants XB
c19 and
XB
c32 both contain the cluster of eight serines but differ in the
amino acids following it (Fig. 1A). Because of these
mutants, only XB
c19 binds catenins, supporting evidence by Stappert
and Kemler (22) that amino acids after the serine cluster are necessary
for
-catenin binding. Jou et al. (21) found that these
residues were not required for
-catenin binding. However, this group
used the two-hybrid system, which may fail to represent in
vivo binding. By attaching the 19 most carboxyl-terminal amino
acids to the nonfunctional XB
c38, we generated the construct
XB
i20. Although it lacks a part of the serine cluster as well as the
following amino acids, XB
i20 does form catenin complexes, suggesting
that the 19 most carboxyl-terminal amino acids of XB-cadherin can
substitute for the missing sequence. Taken together, our data suggest
that the site of
-catenin interaction is located upstream of the
carboxyl-terminal 38 amino acids. Stable
-catenin binding in
vivo, however, requires the cadherin sequences following
downstream. These can be substituted by the 19 most carboxyl-terminal
amino acids in XB
i20 but not by the random sequence in XB
c32.
Most strikingly, we learned from our studies that catenin binding
itself is not sufficient to render the cadherin-catenin complex
adhesive. We could only restore complete cadherin activity by
attachment of the 19 most carboxyl-terminal amino acids, which end in
seven consecutive acidic residues (XBi20), to the nonfunctional XB
c38. The addition of a hydrophobic sequence of equal length (XB
c32) neither restored catenin binding nor cell-cell adhesion. Even the addition of the 19 amino acids that normally follow the catenin-binding site (XB
c19) were not able to restore complete cadherin activity. This mutant the tail of which contains three acidic
residues apart from each other forms cell surface-localized catenin
complexes. However, XB
c19 fails to mediate cell-cell adhesion. Our
data imply that a stretch of acidic amino acids downstream of the
catenin-binding site is required to confer adhesive function to the
already formed cadherin-catenin complex. Importantly, this shows that
the two functions of cadherins, recruitment of cytoplasmic catenins and
adhesion, can be separated from each other.
Functional catenin-XB/U-cadherin and -XBi20 complexes cannot be
completely solubilized by nonionic detergents, indicating that they are
linked to the cytoskeleton (13). Conversely, XB
c19 is extracted with
mild detergents, probably because its complexes are not stabilized by
components of the cytoskeleton.
Complex formation and cytoskeletal linkage seem to depend on the
presence of numerous charged amino acids in the catenin-binding domain
and in the cadherin tail. Negative charges in the catenin-binding region are provided by posttranslational phosphorylation of multiple serine residues, which has been shown to be essential for catenin binding (22). The binding domain of plakoglobin, a -catenin homologue, in the desmosomal cadherin desmoglein bears an accumulation of negatively charged amino acids as well (29, 30). More recently, a
-catenin-binding site has been identified in the amino terminus of
the transcription factor LEF-1. This domain also consists of many
negatively charged amino acids (31, 32).
-Catenin has been shown to
bind to the product of the tumor suppressor gene apc in vivo
(33-36). APC bears multiple consensus sequences for
-catenin
interaction, which lack any similarity with the cadherin cytoplasmic
tail and are devoid of negatively charged residues (35).
-Catenin
also interacts with the APC-
-catenin complex, presumably in a manner
similar to how it binds to the
-catenin-cadherin complex. However,
APC-
-catenin complexes are not induced to interact with the
cytoskeleton when bound to
-catenin. Thus, a protein complex
including
- and
-catenin does not, by default, associate with
cytoskeletal elements. Given that
-
-catenin complexes serve functions in vivo as soluble complexes in the cytoplasm as
well as membrane- and microfilament-associated, cytoskeletal binding of
catenins must be specifically regulated. Our results suggest that this
regulation is performed by cadherin cytoplasmic sequences distinct from
the
-catenin-binding site.
The technical assistance of Ulrike Unsöld is greatly appreciated. We are grateful to Drs. Peter Hausen and Rolf Kemler for the donation of antibodies. We also thank Dr. Geri Gurland for critical reading of the manuscript.