Characterization of ZO-2 as a MAGUK Family Member Associated
with Tight as well as Adherens Junctions with a Binding Affinity to
Occludin and
Catenin*
Masahiko
Itoh
,
Kazumasa
Morita
§, and
Shoichiro
Tsukita
¶
From the
Department of Cell Biology, Faculty of
Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan and the
§ Department of Dermatology, Faculty of Medicine, Kyoto
University, Sakyo-ku, Kyoto 606, Japan
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ABSTRACT |
ZO-2, a member of the MAGUK family, was thought
to be specific for tight junctions (TJs) in contrast to ZO-1, another
MAGUK family member, which is localized at TJs and adherens junctions (AJs) in epithelial and nonepithelial cells, respectively. Mouse ZO-2
cDNA was isolated, and a specific polyclonal antibody was generated
using corresponding synthetic peptides as antigens. Immunofluorescence
microscopy with this polyclonal antibody revealed that, similarly to
ZO-1, in addition to TJs in epithelial cells, ZO-2 was also
concentrated at AJs in nonepithelial cells such as fibroblasts and
cardiac muscle cells lacking TJs. When NH2-terminal dlg-like and COOH-terminal non-dlg-like domains of ZO-2 (N-ZO-2 and
C-ZO-2, respectively) were separately introduced into cultured cells,
N-ZO-2 was colocalized with endogenous ZO-1/ZO-2, i.e. at
TJs in epithelial cells and at AJs in non-epithelial cells, whereas
C-ZO-2 was distributed along actin filaments. Consistently, occludin as
well as
catenin directly bound to N-ZO-2 as well as the
NH2-terminal dlg-like portion of ZO-1 (N-ZO-1) in
vitro. Furthermore, immunoprecipitation experiments revealed that
the second PDZ domain of ZO-2 was directly associated with N-ZO-1. These findings indicated that ZO-2 forms a complex with ZO-1/occludin or ZO-1/
catenin to establish TJ or AJ domains, respectively.
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INTRODUCTION |
Generation and maintenance of specialized membrane domains are
required for cells to exert their physiological functions, and the
underlying molecular mechanisms of these processes are attracting
increasing interest from cell biologists. As components of the
machinery responsible for membrane specialization, a new gene family
was identified which is now called the MAGUK family (membrane-associated guanylate kinase homologues) (for reviews, see
Refs. 1-3). MAGUKs are multidomain proteins that consist of PDZ, SH3,
and GUK (guanylate kinase-like) domains, and through their direct
association with cytoplasmic domains of integral membrane proteins,
they are thought to be directly involved in clustering of integral
membrane proteins to create specialized membrane domains
(4-7).
Simple epithelial cells contain three specialized membrane domains at
the most apical part of lateral membranes for intercellular adhesion,
tight junctions (TJs),1
adherens junctions (AJs) and desmosomes (8). In these domains, occludin/claudin (9-11), cadherin (12-14), and desmoglein/desmocollin (15, 16) were identified as major integral membrane proteins (adhesion
molecules), respectively, but our knowledge regarding how these
integral membrane proteins are sorted into three distinct junctional
membrane domains is still fragmentary. To date, three MAGUKs have been
shown to be associated with these intercellular junctions, and these
molecules are now called ZO-1, ZO-2, and ZO-3.
ZO-1 was first identified as a peripheral membrane protein with a
molecular mass of 220 kDa and was concentrated at TJs in epithelial
cells (17, 18). However, in nonepithelial cells lacking TJs, such as
cardiac muscle cells and fibroblasts, it was precisely colocalized with
cadherins (19, 20). ZO-1 molecule is roughly divided into two
functional portions: the NH2-terminal half, which shows
similarity to Drosophila lethal (1) discs large-1 (dlg)
consisting of three PDZ, one SH3, and one GUK domain; and the
COOH-terminal half with no sequence similarity to dlg (20-22).
Consistent with the subcellular distribution of ZO-1 in epithelial and
nonepithelial cells, its NH2-terminal dlg-like half bound
directly to the cytoplasmic domain of occludin (23, 24) as well as
catenin (24) that associates with the cytoplasmic domain of cadherin
via
catenin (25-32). However, how ZO-1 is excluded out from AJs in
epithelial cells where
catenin is highly concentrated remains
unclear. In addition, the COOH-terminal non-dlg-like half of ZO-1 was
shown to be directly associated with actin filaments in
vitro as well as in vivo (24, 44).
As compared with ZO-1, our knowledge of ZO-2 is still limited. ZO-2
with a molecular mass of 160 kDa was first identified as a ZO-1-binding
protein by immunoprecipitation with anti-ZO-1 mAb (33). Cloning and
sequencing of dog ZO-2 cDNA revealed that it also contained a
dlg-like domain containing three PDZ, one SH3, and one GUK domain at
its NH2-terminal region, followed by a short COOH-terminal
non-dlg-like domain (34, 35). In contrast to ZO-1, ZO-2 was reported to
be absent from intercalated discs (N-cadherin-based AJs) of cardiac
muscle cells and exclusively concentrated at TJs in epithelial cells
using dog ZO-2-specific pAb (34). ZO-3 was also identified in ZO-1
immunoprecipitate as a phosphorylated 130-kDa peptide (36), and its
cDNA was recently cloned (37).
At the initial phase of junction formation of epithelial cells, ZO-1
was precisely colocalized with cadherins in primordial spot-like AJs,
where TJs were not yet assembled, and then at the later stage, ZO-1 was
transferred from AJs to TJs (38). Although these findings suggest the
direct involvement of ZO-1 in the establishment of two distinct
membrane domains, AJs and TJs, in epithelial cells, lack of information
concerning ZO-2 has hampered more direct assessment of the junction
sorting mechanism at the molecular level. In this study, we isolated a
mouse ZO-2 cDNA and characterized its product in detail.
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EXPERIMENTAL PROCEDURES |
Cloning of Mouse ZO-2 cDNA--
Based on the human cDNA
clone X104, which was later recognized as human ZO-2 cDNA (39), a
partial human ZO-2 cDNA fragment (2897-3241) was obtained by
RT-PCR using mRNA from human T84 cells. This fragment was used as a
probe to screen a mouse lung
ZAP cDNA library. Eleven positive
clones were isolated, one of which, clone 10, contained the entire open
reading frame of mouse ZO-2.
Antibodies--
Anti-ZO-2 pAbs, pAb59 and pAb62, were raised in
rabbits using synthetic peptides corresponding to the mouse ZO-2
sequences encoding amino acids 407-419 and 1093-1108, as antigens,
respectively. These pAbs were affinity purified with the GST-ZO-2
fusion protein that was produced in Escherichia coli,
separated by SDS-PAGE, and transferred onto nitrocellulose membranes.
Mouse anti-ZO-1 mAb (T8-754) and rat anti-
catenin mAb (
18) were
generated and characterized previously (20, 40). Rat anti-E-cadherin
mAb (ECCD-2) and rat anti-P-cadherin mAb (PCD-1) were generously
provided by Dr. M. Takeichi (Kyoto University, Kyoto, Japan). Mouse
anti-c-myc tag mAb and rabbit anti-c-myc tag pAb
were purchased from MBL (Nagoya, Japan). Mouse anti-HA-tag mAb was
purchased from Boehringer Mannheim (Indianapolis, IN).
Constructs and Transfection--
For expression of
NH2-terminally HA-tagged proteins in mammalian cells, an
oligonucleotide encoding an HA epitope was subcloned into the
eukaryotic expression vector pME18S producing pME18S-HA. The cDNA
fragment containing the entire open reading frame of mouse ZO-2 was
produced by PCR. The amplified product was digested with
StuI and SalI, then subcloned into
EcoRV/SalI-cleaved pME18S-HA. The cDNA
fragments encoding the NH2-terminal dlg-like portion (1-938), COOH-terminal non-dlg-like portion (939-1167) (see Fig. 1),
and other deletion mutants of the NH2-terminal dlg-like
portion of mouse ZO-2 (see Fig. 6) were generated by PCR using
appropriate primers and subcloned into pME18S-HA. MDCKII and 3Y1 cells
were transfected with each expression vector and selected as described previously (24).
Immunofluorescence Microscopy--
Cells plated on glass
coverslips were rinsed in PBS and fixed with 1% formaldehyde in PBS
for 15 min at room temperature. The fixed cells were processed for
immunofluorescence microscopy as described previously (24).
Production of the NH2- and COOH-terminal Portions of
ZO-2 by Recombinant Baculovirus Infection--
The cDNA fragment
encoding the NH2-terminal portion (1-938) or COOH-terminal
portion of ZO-2 (939-1167) was generated by PCR as described above.
These fragments were subcloned into the pBlueBac vector (Invitrogen)
and then integrated into the baculovirus genome. The recombinant virus
carrying each cDNA was isolated and condensed using a MAXBAC kit
(Invitrogen). Insect Sf9 cells were infected with recombinant
viruses, and the total cell lysate was prepared as described previously
(24).
In Vitro Binding Assay Using GST Fusion Proteins--
GST fusion
proteins with full-length
catenin (GST-
catenin) or the
cytoplasmic domain of occludin (GST-Oc358) were prepared as described
previously (24). Fusion proteins were expressed in E. coli
and purified using glutathione-Sepharose 4B beads (Pharmacia LKB
Biotechnology, Uppsala, Sweden). Aliquots of 200 µl of
glutathione-Sepharose beads slurry containing GST fusion proteins were
washed with 100 volumes of PBS containing 0.1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, and 4 µg/ml leupeptin
by brief centrifugation, and then 2 ml of the lysate of Sf9
cells expressing N-ZO-1 or N-ZO-2 was added, followed by incubation for
3 h at 4 °C. The beads were then washed with 40 volumes of the
same solution, and bound proteins were eluted with 1 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM
glutathione. The amounts of GST fusion proteins in each eluate were
determined by SDS-PAGE. An appropriate amount of each eluate was again
applied to SDS-PAGE to contain the same amount of GST fusion proteins.
For biotinylation of the Sf9 cell lysate, Sulfo-NHS-biotin
(Pierce) was added to the lysate at a final concentration of 0.5 mg/ml
and incubated for 10 min at room temperature. The reaction was stopped
by adding Tris-HCl (pH 8.0) up to 50 mM. To detect biotinylated proteins bound to GST-
catenin, each eluate was separated by SDS-PAGE, transferred onto nitrocellulose membranes, followed by incubation with alkaline phosphatase-conjugated streptavidin.
Immunoprecipitation--
Confluent monolayers of cultured cells
on 9-cm dishes were washed three times with ice-cold PBS, and then
cells were lysed in 2 ml of extraction buffer (PBS containing 0.5%
Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 2 mg/ml
leupeptin). Cell lysates were clarified by centrifugation at
100,000 × g for 30 min and incubated with 100 µl of
protein G-Sepharose bead slurry (Zymed Laboratories
Inc., San Francisco, CA) coupled with mouse anti-HA mAb for
3 h at 4 °C. As a control, nonspecific mouse IgG was coupled to
the beads. After five washes with the extraction buffer,
immunoprecipitates were eluted with SDS-PAGE sample buffer. For
metabolic labeling of transfectants, cells were washed once with
methionine-free medium supplemented with 2% fetal calf serum and then
incubated with 3 ml of the same medium containing 0.2 mCi
[35S]methionine (Amersham Pharmacia Biotech,
Buckinghamshire, UK) for 3 h before lysis.
Gel Electrophoresis and Immunoblotting--
One-dimensional
SDS-PAGE (10-12.5% gel) was performed based on the method of Laemmli
(41), and immunoblotting was performed as described previously
(21).
 |
RESULTS |
Isolation of Mouse ZO-2 cDNA and Generation of Anti-ZO-2
pAb--
When we began this study, only a partial dog ZO-2 cDNA
had been isolated (34). Similarity searches in data bases identified a
human cDNA (X104) which showed marked similarity to dog ZO-2 cDNA (39). Based on this sequence, we isolated a part of human ZO-2
cDNA by PCR using the first strand cDNA generated from total RNA of cultured human epithelial cells (T84). Using this cDNA fragment as a probe, we screened a
ZAP cDNA library of mouse lung, and obtained a full-length cDNA encoding mouse ZO-2 (data are
available from GenBank/EBI/DDBJ under accession number AF113005). Its
open reading frame encoded a protein of 1,167 amino acids with a
calculated molecular mass of 132 kDa. During the course of this study,
however, a full-length cDNA encoding dog ZO-2 was reported (35).
Mouse ZO-2 was 85% identical to dog ZO-2 at the amino acid sequence
level, and consisted of three PDZ, one SH3, and one GUK domain from the
NH2-terminal end (Fig. 1).

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Fig. 1.
ZO-2 constructs. HA-tagged full-length
ZO-2 (F-ZO-2), HA-tagged NH2-terminal dlg-like
portion of ZO-2 (N-ZO-2), and COOH-terminal non-dlg-like
portion of ZO-2 (C-ZO-2) are schematically drawn. A HA
sequence (HA) was tagged to the NH2 terminus of
each molecule. F-ZO-2 contains three PDZ domains (PDZ1-3),
one SH3 domain (SH3), and one guanylate kinase-like domain
(GUK).
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Based on the deduced amino acid sequence of mouse ZO-2, we synthesized
two polypeptides corresponding to the middle and COOH-terminal end
portions of ZO-2 that showed no sequence similarity to ZO-1 and raised
pAbs (pAb59 and pAb62, respectively) in rabbits using them as antigens.
Because both pAbs showed the same properties on immunoblotting as well
as immunofluorescence microscopy, we will report here only the data
obtained with pAb59. As shown in Fig.
2a, the affinity-purified pAb59 specifically recognized
recombinant ZO-2 but not recombinant ZO-1 produced in Sf9 cells
by baculovirus infection.

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Fig. 2.
Expression of ZO-2. a,
specificity of anti-mouse ZO-2 pAb, pAb59. The lysates of Sf9
cells expressing full-length ZO-1 (arrowhead in ZO-1
lysate) or ZO-2 (arrowhead in ZO-2 lysate)
were immunoblotted with anti-ZO-1 mAb or anti-ZO-2 pAb, pAb59.
CBB, Coomassie Brilliant Blue. b, expression of
ZO-1 and ZO-2 in cultured cells. The total lysates of dog epithelial
cells (MDCK), mouse epithelial cells (MTD-1A),
rat fibroblasts (3Y1), mouse fibroblasts (NIH 3T3,
Swiss 3T3, L), and mouse myeloma cells (P3) were
immunoblotted with anti-ZO-1 mAb or anti-ZO-2 pAb, pAb59.
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Subcellular Distribution of ZO-2 in Comparison with ZO-1 in
Cultured Cells and Tissues--
Using the affinity-purified anti-ZO-2
pAb, we first examined the expression levels of ZO-2 in various
cultured cells by immunoblotting and compared them with those of ZO-1
(Fig. 2b). As previously reported, ZO-1 was detected in all
the cell types examined including epithelial, fibroblastic, and myeloma
cells. ZO-2 was also detected in cultured epithelial cells as well as
in fibroblasts such as 3Y1, NIH 3T3, and Swiss 3T3 cells, but not in L
fibroblasts or P3 myeloma cells.
We next examined the subcellular localization of ZO-2 in cultured MDCK
cells and 3Y1 cells using affinity-purified anti-ZO-2 pAb. In MDCK
cells, as expected from previous studies, ZO-2 was precisely
colocalized with ZO-1 in a linear pattern at cell-cell borders, and
computer-generated cross-sectional views confirmed that ZO-2 was
colocalized with occludin at TJs and concentrated more apically than
E-cadherin (data not shown). Interestingly, ZO-2 was also precisely
colocalized with ZO-1 in a punctate or serrated pattern at cell-cell
borders of 3Y1 fibroblasts lacking TJs
(Fig. 3, a and b).
This characteristic distribution of ZO-2 was identical to P-cadherin
(Fig. 3, c and d). This finding prompted us to
re-examine the expression and subcellular distribution of ZO-2 in
cardiac muscle cells. Frozen sections were doubly stained with
anti-ZO-1 mAb and affinity-purified anti-ZO-2 pAb, and intense signals
of ZO-2 as well as ZO-1 were detected from intercalated discs (Fig. 3,
e and f). This ZO-2 signal was specific because it was not observed on staining with pAb preabsorbed with GST-ZO-2 fusion protein (data not shown).

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Fig. 3.
Colocalization of ZO-2 with ZO-1 in
nonepithelial cells. Rat 3Y1 fibroblasts were doubly stained with
mouse anti-ZO-1 mAb (a)/rabbit anti-ZO-2 pAb (b),
or with rat anti-P-cadherin mAb (c)/rabbit anti-ZO-2 pAb
(d). Frozen sections of rat hearts were doubly stained with
mouse anti-ZO-1 mAb (e) and rabbit anti-ZO-2 pAb
(f). Both ZO-1 and ZO-2 were co-concentrated at intercalated
discs (arrows), whereas ZO-2 signal was very weak from
ZO-1-positive blood vessels (arrowheads). Bars,
10 µm.
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Characterization of Dlg-like and Non-dlg-like Domains of ZO-2 in
Vivo and in Vitro--
Previously, we divided ZO-1 into the
NH2-terminal half dlg-like (N-ZO-1) and COOH-terminal half
non-dlg-like (C-ZO-1) portions and characterized them both in
vivo and in vitro (24). In this study, the full-length
ZO-2 (F-ZO-2) was also divided into the NH2-terminal
dlg-like (N-ZO-2) and COOH-terminal non-dlg-like (C-ZO-2) domains (see
Fig. 1). First, F-ZO-2, N-ZO-2, and C-ZO-2 were tagged with HA peptide
at their NH2 ends and introduced into cultured MDCK cells
as well as 3Y1 cells (Fig. 4). Under
transient expression conditions, immunofluorescence microscopy with
anti-HA mAb revealed that in both MDCK and 3Y1 cells, F-ZO-2 as well as N-ZO-2 were recruited to the cell-cell borders where endogenous ZO-2
was concentrated, i.e. TJs in MDCK cells and
P-cadherin-based spot AJs in 3Y1 cells (Fig. 4,
a-d). In marked contrast, C-ZO-2 appeared to be
colocalized with actin filaments. In 3Y1 fibroblasts, C-ZO-2 was
clearly distributed along stress fibers (Fig. 4f), although
in MDCK cells it was distributed diffusely with some concentration at
cell-cell borders (along circumferential actin bundles) and plasma
membranes (along microvilli) (Fig. 4e).

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Fig. 4.
Subcellular distribution of F-ZO-2, N-ZO-2,
and C-ZO-2 in cultured epithelial (MDCK) and
fibroblastic (3Y1) cells. HA-tagged full-length
ZO-2 (F-ZO-2; a and b), NH2-terminal
dlg-like portion of ZO-2 (N-ZO-2; c and d), and
COOH-terminal non-dlg-like portion of ZO-2 (C-ZO-2; e and
f) were introduced into MDCK (a, c, and
e) and 3Y1 cells (b, d, and f), and
transient expression of these proteins was detected using anti-HA mAb.
Transfected cells are surrounded by untransfected cells that are not
visible by immunostaining. Bars, 10 µm.
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We have previously reported that N-ZO-1 directly binds to the
cytoplasmic domain of occludin as well as
catenin in
vitro (24). The binding of N-ZO-2 to occludin and/or
catenin
was compared with that of N-ZO-1 (Fig.
5). N-ZO-1 and N-ZO-2 were produced in Sf9 cells by baculovirus
infection, and the cell lysate of Sf9 cells containing almost
the same amounts of N-ZO-1 or N-ZO-2 was incubated with a GST fusion
protein with the cytoplasmic domain of occludin (Fig. 5a).
CBB staining of the eluates from the GST/occludin fusion protein beads
revealed that the cytoplasmic domain of occludin directly bound to
N-ZO-2 as well as N-ZO-1. Next, we examined the binding of N-ZO-2 to
catenin, but in this case the electrophoretic mobilities of
recombinant N-ZO-2 (and also that of N-ZO-1) were almost the same as
that of GST-
catenin fusion protein, making it difficult to estimate
the amount of bound N-ZO-2 (and of bound N-ZO-1) by CBB staining (Fig.
5b). Thus, the total proteins of Sf9 cells containing
almost the same amounts of N-ZO-1 or N-ZO-2 were biotinylated, then the
in vitro binding assay with GST-
catenin fusion protein
was performed. The amounts of bound N-ZO-1 and N-ZO-2 were estimated by
detection with alkaline phosphatase-avidin. As shown in Fig.
5b, GST-
catenin fusion proteins bound directly to N-ZO-2
as well as N-ZO-1.

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Fig. 5.
Association of N-ZO-2 with the cytoplasmic
domain of occludin as well as catenin.
a, occludin binding assay. GST/Occ, GST fusion
protein with the cytoplasmic domain of occludin (G);
N-ZO-1 Sf9-lysate, lysate of Sf9 cells
expressing N-ZO-1 (arrow); N-ZO-2
Sf9-lysate, lysate of Sf9 cells expressing N-ZO-2
(arrowhead); GST/Occ-bound N-ZO-1, glutathione
eluate from the mixture of GST/Occ and N-ZO-1 Sf9-lysate;
GST/Occ-bound N-ZO-2, glutathione eluate from the mixture of
GST/Occ and N-ZO-2 Sf9-lysate; GST+N-ZO-1,
glutathione eluate from the mixture of GST and N-ZO-1
Sf9-lysate; GST+N-ZO-2, glutathione eluate from the
mixture of GST and N-ZO-2 Sf9-lysate. b, catenin
binding assay. Biotinylated N-ZO-1 Sf9-lysate,
biotinylated lysates of Sf9 cells expressing N-ZO-1
(arrow); Biotinylated N-ZO-2 Sf9-lysate,
biotinylated lysates of Sf9 cells expressing N-ZO-2
(arrowhead); GST/ cat, GST fusion protein with
catenin (G); GST/ cat-bound N-ZO-1,
glutathione eluate from the mixture of GST/ cat and biotinylated
N-ZO-1 Sf9-lysate; GST/ cat-bound N-ZO-2,
glutathione eluate from the mixture of GST/ cat and biotinylated
N-ZO-2 Sf9-lysate; GST+N-ZO-1, glutathione eluate
from the mixture of GST and biotinylated N-ZO-1 Sf9-lysate;
GST+N-ZO-2, glutathione eluate from the mixture of GST and
biotinylated N-ZO-2 Sf9-lysate. Each eluate was examined by
Coomassie Brilliant Blue staining (CBB staining) or by
detection with alkaline phosphatase-avidin (AP-avidin).
Bars indicate molecular masses of 200, 116, 97, 66, 45, and
31 kDa, respectively, from the top.
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Interaction of ZO-2 with ZO-1--
Because ZO-2 was first
identified in the ZO-1 immunoprecipitate (33), ZO-1 is thought to be
directly associated with ZO-2. Recently, the ZO-2 binding domain on
ZO-1 was narrowed down to its PDZ2 domain (44). We attempted to
identify the ZO-1 binding domain on ZO-2. First, we examined the
in vitro binding of GST fusion proteins with N-ZO-1 or
C-ZO-1 with recombinant N-ZO-2 or C-ZO-2 produced in Sf9 cells,
but we detected no significant binding. These findings suggested that
some modifications on ZO-1 or ZO-2 molecules within cells are required
for the ZO-1/ZO-2 interaction. Therefore, we introduced HA-tagged
F-ZO-2 cDNA into EL cells (L cells transfected with E-cadherin)
expressing myc-tagged N-ZO-1 (NZ-EL cells) or myc-tagged C-ZO-1 (CZ-EL
cells), and F-ZO-2 in the total cell lysate was immunoprecipitated with
anti-HA mAb. As shown in Fig.
6a by immunoblotting with anti-myc mAb, N-ZO-1 but not
C-ZO-1 was co-immunoprecipitated with F-ZO-2, indicating that the
NH2-terminal dlg-like domain of ZO-1 bound to F-ZO-2. When
HA-tagged C-ZO-2 cDNA was introduced into NZ-EL cells or CZ-EL
cells, neither N-ZO-1 or C-ZO-1 was co-immunoprecipitated with C-ZO-2,
indicating that the NH2-terminal dlg-like domain of ZO-2 is
responsible for ZO-1 binding (data not shown). Then, to further narrow
down the domain responsible for the ZO-1 binding, various deletion
constructs of N-ZO-2 were introduced into NZ-EL cells (Fig.
6b). Each construct was tagged with HA at its
NH2-end. Stable transfectants were metabolically labeled
with [35S]methionine and solubilized, and then mutant
ZO-2 produced from these constructs was immunoprecipitated with anti-HA
mAb. As shown in Fig. 6b, N-ZO-1 was co-immunoprecipitated
only when the introduced N-ZO-2 constructs contained the PDZ2 domain,
indicating that the PDZ2 domain of ZO-2 is responsible for ZO-1/ZO-2
interaction.

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Fig. 6.
ZO-1/ZO-2 interaction. a,
association of F-ZO-2 with N-ZO-1. F-ZO-2 (with HA-tag) cDNA was
introduced into NZ-EL (NZ-EL) or CZ-EL (CZ-EL)
cells that expressed E-cadherin and N-ZO-1 or C-ZO-1 (with myc-tag),
respectively. When F-ZO-2 was immunoprecipitated with anti-HA mAb,
N-ZO-1 (N-ZO-1) but not C-ZO-1 was co-immunoprecipitated
with F-ZO-2 (arrowhead), which was confirmed by
immunoblotting with anti-myc pAb. Arrows, IgG. b,
domain of ZO-2 responsible for ZO-1 interaction. Various HA-tagged
deletion N-ZO-2 constructs (as schematically shown above the panel)
were introduced into NZ-EL cells. After stable transfectants were
metabolically labeled with [35S] methionine, each ZO-2
construct (1-7) was immunoprecipitated with anti-HA mAb and
examined by autoradiography. N-ZO-1 (arrowheads) was
co-immunoprecipitated only with bands 2, 5 and 6 which contained the PDZ2 domain.
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 |
DISCUSSION |
ZO-1 is expressed not only in epithelial/endothelial cells but
also in nonepithelial/endothelial cells such as cardiac muscle cells,
fibroblasts, and astrocytes (19, 20, 42) and that, in these
nonepithelial/endothelial cells, ZO-1 is precisely colocalized with
cadherins (19, 20). In contrast to the ubiquitous expression and the
peculiar subcellular distribution of ZO-1, ZO-2, another MAGUK family
member, was reported to be specific for TJs, and to be absent from AJs
in cardiac muscle cells (34). However, in the present study using
anti-ZO-2 pAb, we found that ZO-2 was precisely co-concentrated with
ZO-1 at intercalated discs (AJs). At present, the reason for this
discrepancy remains unclear, but the following observations favored the
notion that ZO-2 is very similar to ZO-1 in terms of AJ-association in
nonepithelial/endothelial cells. First, a human cDNA called X104,
which was later recognized to be human ZO-2 cDNA, was ubiquitously
detected in various tissues and was abundant in heart (39). Second, our
anti-ZO-2 pAb also detected concentration of ZO-2 at the
P-cadherin-based spot-like AJs in cultured fibroblasts, which had not
been examined in the previous report (34). Third, when HA-tagged F-ZO-2
and N-ZO-2 were introduced into cultured fibroblasts, it was correctly
targeted to the P-cadherin-based spot-like AJs.
Our previous study suggested that ZO-1 functions as a cross-linker
between occludin and actin filaments in epithelial/endothelial cells or
between
catenin and actin filaments in nonepithelial/endothelial cells (24). The present study revealed that the cytoplasmic domain of
occludin and
catenin also bound to N-ZO-2 in vitro. Exogenously expressed C-ZO-2 was distributed along stress fibers in
cultured fibroblasts similarly to exogenously expressed C-ZO-1. From
these observations, we concluded that ZO-2 is very similar to ZO-1 also
as a cross-linker. At present, it remains unknown why two similar
cross-linkers, ZO-1 and ZO-2, exist in intercellular junctions such as
AJs and TJs. Furthermore, the recent knockout study of occludin
revealed that ZO-1 is still exclusively concentrated at
occludin-deficient TJs (43). Preliminary experiments revealed that ZO-2
also remained at occludin-deficient TJs (data not shown). These
findings indicate that ZO-1 as well as ZO-2 are recruited to normal TJs
through direct or indirect interactions not only with occludin but also
with other TJ-specific membrane proteins such as the recently
identified claudins (10).
The possible interaction of ZO-1 and ZO-2 would make the relationship
of these two similar cross-linker proteins more complex. Recent
immunoprecipitation analyses indicated that the PDZ2 domain of ZO-1 was
responsible for ZO-1/ZO-2 interaction although it was not clear whether
this interaction was direct or indirect (44). L cells and their
transfectants gave a good model in which to examine the ZO-1/ZO-2
interaction because they lack endogenous expression of ZO-2. In the
present study, immunoprecipitation experiments using metabolically
labeled cells demonstrated that N-ZO-1 binds to the PDZ2 domain of
ZO-2. Of course, although the possibility cannot be completely excluded
that a third protein mediates this binding, these findings favored the
notion that ZO-1 and ZO-2 directly form a heterodimer (or oligomer)
through PDZ2/PDZ2 interaction. This type of PDZ/PDZ interaction has
been reported between neuronal nitric oxide synthase and PSD-95 (or PSD-93) (45).
In this study, two intercellular-associated MAGUK family members, ZO-1
and ZO-2, were compared in detail. The elucidation of the molecular
mechanism behind the peculiar behavior of ZO-1 and ZO-2,
i.e. their respective localization at TJs and AJs in epithelial/endothelial and nonepithelial/endothelial cells, is necessary to better understand the molecular mechanism not only underlying the establishment of intercellular junction domains but also
that behind the polarization of epithelial/endothelial cells. Because
information concerning the molecular components of intercellular
junctions is rapidly accumulating, further analyses of ZO-1 and ZO-2
including knocking-out their genes will lead to a better understanding
of the physiological functions of MAGUK family members in general.
 |
ACKNOWLEDGEMENTS |
We thank all the members of our laboratory
(Dept. of Cell Biology, Faculty of Medicine, Kyoto University) for
helpful discussions. We also thank K. Kamikubo-Tsuchihashi for
excellent technical assistance.
 |
FOOTNOTES |
*
This study was supported in part by a Grant-in-Aid for
Cancer Research and a Grant-in-Aid for Scientific Research (A) from the
Ministry of Education, Science and Culture of Japan (to S. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF113005.
¶
To whom correspondence should be addressed: Dept. of Cell
Biology, Kyoto University Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan. Tel.: 81-75-753-4372; Fax: 81-75-753-4660; E-mail: htsukita{at}mfour.med.kyoto-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
TJ, tight junction;
AJ, adherens junction;
N-ZO-2, NH2-terminal dlg-like domain
of ZO-2;
C-ZO-2, COOH-terminal non-dlg-like domain of ZO-2;
F-ZO-2, full-length ZO-2;
mAb, monoclonal antibody;
pAb, polyclonal antibody;
RT-PCR, reverse transcriptase polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline;
GST, glutathione S-transferase;
HA, hemagglutinin;
MDCK, Madin-Darby canine kidney.
 |
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