Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, 10021
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Abstract |
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Cadherin cell-cell adhesion molecules form
membrane-spanning molecular complexes that couple
homophilic binding by the cadherin ectodomain to the
actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic
binding activity of the ectodomain into strong adhesion.
Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to
substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened
adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy,
and B.M. Gumbiner. Curr. Biol. 7:308-315). In this
study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained
the juxtamembrane 94-amino acid region of the cytoplasmic tail, but not the -catenin-binding domain,
clustered upon attachment to substrata coated with
CEC1-5. Like wild-type C-cadherin, this clustering was
ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering
when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669
cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding
to the juxtamembrane region revealed that the major
associated protein is p120ctn. These findings identify the
juxtamembrane region of the cadherin cytoplasmic tail
as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility
that p120ctn is involved in clustering and cell adhesion.
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Introduction |
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CADHERIN cell adhesion molecules are fundamental
determinants of tissue organization in developing
and adult organisms (Takeichi, 1995; Gumbiner,
1996
). Classical cadherins (including E-, N-, and Xenopus
C-cadherin) are membrane-spanning glycoproteins that interact with cytoplasmic proteins (catenins) capable of associating with the actin cytoskeleton (Takeichi, 1991
, 1995
;
Yap et al., 1997a
). There is compelling evidence that protein interactions mediated by both the cadherin ectodomain and cytoplasmic tail participate in adhesion. The cadherin ectodomain supports the Ca2+-dependent, homophilic
binding that ultimately forms the basis for cadherin adhesiveness. Homophilic binding activity is retained in purified ectodomains of Xenopus C-cadherin and, using a sensitive laminar flow assay, adhesive activity was also
detected in CHO cells expressing mutants of C-cadherin
lacking the cytoplasmic tail (Brieher et al., 1996
). However, tailless mutants supported adhesion that was considerably weaker than that mediated by wild-type C-cadherin
(Brieher et al., 1996
). Nor did tailless mutants of E-cadherin support adhesion of L cell fibroblasts measured in aggregation assays (Nagafuchi and Takeichi, 1988
, 1989
;
Ozawa et al., 1990
). Therefore, additional molecular mechanisms involving the cytoplasmic tail must strengthen the
weak intrinsic binding activity of the cadherin ectodomain
to support physiological cell adhesion.
Earlier studies identified catenin-binding interactions as
one important basis for this adhesive activity. -catenin
binds with high affinity to the COOH-terminal region of
the cadherin cytoplasmic tail and serves as an anchor for
-catenin which can, in turn, associate with actin filaments
(Aberle et al., 1994
; Hulsken et al., 1994
; Funayama et al.,
1995
; Jou et al., 1995
; Rimm et al., 1995
) and potentially
other junctional proteins, such as
-actinin (Knudsen et al.,
1995
). Cadherin mutants lacking the catenin-binding site
were poorly adhesive in aggregation assays when expressed in L cell fibroblasts (Nagafuchi and Takeichi, 1988
, 1989
; Ozawa et al., 1990
). Furthermore, mutant cadherins bearing the cytoplasmic tail or the catenin-binding
region alone exerted potent dominant-negative effects
upon adhesion when expressed in Xenopus embryos (Kintner, 1992
), cultured cells (Fujimori and Takeichi, 1993
),
and in mouse intestinal epithelia (Hermiston and Gordon,
1995
). This inhibitory effect was attributed, at least in part,
to titration of
-catenin away from wild-type cadherins by
the mutant molecules (Kintner, 1992
). Genetic evidence
implicating catenins in strong cell adhesion was also obtained in studies of cancer cells (Hirano et al., 1992
;
Watabe et al., 1994
) and embryos (Kofron et al., 1997
;
Torres et al., 1997
) lacking
-catenin and in studies of armadillo (the homologue of
-catenin) in Drosophila (Cox
et al., 1996
). Taken together, these findings have led to the
hypothesis that a protein complex consisting of
- and
-catenin linked to the distal region of the cytoplasmic tail
plays a fundamental role in cadherin function, perhaps
through association with the actin cytoskeleton (Takeichi,
1991
, 1995
). The precise molecular mechanism by which
this complex determines adhesive strength has yet to be
fully elucidated.
Additionally, however, there is evidence that other regions of the cytoplasmic tail, particularly the juxtamembrane region proximal to the catenin-binding site, can influence cadherin function (Kintner, 1992; Riehl et al.,
1996
; Chen et al., 1997
). Cadherin mutants bearing portions of the juxtamembrane region, but not the distal catenin-binding site, also inhibited adhesion when expressed in
Xenopus embryos (Kintner, 1992
) whereas a mutant of
vascular endothelial (VE)1 cadherin possessing the membrane-proximal region but not the distal catenin-binding
site supported aggregation of tissue culture cells (Navarro
et al., 1995
). The juxtamembrane region is also reported to
influence the motility of cultured cells (Chen et al., 1997
)
and neurons (Riehl et al., 1996
). Therefore, although there
is clear and compelling evidence that catenin-binding by the distal cytoplasmic tail plays an important role in adhesion, other regions of the cytoplasmic tail may also have
yet-to-be defined functional contributions.
One mechanism by which cadherin ectodomain and cytoplasmic tail may cooperate in adhesion is through lateral
clustering of cadherin molecules. Recently we demonstrated that the distribution of the cadherin ectodomain
presented at the cell surface significantly influences adhesive function (Yap et al., 1997b). We used the FKBP-FK1012 protein oligomerization system (Spencer et al.,
1993
) to force clustering of a chimeric molecule in which
the cytoplasmic tail of Xenopus C-cadherin was replaced
by tandem repeats of the FK506-binding protein, FKBP12.
Strong adhesion was generated in this system by clustering
of the ectodomain alone independent of possible contributions from cytoskeletal interactions or signaling events
mediated by the normal cadherin cytoplasmic tail. Therefore, lateral clustering is a potential mechanism to convert
the homophilic binding activity of the ectodomain into a
state capable of supporting physiological cell adhesion.
We also found that native C-cadherin molecules clustered in a ligand-dependent fashion when cells attached to
substrata coated with the adhesive ectodomain of C-cadherin expressed as a recombinant protein (CEC1-5). Cadherin clustering correlated with a significant strengthening
of adhesion to CEC-coated substrata (Yap et al., 1997b).
However, those studies suggested that some component of the cytoplasmic tail was necessary for clustering of native
cadherins to occur, since the ectodomain-FKBP12 chimera
failed to cluster in the absence of FK1012. This suggested
that the cytoplasmic tail might contribute to adhesion by
driving clustering of the ectodomain presented at the cell
surface (Yap et al., 1997b
). Those studies did not identify
the region of the cytoplasmic tail responsible for clustering
nor was it known whether catenins were necessary for cadherin clustering. In this paper, we therefore sought to
identify the cytoplasmic region responsible for cadherin clustering, test its role in adhesion and identify interacting proteins that might be involved.
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Materials and Methods |
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Cadherin Mutants and Transfected Cell Lines
A summary of the C-cadherin mutants is shown in Fig. 1. Construction of
the tailless truncation mutant (CT) that lacks the complete predicted cytoplasmic domain and bears a myc-tag at the COOH terminus has been described previously (Brieher et al., 1996). To generate a protein lacking the
catenin-binding site (CT669), the cytoplasmic domain was truncated at
amino acid position 669 that deletes the terminal 56 amino acids predicted
to mediate binding of
-catenin (Nagafuchi and Takeichi, 1988
, 1989
;
Stappert and Kemler, 1994
). First, oligonucleotide linkers containing the cloning sites 5'-NdeI-BamHI-NotI-3' were inserted into the mammalian expression vector pcDNA3, and a BamHI-NotI fragment containing a
6×-myc tag from pCS2 (Fagotto et al., 1996
) was ligated into the BamHI-
NotI sites. An EcoRV-NdeI fragment from pBS/7B3 containing the signal
sequence, ectodomain, transmembrane region and cytoplasmic tail of
C-cadherin to amino acid position 669 (Levine et al., 1994
) was then inserted to yield the predicted truncation mutant containing a COOH-terminal 6×-myc tag.
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To construct a mutant molecule with an internal deletion of the juxtamembrane region of the cytoplasmic tail, replaced by a 5×-myc sequence
to act as an epitope tag and spacer (CT-CAT), an NdeI-XbaI fragment
from pBS/7B3 was isolated encoding the catenin-binding region of the
C-cadherin cytoplasmic tail (from amino acid 670) and ligated into pBS/
ECFK (Yap et al., 1997b), which contains an NdeI site at the predicted
transmembrane-cytoplasmic junction, to yield a mutant lacking the membrane-proximal region of the cadherin cytoplasmic tail (pBS/CT-CAT). A
5×-myc tag was isolated from pCS2 by polymerase chain reaction using
primers that inserted 5'- and 3'-NdeI sites. The PCR fragment was then ligated into pBS/CT-CAT and the complete construct transferred to
pCDNA3 by directional cloning using EcoRV and XbaI.
To construct a chimeric protein containing the ectodomain and transmembrane region of the IL2 receptor -subunit fused to the juxtamembrane region of the C-cadherin cytoplasmic tail (IL2R-669), DNA encoding the cytoplasmic tail from the predicted membrane-cytoplasmic junction to amino acid position 669 was isolated by the polymerase chain
reaction using primers that inserted 5'-HindIII and 3'-BamHI sites for
cloning purposes. The PCR product was then inserted into pCV-IL2L,
which contains the ectodomain and transmembrane region of the interleukin 2 receptor
-subunit (LaFlamme et al., 1992
).
All PCR products and ligation sites were sequenced using the Sequenase II kit (following the manufacturer's instructions). Plasmids were expressed in CHO cells using lipofectamine (following the manufacturer's instructions). For stable transfections, cell lines were picked using G418 selection (500-800 µg/ml). Two lines of C-CHO cells were used. Both cell lines spread on CEC1-5-coated substrata, but C-CHO21 cells express cadherin levels ~4× greater than C-CHO12 cells and were more strongly adhesive in flow assays.
GST Fusion Protein Constructs
The polymerase chain reaction was performed to obtain fragments encoding either the full-length (nucleotides 2185-2643) or the juxtamembrane region (nucleotides 2185-2415) of the C-cadherin cytoplasmic tail. In both cases a sense primer was used with an EcoRI site at its 5' site, which allowed subcloning of the fragments into the EcoRI-SmaI sites of pGEX4T-3 (Pharmacia Biotech Sverige, Uppsala, Sweden). All constructs were sequenced.
GST fusion proteins containing the full-length cytoplasmic tail (GST-FL) and the juxtamembrane region of the cytoplasmic tail (GST-Prox)
were expressed in BL21 bacteria (Smith and Johnson, 1987), lysed in PBS/
1% Triton using a French press and purified with glutathione-agarose
beads (Sigma Chemical Co., St. Louis, MO). After elution with glutathione and extensive dialysis fusion proteins were bound to fresh glutathione beads to use in the isolation experiments.
Isolation of the 92-kD Protein and Microsequencing
Cells were metabolically labeled overnight using a mixture of [35S]methionine and [35S]cysteine in methionine- and cysteine-free MEM supplemented with 10% regular MEM. Cells were lysed in 1% NP-40 buffer (20 mM Tris, 4 mM EDTA, 150 mM NaCl, pH 7.6) and the soluble fraction was precleared for 3 h at 4°C with glutathione beads. Subsequently, the lysate was incubated with the fusion proteins immobilized on the glutathione beads (5 µg/sample). After extensive washing, the beads and associated proteins were analyzed by 6% SDS-PAGE under reducing conditions.
To obtain sufficient quantities of the 92-kD protein associated with
GST-Prox, cells from 260 plates (15-cm diam) were lysed in 1% NP-40
buffer and processed as described above. A total of 100 µg of GST-Prox
was used to precipitate proteins from the pooled lysate. After extensive
washing the beads were loaded on a 5% SDS-PAGE gel, blotted to nitrocellulose from which the 92-kD protein was excised after staining with
Ponceau S. This band was further processed for a combined analysis by
mass spectrometry and internal amino acid sequencing essentially as described previously (Erdjument-Bromage et al., 1994). In brief, nitrocellulose-bound protein was trypsin digested in situ and this peptide mixture
was subjected to matrix-assisted laserdesorption time-of-flight mass
(MALDITOF) spectrometry (Reflex III; Bruker Franzen, Germany). The
32 major peptides were used to search the NRDB protein database (European Bioinformatics Institute, Hinxton, UK) using the Peptide Search algorithm (Mann et al., 1993
). Two peptides with mixed mass spectra were
subjected to NH2-terminal sequence analysis, using an applied biosynthesis 477A automated sequenator (Perkin-Elmer Corp., Norwalk, CT) as designed by Tempst et al. (1994)
.
Adhesion Assays
Recombinant C-cadherin ectodomain (CEC1-5) was purified from conditioned media as previously described (Brieher et al., 1996). For adhesion
assays glass coverslips and capillaries were coated with CEC1-5 (10 µg/ml
in 100 mM NaCl, 20 mM Hepes, and 1 mM CaCl2, pH 7.2) for 8 h and then
blocked with 10 mg/ml BSA (overnight at 4°C). Laminar flow adhesion assays were performed as previously described with minor modifications
(Brieher et al., 1996
; Yap et al., 1997b
). All adhesion assays were performed in the presence of the RGD-containing peptide, GRGDTP (1 mg/ml;
Sigma Chemical Co.) to inhibit possible background integrin-mediated
adhesiveness (Yap et al., 1997b
). Cells expressing wild-type or mutant
cadherins were isolated by incubation with 0.01% crystalline trypsin in Hanks balanced salt solution supplemented with 1 mM CaCl2 (HBSS/ Ca2+; at 37°C for 10 min), conditions that preserve cellular cadherins (Takeichi, 1977
). Enzymatic digestion was stopped by addition of soybean
trypsin inhibitor (1 mg/ml), cells collected by centrifugation and resuspended in HBSS/Ca2+. Aggregation assays were performed based on previously described methods (Nagafuchi and Takeichi, 1988
, 1989
). In brief,
freshly isolated cells (1-ml aliquots; 2.5 × 105 cells/ml in HBSS/Ca2+) were
pipetted into agarose-coated wells of 12-well plates and agitated on an orbital shaker. The numbers of single cells remaining after 45 min were expressed as percentages of the numbers of single cells in the cell suspension
immediately before aggregation (Nt/No).
Immunofluorescence Microscopy
Specimens were prepared for immunofluorescent staining by fixation in paraformaldehyde (3% in PBS containing 1 mM CaCl2 and 1 mM MgCl2; 30 min) and permeabilized with Triton X-100 (0.25% in PBS, room T°, 5 min). Reactive aldehyde groups were blocked with glycine (100 mM, 30 min). Nonspecific binding was blocked with 5% nonfat dried milk (in PBS) and all antibody reactions and intermediate washes were performed in blocking buffer. Specimens were incubated with primary antibodies overnight at 4°C, washed, and then incubated with TR- or FITC-conjugated secondary antibodies (room temperature, 1 h). Specimens were examined with a Zeiss Axioskop equipped with ×63 and ×100 plan-APOCHROMAT objectives and photographed using Kodak Elite 400 film. Some samples were examined by confocal laser scanning microscopy using a Biorad MRC 600 confocal microscope mounted on a Zeiss Axioskop equipped with ×40 and ×100 plan-APOCHROMAT objectives and images acquired using the COMOS software supplied with the MRC 600. All images were compiled for publication using Adobe Photoshop.
Western Blotting and Immunoprecipitations
For Western blotting, cells were extracted in 1% NP-40 lysis buffer (1%
NP-40, 10 mM Hepes, 150 mM NaCl, 1.5 mM EDTA, pH 7.4) supplemented with protease inhibitors as described previously (Brieher and
Gumbiner, 1994). Samples containing equal quantities of total protein
were separated by SDS-PAGE and transferred to nitrocellulose. For immunoprecipitation studies cells were lysed with 1% NP-40 extraction
buffer, aliquots of NP-40 soluble supernatants containing equal quantities
of protein were immunoprecipitated with a polyclonal antibody directed
against the C-cadherin ectodomain (Yap et al., 1997b
) or the anti-p120ctn
mAb and collected with protein A- or protein G-Sepharose beads. Beads
were washed in 1% NP-40 lysis buffer, boiled in SDS-Laemmli buffer containing 50 mM DTT, and separated by SDS-PAGE for immunobloting.
Antibodies
Cadherins were detected using (a) a pAb directed against the conserved
cytoplasmic tail of mouse E-cadherin (Marrs et al., 1993) that recognizes
other cadherins, including Xenopus C-cadherin (Yap et al., 1997b
; unpublished data; a generous gift from Dr. J. Marrs, University of Indiana, Indianapolis); and (b) a polyclonal Ab (Yap et al., 1997b
) or mAb 6B6 (Brieher and Gumbiner, 1994
) directed against the ectodomain of C-cadherin.
-catenin was detected using a mAb (Transduction Laboratories; Lexington, KY) or with a polyclonal Ab directed against the NH2 terminus (11).
p120ctn mAb was purchased from Transduction Labs. mAB 9E10 (Evan et
al., 1985
) was used to detect myc-tagged proteins. The ectodomain of the
human IL2 receptor
-subunit was detected with mAb 3G10 (Boehringer Mannheim Corp., Indianapolis, IN).
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Results |
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Expression and Characterization of C-cadherin Mutants
To examine the contribution of the cadherin cytoplasmic
tail to clustering, a series of C-cadherin mutants were designed (Fig. 1). In addition to CT, a mutant lacking the
complete cytoplasmic tail, two further mutants were constructed to dissect potential contributions of regions
within the tail. (a) A truncation mutant (CT669) lacking
the terminal 56 amino acids predicted to mediate catenin
binding (Nagafuchi and Takeichi, 1988, 1989
; Ozawa et al.,
1990
; Stappert and Kemler, 1994
) was designed in an attempt to detect any activity associated with the 94-amino
acid juxtamembrane region of the cytoplasmic tail. (b)
Conversely, to assess the potential specific adhesive contribution of the catenin-binding site, the terminal 56 amino
acids were retained in the mutant CT-CAT, but the juxtamembrane region was replaced by a 5x-myc tag, acting
as a spacer of approximately similar molecular mass to the
deleted region of the cytoplasmic tail. Upon stable transfection in CHO cells CT669 and CT-CAT were expressed
as polypeptides of molecular mass 125 and 116 kD, respectively (Fig. 2 A), consistent with the predicted contribution
of the myc tags to molecular mass.
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To confirm the predicted effects of these deletions on
catenin-binding activity, we tested the ability of wild-type
and mutant C-cadherin molecules to coimmunoprecipitate
-catenin. As shown in Fig. 2 B, a polyclonal antibody directed against the cadherin ectodomain immunoprecipitated wild-type C-cadherin, CT669, and CT-CAT, and
lesser amounts of precursor proteins, from CHO cells stably transfected with these molecules. Immunoblots identified
-catenin in cadherin immunoprecipitates from C-CHO
and CT-CAT-CHO cells, but not from CT669-CHO cells.
These studies were performed under mild buffer conditions that do not affect the high affinity cadherin-
-catenin interaction (McCrea and Gumbiner, 1991
). In addition, expression of cadherins generally increases total
cellular
-catenin levels, presumably due to metabolic stabilization of
-catenin bound to cadherins at the cell membrane (Kowalczyk et al., 1994
; Finnemann et al., 1997
). This
phenomenon was observed in C-CHO cells and CT-CAT-CHO cells, but CT669-CHO cells and CT-CHO cells showed
total cellular
-catenin levels comparable to parental CHO
cells (Fig. 2 A), providing indirect biochemical evidence that CT669 did not retain significant
-catenin-binding activity. These biochemical data are further supported by the
observation (described below) that
-catenin failed to
colocalize with CT669 in immunofluorescence studies (Fig.
3). Taken together, these findings indicated that
-catenin-binding activity was lost in the CT669 mutant, but retained in the CT-CAT mutant, as predicted from earlier
mapping studies performed with other classical cadherin molecules.
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Role of Cytoplasmic Domains in Lateral Clustering
To assess clustering activity cells bearing wild-type or mutant cadherins were allowed to attach to glass substrata
coated with CEC1-5, then fixed and examined for cadherin clustering by immunofluorescence microscopy. As
we previously reported (Yap et al., 1997b), wild-type
C-cadherin localized in prominent clusters at the cell-substrate interface (Fig. 3 A).
-catenin colocalized with many
of these C-cadherin clusters (Fig. 3 B; and yellow overlap staining in Fig. 3 C), as well as in a more diffuse pattern,
some of which is likely to be in a cytoplasmic pool. In contrast, no clustering of the tailless CT mutant was detectable, even with prolonged periods of attachment (Fig. 3
G). This is consistent with our earlier observation that the
cadherin-FKBP12 chimeric protein, which lacks the normal cytoplasmic tail, failed to cluster in the absence of
FK1012 (Yap et al., 1997b
).
The CT669 cadherin mutant also localized in prominent
clusters, as revealed by staining for the myc epitope tag
(Fig. 3 D). However, in these cells the weak -catenin
staining remained diffuse and did not colocalize with
CT669 in clusters (Fig. 3 E), a finding consistent with the
lack of biochemical association between these proteins. As
previously reported for wild-type cadherin (Yap et al.,
1997b
), clustering of CT669 was ligand specific. Clustering of CT669 only occurred in cells attached to CEC1-5 (Fig. 3
D), but was not observed when cells were plated onto
poly-L-lysine (Fig. 3 F) or plasma fibronectin (not shown).
In contrast, no clustering of the CT-CAT mutant was detected at any time (Fig. 3 H). Therefore, the juxtamembrane region of the cytoplasmic tail appeared to support
ligand-specific accumulation of C-cadherin in clusters.
To determine whether the juxtamembrane region of the
cytoplasmic tail was sufficient by itself to induce lateral
clustering, we constructed a fusion protein consisting of
the juxtamembrane 94 amino acids of the C-cadherin cytoplasmic tail fused to the ectodomain and transmembrane
region of the IL2-receptor -subunit (IL2R-669). This,
and a control protein consisting of the IL2R elements
alone, were transiently expressed in CHO cells. Surface localization of the expressed proteins was determined by immunofluorescent staining using a mAb directed against
the IL2R ectodomain in fixed, nonpermeabilized cells attached to poly-L-lysine-coated substrata. Cells expressing
the IL2R control protein showed only uniformly diffuse
staining (Fig. 4 B). In contrast, cells expressing IL2R-669 showed clustered staining in prominent foci at the cell surface (Fig. 4 A). Clusters of IL2R-669 also stained with a
pAb directed against the cadherin cytoplasmic tail but
only after permeabilization of the cells (not shown), indicating that the protein was being correctly expressed at the
cell surface. Therefore, the juxtamembrane region appeared capable of mediating lateral clustering even in the
absence of both the ectodomain or catenin-binding region. Interestingly, IL2R-669 clustered independently of ligand,
in contrast to the clustering of C-cadherin and CT669 that
depended on cell attachment to CEC1-5.
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Functional Activity of Cadherin Cytoplasmic Mutants
Recently. we observed that the strength of adhesion between C-CHO cells and substrata coated with CEC1-5 depended on the duration that cells were initially allowed to
attach to substrate under stasis (temporal strengthening)
and that this correlated with cadherin clustering (Yap et al.,
1997b). To explore the mechanism underlying this correlation, we tested which of the C-cadherin mutants were capable of supporting temporal strengthening. As positive
controls, we used C-CHO cell lines expressing wild-type C-cadherin at levels similar to those of the mutant cadherins.
Temporal strengthening of wild-type C-cadherin is demonstrated in Fig. 5. Measured in a sensitive laminar flow
adhesion assay, C-CHO cells resisted detachment from
substrata coated with CEC1-5 to a significantly greater extent after 40 min static attachment than after an initial 10-min attachment period. As shown previously (Brieher et al.,
1996), adhesion of CT-CHO cells (expressing the tailless
C-cadherin mutant) was weak but consistently greater than parental CHO cells (Fig. 5). However, in contrast to
the significant strengthening displayed by C-CHO cells,
CT-CHO cells showed no evidence of temporal strengthening over the range of attachment times studied in these
experiments. Thus, although the ectodomain alone can
support a measurable level of adhesion when expressed in
cells, the cytoplasmic tail is necessary for temporal strengthening to occur.
|
We then examined the adhesive activity of CT669-CHO and CT-CAT-CHO cells using the laminar-flow assay. As shown in Fig. 6, both CT669-CHO and CT-CAT-CHO cells displayed greater adhesive activity than parental CHO cells. Strikingly, the adhesive activity of CT669-CHO cells was very similar to that displayed by control C-CHO cells. Like C-CHO cells, CT669-CHO cells demonstrated considerable temporal strengthening of adhesion to levels comparable to C-CHO cells. Similar patterns of adhesive behavior were shown by other independent clones of CT669-CHO cells, including lines that expressed lower levels of mutant cadherin protein (not shown). In contrast, adhesion of CT-CAT-CHO cells was weaker than that displayed by C-CHO and CT669-CHO cells (Fig. 6). Comparison of adhesive activity after 10 and 40 min of initial static attachment showed some temporal strengthening in CT-CAT-CHO cells, but to levels of adhesive strength significantly less than either C-CHO or CT669-CHO cells. Therefore, these findings show that the juxtamembrane region of the cytoplasmic tail was capable of strengthening adhesion and supporting strong resistance to detachment.
|
The degree of adhesive activity demonstrated by CT669
was surprising in light of previous studies that demonstrated that mutant E-cadherin molecules lacking the catenin-binding region retained little, if any, adhesive activity
when expressed in L cell fibroblasts (Nagafuchi and Takeichi, 1988, 1989
; Ozawa et al., 1990
). Those studies were
performed in aggregation assays that may measure somewhat different parameters of adhesion than the flow assay used in our studies (Kuo et al., 1997
). Therefore, we assessed the adhesive activity of the C-cadherin mutants in
aggregation assays (Fig. 7). Untransfected parental CHO
cells did not aggregate under the conditions used in this
study, in contrast to C-CHO cells that showed significant
aggregation that was Ca2+-dependent (not shown).
CT669-CHO cells also exhibited aggregation activity that
was consistently greater than CT-CAT-CHO cells but less
pronounced than C-CHO cells. Aggregation of CT669-CHO and CT-CAT-CHO cells was Ca2+-dependent (not
shown). Therefore, in our hands significant adhesive activity of CT669 is detectable in aggregation assays as well in
the laminar flow detachment assay.
|
Identification of Protein Interactions Associated with the Juxtamembrane Region of the Cadherin Cytoplasmic Tail
The clustering activity associated with the CT669 and
IL2R-669 mutants could be mediated either by direct lateral interactions between the juxtamembrane regions of
adjacent cadherin molecules or by intermediary proteins
that bind to the juxtamembrane region. First, we used bacterially expressed GST- and MBP-fusion proteins bearing
the full-length and juxtamembrane cytoplasmic tail regions to test for direct interactions between cytoplasmic
domains. Blot overlay assays as well as in vitro reconstitution assays were performed to test if MBP-FL could bind
directly to GST-FL and/or GST-Prox. However, we were
unable to detect an association despite using a range of
different incubation conditions (data not shown). This result is consistent with biochemical studies that have shown that the E-cadherin cytoplasmic domain/-catenin complex behaves in isolation as a heterodimer (Weis, W., personal communication).
Therefore, we attempted to identify proteins that associate with the membrane proximal region. GST, GST-FL, or GST-Prox bound to glutathione beads were used to precipitate proteins from metabolically labeled HCT116 cells, a human colon carcinoma cell line. Human cells were chosen because the extensive human protein database allows for more reliable identification of any associated proteins after microsequencing.
Both GST-FL and GST-Prox, but not GST, precipitated
a protein from HCT116 cell lysates that migrated ~92 kD.
Moreover, this protein was the major labeled band associated with GST-Prox (Fig. 8 A). Although the molecular
mass of this band was similar to that of -catenin, GST-prox lacks the predicted
-catenin-binding sequence and,
as shown in Fig. 2,
-catenin did not coimmunoprecipitate with CT669 from CHO cells. To confirm that the 92-kD
band associated with GST-Prox was not
-catenin, samples from the in vitro binding assay were immunoblotted
with a mAb directed against
-catenin. As expected
-catenin was bound by the GST-FL but the similar sized band
associated with GST-prox was not recognized by the
-catenin antibody (Fig. 8 B).
|
The procedure using GST-Prox was scaled up to purify
large amounts of the 92-kD polypeptide (Fig. 9 A), which
were transferred to nitrocellulose. After trypsin digestion
the peptide mixture was subjected to mass spectrometry
analysis resulting in the recovery of 32 major peptide
masses, which were used to search the NRDB protein database for possible matches. One protein in the database, human p120ctn, matched to 27 of the 32 peptide masses
(Fig. 9 B). p120ctn is a src substrate that has recently been
demonstrated to bind to the cadherin complex (Reynolds
et al., 1994; Shibamoto et al., 1995
; Staddon et al., 1995
).
Some of the peptides showed mixed mass spectra after
separation by HPLC, suggesting the presence of other
peptide sequences. Two of these peptides were therefore subjected to NH2-terminal protein sequence analysis to
determine the possible presence of another protein. However, both sequences matched perfectly with p120ctn.
|
To confirm that p120ctn binds to GST-FL and GST-Prox,
western blot analysis of the proteins in HCT116 lysates
that associated with the different GST-fusion proteins was
performed. Indeed, both GST-FL and GST-Prox, but not
GST alone, bind the 92-kD isoform of p120ctn, the major
isoform expressed in HCT116 cells (Mo and Reynolds, 1996), using a mAb to p120ctn that recognizes all major isoforms (results not shown).
We then sought to confirm that this association also occurred in CHO cells. First, GST fusion proteins bound to glutathione beads were used to isolate proteins from lysates of untransfected CHO cells. As shown in Fig. 10 A both the high molecular mass and lower molecular mass isoforms of p120ctn bound to GST-FL and GST-Prox, but not to GST alone. We then wanted to determine whether p120ctn indeed associated with CT669. Therefore, p120ctn was immunoprecipitated from CHO cells transfected with full-length or mutant C-cadherin. Samples were analyzed by immunoblotting using a pAb that recognized the extracellular domain of C-cadherin. Both full-length C-cadherin and CT669 were found to be associated with p120ctn whereas neither CT-CAT nor CT were detected in the p120ctn immunoprecipitates (Fig. 10 B). Similar amounts of C-cadherin protein were expressed in the different cell lysates (Fig. 10 B), excluding the possibility that the lack of detectable association with CT-CAT was due to lower expression levels in the cells.
|
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Discussion |
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This study identifies the 94-amino acid juxtamembrane region of the cytoplasmic tail as a functionally important
part of a classical cadherin molecule. This region is sufficient to support lateral clustering and exerts a significant
influence on adhesive activity. These experiments derive
from our previous demonstration that wild-type C-cadherin expressed in CHO cells clusters in a ligand-specific
manner upon attachment to CEC1-5-coated substrata (Yap et al., 1997b). A role for the cytoplasmic tail in cadherin clustering was inferred from the observation that a
chimeric molecule in which the cadherin cytoplasmic tail
was replaced by tandem repeats of the FK506-binding protein, FKBP12, failed to cluster (Yap et al., 1997b
). Two observations in the current study now identify the juxtamembrane region of the cytoplasmic tail as sufficient to mediate
clustering. First, CT669, a mutant that retains the juxtamembrane region of the cytoplasmic tail, clustered in a
ligand-specific manner when cells attached to substrata
coated with CEC1-5. In contrast, cadherin mutants lacking
the complete cytoplasmic tail (CT) or the juxtamembrane
region alone (CT-CAT) failed to cluster. Second, the
IL2R-669 chimera, but not the parent IL2R
protein, accumulated in surface clusters when expressed in cells.
Therefore, this juxtamembrane region was capable of inducing clustering even without the cadherin ectodomain
and transmembrane regions. Taken together these findings indicate that the juxtamembrane region of the cadherin cytoplasmic tail is sufficent for clustering activity.
Our analysis of the cadherin mutants also provides compelling evidence that this juxtamembrane region of the cytoplasmic tail contributes to adhesive function. Thus, the
CT669 mutant supported significant levels of adhesion
measured in two separate assay systems: a laminar flow
detachment assay and cell aggregation assays. Compared
with the tailless mutation (CT) that supports measurable
adhesion (Brieher et al., 1996), CT669 demonstrated substantially greater levels of adhesive activity in both assays and also showed temporal strengthening in the laminar
flow assay. Cells expressing CT669, like C-CHO cells, also
spread more extensively on CEC-coated substrata than either CT-CHO- or CT-CAT-CHO cells. How then might
significant adhesive strength be supported without the
important contribution of the
-catenin-binding region?
Recently, we demonstrated that forced clustering of the
cadherin ectodomain alone can substantially strengthen
adhesion independently of cytoskeletal or signaling events
mediated by the cytoplasmic tail (Yap et al., 1997b
).
Therefore, we propose that the juxtamembrane region
contributes to adhesion by supporting the accumulation of
cadherin molecules in clusters after initial ligand binding.
In this scheme, changes in adhesive strength would occur
through the redistribution of adhesive receptors on the
cell surface, a process driven, in turn, by the clustering activity of the juxtamembrane cytoplasmic tail.
A role for the juxtamembrane region in cell adhesion
had previously been suggested by reports that a truncation
mutant of VE-cadherin retaining this region could support
aggregation (Navarro et al., 1995) and by the potent dominant inhibitory activity of this region alone when expressed in early Xenopus embryos (Kintner, 1992
). Our
current findings, indeed, provide a potential explanation for this inhibitory effect in Xenopus embryos. Overexpression of the juxtamembrane region alone could inhibit adhesion by competing with corresponding regions of wild-type
cadherins for protein-protein interactions that mediate
clustering.
Many studies have established a clear role for catenins
and the -catenin-binding domain in cadherin-based adhesion (Nagafuchi and Takeichi, 1988
, 1989
; Ozawa et al.,
1990
; Hirano et al., 1992
; Kintner, 1992
; Hermiston and
Gordon, 1995
; Lee and Gumbiner, 1995
; Cox et al., 1996
;
Kofron et al., 1997
; Torres et al., 1997
). Earlier studies,
based on cell aggregation assays, did not, however, identify a discrete functional role for the juxtamembrane region (Nagafuchi and Takeichi, 1988
, 1989
; Ozawa et al.,
1990
). Several factors may account for this disparity between the current and earlier reports. First, our laminar
flow detachment assay may measure different biophysical
parameters of adhesion than aggregation assays. In particular, the kinetics (on- and off-rates) of the adhesive interaction may influence aggregation assays more than the
flow detachment assay (Kuo et al., 1997
). Nonetheless, the observation that CT669 mediated aggregation to a significant extent indicates that assay characteristics cannot
solely account for the discrepancy with earlier findings.
Second, Xenopus C-cadherin may differ from the mammalian E-cadherins studied in earlier reports. There is, indeed, accumulating evidence that cadherins differ in their
functional properties (Levine et al., 1994
; Marrs et al.,
1995
; Islam et al., 1996
) that may be due both to differences in homophilic binding characteristics (Levine et al.,
1994
) and also to apparently small divergent regions of the
cytoplasmic tails (Marrs et al., 1995
). Third, these cadherins were studied in different cellular expression systems. CHO cells seem to support higher levels of cadherin
protein expression than mouse L cell fibroblasts (unpublished results) that can influence adhesive activity (Angres
et al., 1996
; Yap et al., 1997b
). Interestingly, in another
study that used CHO cells an adhesive contribution of the
juxtamembrane region of VE-cadherin was also reported
(Navarro et al., 1995
). In all probability a combination of
these factors are likely to have facilitated detection of the
functional activity of the juxtamembrane region in the current experiments.
Since the CT669 and IL2R-669 mutants do not bind
-catenin, this further suggests that
- and
-catenin are
not required for cadherin clustering. In this regard, we also
found that a construct lacking the juxtamembrane region
but retaining the
-catenin-binding site of the cytoplasmic
tail (CT-CAT) failed to cluster, implying that the juxtamembrane region of the cytoplasmic tail might be solely
responsible for cadherin clustering. However, our CT-CAT mutant, containing a 5x-myc tag spacer, was relatively weakly active in functional assays, compared with
earlier findings that the
-catenin-binding region was sufficient to support significant cell aggregation activity
(Ozawa et al., 1990
). Consequently, we cannot be certain
that our CT-CAT mutant retained all the functional activity of the catenin-binding region. Nonetheless, CT-CAT
possessed strong catenin-binding activity, demonstrating
that
-catenin-binding alone is not sufficient to mediate
cadherin clustering. Therefore, although we cannot exclude an additional role of the
-catenin-binding region,
our data provide strong evidence that the juxtamembrane region of the cytoplasmic tail mediates distinct interactions that are experimentally sufficient to induce cadherin
clustering.
The juxtamembrane region of the cadherin cytoplasmic
tail could support clustering either through direct lateral
interactions or through the participation of intermediary
binding proteins. To date, we have been unable to identify
any biochemical evidence for direct lateral interactions between the juxtamembrane regions of cadherin molecules
using purified proteins that have been studied under a variety of binding conditions. Although this does not definitively exclude a role for direct lateral interactions, it seems
more likely that other, intermediary binding proteins are necessary for clustering. Our current experiments suggest
that p120ctn is a possible candidate to perform this role. It
was isolated as the major protein that bound to the juxtamembrane region of the cadherin cytoplasmic tail, using
a biochemical approach designed to identify as many associating proteins as possible. Further experiments will be
needed to address whether p120ctn is required for cadherin
clustering. p120ctn has been shown previously to interact
with the cadherin complex independently of the -catenin-binding site (Reynolds et al., 1994
; Shibamoto et al.,
1995
; Staddon et al., 1995
; Lampugnani et al., 1997
). However, the role of p120ctn in cadherin function has not been
established. Although this protein is a substrate for several
kinases and the amount associated with the cadherin complex may vary in different cell types, it is unclear whether
this variation is regulated nor what role it plays in adhesion (Daniel and Reynolds, 1997
).
Interestingly, the clustering activity of the juxtamembrane region also appears to be regulated by the extracellular domain. Whereas clustering of the IL2R-669 chimera
occurred in a ligand-independent manner, clustering of
wild-type C-cadherin and of the CT669 cadherin mutant
was strictly ligand-dependent. In one scenario, the unligated ectodomain might inhibit the clustering activity of
the cytoplasmic tail, which is then disinhibited upon ligand
binding to the ectodomain. A similar sequence of events,
where ligand-binding releases an inhibitory influence of
the ectodomain, has been implicated in the ligand-dependent targeting of 1-integrins into focal adhesions (LaFlamme et al., 1992
) and activation of signaling by the
FGF receptor (Webster and Donoghue, 1997
).
These and other recent findings (Brieher et al., 1996;
Yap et al., 1997b
) point to a model of cadherin function in
which the homophilic binding activity of the ectodomain is
strengthened by the clustering activity of the cytoplasmic
tail (Yap et al., 1997a
). This clustering activity may be essential to convert the apparently weak intrinsic binding activity of the ectodomain into a state capable of supporting
physiological adhesion. Furthermore, alterations in the degree of clustering (the distribution of adhesive receptors
presented at the cell surface) supported by the cadherin cytoplasmic tail provide a potential mechanism by which
surface adhesiveness can be regulated in response to cellular signals.
Finally, our experiments raise questions about the mechanistic contributions to adhesion served by the juxtamembrane and catenin-binding regions of the cytoplasmic tail.
It is possible that both regions participate in the same or
similar molecular mechanisms for cell adhesion. Alternatively, it is interesting to consider the possibility that these
regions might mediate mechanistically distinct contributions to adhesion. For example, the juxtamembrane region
may be principally responsible for clustering, whereas the
catenin-binding region may be the major link to the actin
cytoskeleton. Actin association might stabilize clusters or
be more important for supporting morphogenetic responses to adhesion, such as compaction and epithelialization (Watabe et al., 1994). In addition, the catenin complex
is likely to participate in signal transduction mechanisms
that regulate cadherin-based adhesion in response to
growth factors and other physiological cues (reviewed in
Yap et al., 1997a
). With the identification of the juxtamembrane region as a distinct contributor to cadherin function, it should be possible to dissect more thoroughly
the specific mechanistic contributions of cadherins and
catenins and determine how they are integrated to form
fully functional adhesive complexes at the cell surface.
![]() |
Footnotes |
---|
Dr. Yap's present address is Department of Physiology and Pharmacology, The University of Queensland, St. Lucia, Brisbane, Australia 4072. E-mail: yap{at}plpk.uq.oz.au
Received for publication 6 October 1997 and in revised form 17 March 1998.
A.S. Yap and C.M. Niessen contributed equally to the work described in this paper.We thank Dr. James Marrs for a generous gift of antibodies and Dr. Susan
LaFlamme for the IL2 receptor plasmid. This work would not have been
possible without the support, imagination, and intellectual energy of our
colleagues, Bill Brieher, Francois Fagotto, Cara Gottardi, Kathleen
Guger, and all the members of the Gumbiner lab who showed us the way
out of the harbor.
We thank the Sloan-Kettering Structural Chemistry Laboratory (supported by grant 5P309CA05746 from the NCI), especially Drs. Hediye Erdjument-Bromage and Paul Tempst, for carrying out the combined analysis of mass spectrometry and amino acid sequencing. Confocal microscopy was performed at the Confocal Microscopy Facility of the University of Queensland, established by grants from the Australian Research Council, and maintained by Mr. Colin Macqueen. This work was supported by National Institutes of Health grant GM52717 awarded to B.M. Gumbiner, the Cancer Center Support grant NCI-P30-CA-08748, and in part by funds from the Dana Fund. A.S. Yap was also supported by the National Health and Medical Research Council of Australia. C.M. Niessen was supported by The Dutch Cancer Society.
![]() |
Abbreviations used in this paper |
---|
CT, cytoplasmic tail; VE, vascular endothelial.
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