From the Department of Medicine and Pathophysiology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan
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ABSTRACT |
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The gap junction protein connexin-43 is normally
located at the intercalated discs of cardiac myocytes, and it plays a
critical role in the synchronization of their contraction. The
mechanism by which connexin-43 is localized within cardiac myocytes is
unknown. However, localization of connexin-43 likely involves an
interaction with the cytoskeleton; immunofluorescence microscopy showed
that in cardiac myocytes, connexin-43 specifically colocalizes with the
cytoskeletal proteins ZO-1 and -spectrin. In transfected HEK293
cells, immunoprecipitation experiments using coexpressed epitope-tagged
connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with
-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity
binding assays with deleted ZO-1 and connexin-43 fusion proteins.
Immunoblot analysis of associated proteins showed that the C-terminal
domain of connexin-43 binds to the N-terminal domain of ZO-1. The role
of this linkage in gap junction formation was examined by a
dominant-negative assay using the N-terminal domain of ZO-1.
Overexpression of the N-terminal domain of ZO-1 in
connexin-43-expressing cells resulted in redistribution of connexin-43
from cell-cell interfaces to cytoplasmic structures; this intracellular
redistribution of connexin-43 coincided with a loss of electrical
coupling. We therefore conclude that the linkage between connexin-43
and
-spectrin, via ZO-1, may serve to localize connexin-43 at the
intercalated discs, thereby generating functional gap junctions in
cardiac myocytes.
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INTRODUCTION |
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Gap junctions are aggregates of channels at cell-cell interfaces (1-3). Each channel is formed through the docking of two hemichannels located in opposing cell membranes, and each hemichannel is composed of a connexin homohexamer. By permitting the direct exchange of ions and small molecules between cells, these channels play a major role in a wide variety of cellular processes, including embryogenesis, cellular differentiation and development, and electrical coupling. In heart, gap junctions are a prominent feature of intercalated discs, which connect myocytes in an end-to-end orientation; the coupling provided by gap junctions serves to synchronize the activity of cells, thus providing an isochronous front for the wave of excitation that sweeps through ventricular muscle (4, 5).
How gap junctions are localized at the intercalated discs in cardiac
myocytes is unknown, but a component of gap junctions, connexin-43, may
interact with specific elements of the cytoskeleton that restrict its
diffusion in the plane of the membrane. Recent studies indicate that a
number of membrane proteins are anchored by cytoskeletal elements such
as PSD-95/SAP90 (6), ankyrin (7), and -spectrin (8). ZO-1, which has
been identified at vertebrate tight junctions (9, 10), is thought to
play a role in tissue compartmentalization and in maintaining the
apical-basolateral polarity of epithelial cells. In cardiac myocytes,
ZO-1 appears at the intercalated discs in the immediate vicinity of the
plasma membrane (11-13). Since ZO-1 is tightly associated with
-spectrin (11, 12), which is an actin-linking protein (14), it is possible that ZO-1 acts as an adapter mediating the anchoring of
membrane proteins to the cytoskeleton.
We recently demonstrated that when connexin-43 proteins are expressed in HEK293 cells, which otherwise lack gap junctional communication, the cells are able to form gap junctions at cell-cell interfaces (15). This finding serves as the basis for our continued investigation of the functional role of selected protein domains in gap junctional communication as well as the mechanism of gap junction formation. In this study, we examined how the gap junction protein connexin-43 localizes to the intercalated discs in cardiac myocytes. Two assay systems were used to determine the linkage between connexin-43 and cytoskeletal proteins: a biochemical approach involving coprecipitation and binding of tagged proteins and a functional approach involving a dominant-negative assay using a deletion construct of ZO-1.
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EXPERIMENTAL PROCEDURES |
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Construction of Epitope-tagged Connexin-43 and ZO-1 cDNAs-- Full-length rat connexin-43 cDNA (16) and mouse ZO-1 cDNA (12) were cloned by reverse transcription-PCR1 using mRNA isolated from rat or mouse heart (see Fig. 1). For immunoprecipitation, cDNAs were modified to encode the epitopes for monoclonal antibodies at the C-terminal ends of the proteins. For the construction of rat connexin-43 cDNA, the sense PCR primer was designed to contain the known sense sequences of connexin-43 and an EcoRI site at the 5'-end. The antisense PCR primer was designed to delete the endogenous stop codon in the cDNA encoding connexin-43 and replace it with a FLAG tag encoding nucleotides with an EcoRI site at the 5'-end. Thus, the sequence at the C terminus of connexin-43 that normally ends as DLEI now became DLEI-GSDYKDDDDKN, which binds to a mouse monoclonal anti-FLAG IgG antibody. The PCR product called connexin-43-FLAG was excised by EcoRI and ligated into Bluescript KS(+); its sequence was then verified by nucleotide sequencing.
For the construction of mouse ZO-1 cDNA, four domains of ZO-1 (d1, amino acids 1-362; d2, amino acids 358-839; d3, amino acids 836-1257; and d4, amino acids 1254-1745) were first amplified by PCR. The sense PCR primers were designed to contain an EcoRI site at the 5'-end. The antisense PCR primers were, in turn, designed to contain the c-Myc tag encoding nucleotides with an EcoRI site at the 5'-end. The sequence at the C terminus of ZO-1 was GSEQKLISEEDL, which binds to a mouse monoclonal anti-c-Myc IgG antibody. The PCR products were excised with EcoRI and then ligated into Bluescript KS(+). After verification of the nucleotide sequences, the four domains were excised and ligated to produce the full-length ZO-1-Myc cDNA using the following endogenous restriction endonuclease sites: AccI (nucleotide 1075) in d1 and d2, AccI (nucleotide 2510) in d2 and d3, and BamHI (nucleotide 3762) in d3 and d4. For the transfection experiment, the full-length cDNA encoding modified connexin-43-FLAG was ligated into the EcoRI site of a pcDNA3 mammalian expression vector containing a neomycin (G418)-resistant gene as a dominant selectable marker (Invitrogen). The full-length cDNAs encoding modified ZO-1-Myc and d1 of ZO-1, called ZO-1-d1-Myc, were ligated into the EcoRI site of a pZeoSV expression vector (Invitrogen) containing a Zeocin-resistant gene as a dominant selectable marker.Construction and Purification of T7-tagged Connexin-43 Fusion
Proteins--
To facilitate cloning, cDNAs encoding cytoplasmic
domains of connexin-43 (N, amino acids 1-23; II-III, amino acids
95-150; C1, amino acids 227-382; C2, amino acids 227-302; and C3,
amino acids 303-382) were amplified by PCR using sense primers with an
EcoRI site at 5'-end and antisense primers with an
XhoI site at the 5'-end (Fig.
1). Each fragment was excised with
EcoRI and XhoI and then ligated in frame into a
pET28a vector (Novagen). Clones were transfected into Escherichia
coli BL21(DE3) pLysS; overnight cultures were diluted 1:10,
incubated for 2 h, and induced for 3 h with 1 mM
isopropyl--D-thiogalactopyranoside. Expressed proteins
carrying T7 and His epitope tags encoded in the N-terminal site of the
vector were affinity-purified on His-Bind columns (Novagen) as
described in the manufacturer's instructions.
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Construction and Purification of GST-ZO-1 Fusion
Proteins--
Four domains of ZO-1-Myc (d1, d2, d3, and d4) in
Bluescript KS(+) were excised using EcoRI and then ligated
in frame into pGEX-3X vectors (Amersham Pharmacia Biotech). After
expression in E. coli DH5, GST-ZO-1 domain fusion
proteins were purified by affinity chromatography on
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) as described in
the manufacturer's instructions.
Cell Culture-- Rat neonatal cardiac myocytes were prepared as described previously (17). Briefly, the hearts were isolated from 1-day-old HLA-Wistar rats. The ventricular portions were minced, and the cells were dispersed by digestion with 0.1% collagenase at 37 °C. The dispersed cells were resuspended in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum and 10 µg/ml bromodeoxyuridine and preplated onto culture dishes for 30 min to remove fibroblasts. Cells were then plated on glass coverslips to an initial density of 1 × 105/ml and maintained at 37 °C under a 5% CO2 atmosphere. Gap junction-incompetent HEK293 cells were grown in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum and penicillin at 37 °C under a 5% CO2 atmosphere.
Stable Coexpression of Connexin-43-FLAG and Truncated ZO-1-Myc in HEK293 Cells-- HEK293 cells were transfected with the pcDNA3 vector containing connexin-43-FLAG cDNA using the calcium phosphate precipitation technique. For purposes of selection, the transfected cells were grown in medium containing 800 µg/ml G418. Each of the clones selected with G418 was further analyzed by Northern blot and immunoblot analyses. Clones expressing connexin-43-FLAG were then transfected with pZeoSV vectors containing ZO-1-Myc or ZO-1-d1-Myc cDNAs, and the transfected cells were grown for selection in medium containing 250 µg/ml Zeocin and 400 µg/ml G418. Each clone selected with Zeocin and G418 was analyzed by Northern blot and immunoblot analyses.
Transient Coexpression of ZO-1-Myc or ZO-1-d1-Myc with Plasmid pGreen Lantern-1 in Cardiac Myocytes-- Cardiac myocytes plated on coverslips were cotransfected with plasmid pGreen Lantern-1 (Life Technologies, Inc.) and pZeoSV vectors containing either ZO-1-Myc or ZO-1-d1-Myc cDNAs at a ratio of 1:5. Twenty-four hours after transfection, the myocytes were examined under a fluorescence microscope to identify GFP-positive cells, which presumably coexpressed ZO-1-Myc or ZO-1-d1-Myc proteins. Transfected GFP-positive cell pairs were then subjected to electrophysiological analysis.
Immunofluorescence Analysis--
Rat cardiac myocytes were grown
on glass coverslips for 3 days, fixed with 3% paraformaldehyde for 10 min, and then permeabilized with 0.1% Triton X-100 for 10 min. After
blocking with 5% bovine serum albumin in phosphate-buffered saline for
30 min, cells were incubated for 2 h with mouse monoclonal
anti-connexin-43 IgG antibodies (Transduction Laboratories, Inc.),
rabbit monoclonal anti-ZO-1 IgG antibodies (Zymed
Laboratories Inc.), or rabbit monoclonal anti--spectrin IgG
antibodies (Transformation Research, Inc.). Primary antibody-bound
connexin-43 complexes were visualized by incubation with biotinylated
anti-mouse IgG antibodies (Vector Labs, Inc.) for 1 h, followed by
incubation with fluorescein isothiocyanate-conjugated streptavidin
(Vector Labs, Inc.) for an additional 1 h. Primary antibody-bound
ZO-1 complexes were visualized using biotinylated anti-rabbit IgG
antibodies (Cappel Inc.), followed by fluorescein isothiocyanate-conjugated streptavidin. Primary antibody-bound
-spectrin complexes were visualized using rhodamine-conjugated anti-rabbit IgG antibodies (Cappel Inc.). Coverslips were then mounted
in Mowiol 4-88 (Vector Labs, Inc.). The cells were photographed on an
Olympus Provis AX80 microscope fitted with the appropriate filters.
Protein Immunoblot Analysis-- Affinity-purified, T7-tagged connexin-43 domain fusion proteins and GST-ZO-1 domain fusion proteins were solubilized in SDS loading buffer, resolved on SDS-polyacrylamide gels, and transferred to nitrocellulose by electrophoresis. Transfected HEK293 cells were harvested, pelleted with a microcentrifuge, and then incubated in lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The lysates were then solubilized in SDS loading buffer and transferred to nitrocellulose. The nitrocellulose blots were incubated with primary antibodies against target proteins. They were then washed three times with Tris-buffered saline containing 0.1% Tween 20; incubated with peroxidase-labeled, affinity-purified antibodies against primary antibodies; washed again; and then developed using an enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech).
Coimmunoprecipitation Analysis--
Cells overexpressing
connexin-43-FLAG and ZO-1-Myc were collected, washed with
phosphate-buffered saline, and incubated in lysis buffer for 30 min at
4 °C. Cell lysates were incubated with 0.1% albumin-coated protein
A-Sepharose for 2 h at 4 °C and then clarified by
centrifugation at 15,000 × g for 15 min. The
supernatants were incubated in a rotating vessel with monoclonal
anti-FLAG or anti-c-Myc antibodies bound to protein A-Sepharose for
2 h at 4 °C. After incubation, immunoprecipitates were
extensively washed with lysis buffer. Samples were divided into three
equal aliquots and subjected to immunoblot analysis with anti-FLAG, anti-c-Myc, or anti--spectrin IgG antibodies. ECL and
peroxidase-conjugated secondary antibodies against mouse IgG antibodies
were used to visualize primary antibody-antigen complexes.
Affinity Binding Assay-- GST or GST-ZO-1 domain fusion proteins (d1, d2, d3, and d4), bound to glutathione-Sepharose 4B beads, were extensively washed with phosphate-buffered saline and then with binding buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 5% bovine serum, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). T7-tagged connexin-43 domain fusion proteins (N, II-III, C1, C2, and C3) were purified on His-Bind columns, concentrated using an Amicon concentrator 10, and re-equilibrated to a concentration of ~1 µg/µl in binding buffer. Approximately 100 µl of GST-ZO-1 domain fusion proteins, bound to glutathione beads, were incubated in a rotating vessel with 100 µl of purified T7-tagged connexin-43 domain fusion proteins for 2 h at 4 °C. The beads were then extensively washed with binding buffer. Finally, associated proteins were eluted with SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. After transfer to nitrocellulose, immunoblot analysis was performed either with anti-T7 tag IgG antibodies against connexin-43 domain fusion proteins or with anti-c-Myc IgG antibodies against GST-ZO-1 domain fusion proteins. Primary antibody-antigen complexes were visualized using ECL and peroxidase-conjugated secondary antibodies against mouse IgG antibodies.
Electrophysiology--
Gap junctional current
(Ij) was measured using a Geneclamp 500 amplifier (Axon Instruments, Inc.) and a double whole-cell patch-clamp
procedure (15, 18, 19). Pairs of stable HEK293 cells, overexpressing
connexin-43-FLAG or connexin-43-FLAG plus ZO-1-d1-Myc, were obtained by
freshly dissociating pure populations from confluent cultures and
aliquoting them onto 1-cm-diameter glass coverslips. Pairs of cardiac
myocytes, overexpressing ZO-1-Myc or ZO-1-d1-Myc (GFP-positive), were
selected by fluorescence microscopy. The coverslip was transferred to
the stage of a Nikon Diaphot microscope, where experiments was
performed at room temperature while exchanging the bath solution (133 mM NaCl, 3.6 mM KCl, 1.0 mM
CaCl2, 0.3 mM MgCl2, 16 mM glucose, and 3.0 mM HEPES (pH 7.2)). Patch
pipettes were made on a Narishige NA-9 vertical puller and filled with
solution containing 10 nM free Ca2+ (135 mM CsCl, 0.5 mM CaCl2, 2 mM MgCl2, 5.5 mM EGTA, and 5.0 mM HEPES (pH 7.2)). High resistance seals
(>109 ohms) were formed on each cell with the aid of
gentle suction, and access to the cell interior was subsequently
obtained by briefly applying strong suction to the patch pipette. Cells
were voltage-clamped at holding potentials of 40 mV, and 10-mV
voltage pulses were applied to each cell of the pair. Within each pair
of cells, Ij values were measured as the
currents recorded from one cell when voltage steps
(Vj) were applied to the other cell. Junctional conductance (Gj) was calculated from the
following equation: Gj = Ij/Vj.
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RESULTS |
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Localization of Connexin-43, ZO-1, and -Spectrin in Cardiac
Myocytes--
Immunofluorescent localization of connexin-43, ZO-1, and
-spectrin was examined in cultured cardiac myocytes. Immunolabeling of the intercalated disks indicated that all three proteins colocalized in this region (Fig. 2).
-Spectrin
immunolabeling also appeared as a striation pattern throughout the
cytoplasm.
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Association among Connexin-43, ZO-1 and -Spectrin--
To
examine whether connexin-43 forms a complex with ZO-1 and
-spectrin,
a biochemical assay involving the expression of tagged proteins in
HEK293 cells and the subsequent coprecipitation of these proteins was
used. We cotransfected HEK293 cells with pcDNA3 vectors containing
connexin-43-FLAG cDNAs and with pZeoSV vectors containing ZO-1-Myc
cDNAs. The resultant cell line overexpressed connexin-43-FLAG and
ZO-1-Myc proteins, which were detected at cell-cell interfaces by
immunofluorescent assay (Fig.
3A). These transfected cells
were then solubilized with 1% Triton X-100, and the soluble cell
lysate was immunoprecipitated with either anti-FLAG or anti-c-Myc IgG
antibodies. The precipitates were then immunoblotted with anti-c-Myc,
anti-FLAG, or anti-
-spectrin IgG antibodies. As shown in Fig.
3B, ZO-1-Myc and
-spectrin could be immunoprecipitated by
anti-FLAG IgG antibodies, which indicates that ZO-1-Myc and
-spectrin associate with connexin-43-FLAG. Similarly,
connexin-43-FLAG and
-spectrin could be immunoprecipitated by
anti-c-Myc IgG antibodies. Taken together, these results indicate that
connexin-43 forms a complex with ZO-1 and
-spectrin in the transfected cells.
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Identification of Associating Regions of Connexin-43 and ZO-1-- To define the regions mediating the association between connexin-43 and ZO-1, binding assays were performed using the batch method and bacterially expressed fusion proteins. Purified GST-ZO-1 fusion proteins, encoding distinct domains of ZO-1-Myc (d1, d2, d3, and d4) and coupled to glutathione-Sepharose 4B beads, served as the substrate for binding purified T7-tagged connexin-43 fusion proteins (N, II-III, C1, C2, and C3). After incubation and washing, the proteins associated with ZO-1 fusion proteins were resolved by SDS-polyacrylamide gel electrophoresis. Immunoblots of proteins eluted with anti-T7 tag IgG antibodies revealed that the C1 and C3 domains of connexin-43 bind to d1 of ZO-1 (Fig. 4A).
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Effects of a Dominant-negative Construct of ZO-1 on Gap Junction
Formation--
The biochemical approach revealed a direct
protein-protein linkage among connexin-43, ZO-1, and -spectrin, but
it left open the question of the functional significance of this
interaction. We previously demonstrated that when connexin-43 is
expressed in HEK293 cells, gap junctions between transfected cell pairs are reconstituted (15). Using immunoblotting and anti-ZO-1 IgG antibodies, we have now identified endogenous ZO-1 in HEK293 cells (Fig. 5). Therefore, we hypothesized that
endogenous ZO-1 may play a role in the clustering of expressed
connexin-43 at cell-cell interfaces and in the construction of the
functional gap junctions in vivo. To test this idea, we used
a dominant-negative approach (20). Biochemical assays had already
revealed that the N-terminal region of ZO-1 (d1) binds to connexin-43,
but not to
-spectrin (Fig. 4). In an attempt to disrupt the
interaction between endogenous ZO-1 and expressed connexin-43, we
generated stable HEK293 cells that overexpressed connexin-43-FLAG alone
or in combination with ZO-1-d1-Myc (Fig. 5). The effect of ZO-1-d1-Myc
on gap junctions reconstituted from expressed connexin-43-FLAG was then
analyzed. Immunoblots showed that connexin-43-FLAG proteins were
expressed to almost the same degree in both cell lines and that
ZO-1-d1-Myc proteins were expressed in larger amounts than endogenous
ZO-1 (Fig. 5A). The distributions of connexin-43-FLAG and
ZO-1-d1-Myc were then localized by immunofluorescence using anti-FLAG
and anti-c-Myc IgG antibodies, respectively. In cells not expressing ZO-1-d1-Myc, the connexin-43-FLAG proteins were distributed at cell-cell interfaces (Fig. 5B). On the other hand,
connexin-43-FLAG exhibited a markedly different pattern of localization
in cells coexpressing ZO-1-d1-Myc; aggregates were observed in the
perinuclear region, which is consistent with the distribution of
coexpressed ZO-1-d1-Myc in cytoplasmic structures. Localization of
endogenous N-cadherin at the cell-cell interfaces was not affected by
ZO-1-d1-Myc. These results indicate that ZO-1-d1-Myc lacks the ability
to localize at the cell-cell interfaces, and moreover, it inhibits the
transport of connexin-43-FLAG to the cell membrane.
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Effects of Dominant-negative Constructs of ZO-1 on Gap Junctional Conductance-- To assess the effect of ZO-1-d1-Myc on the conductance properties of connexin-43-mediated gap junctions, whole-cell voltage-clamp recordings were obtained from pairs of transfected HEK293 cells (Fig. 6A). Repetitive 10-mV pulses were applied to pairs of voltage-clamped cells, and Gj was measured as described under "Experimental Procedures" (19). In control HEK293 cells, Gj was typically below the level of detectability (<20 picosiemens). However, expression of connexin-43-FLAG markedly increased Gj to 45.9 ± 18.6 nanosiemens (n = 16), which is compatible with the reported conductance of connexin-43-expressing SKHep1 cell pairs (21, 22). Exposing voltage-clamped cell pairs to solution containing octanol, an inhibitor of gap junctional communication (19), reduced the junctional conductance. Thus, the gap junctions reconstituted from expressed connexin-43-FLAG proteins appear to be functional. In contrast, when ZO-1-d1-Myc was coexpressed with connexin-43-FLAG, Gj was decreased to control levels (Fig. 6A).
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DISCUSSION |
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In this study, biochemical analysis demonstrated that connexin-43 is directly linked to ZO-1 in cardiac myocytes and that the linkage involves an interaction between the C-terminal domain of connexin-43 and the N-terminal domain of ZO-1. Furthermore, dominant-negative assays using a deletion construct of ZO-1 showed that overexpression of the N-terminal domain of ZO-1 inhibits localization of connexin-43 at cell-cell interfaces and that there is a loss of electrical coupling between transfected cell pairs. We therefore propose that the linkage between ZO-1 and connexin-43 may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.
Cytoskeletal proteins play a major role in the regional localization of membrane proteins (6, 8). The N-terminal domain of ZO-1, which participates in the protein-protein interaction with connexin-43, contains repeats of the 90-amino acid PDZ motif (24, 25). PDZ domains have been detected in enzymes and structural proteins concentrated at specialized cell-cell junctions, such as neural synapses and epithelial tight junctions; they were found to bind to a variety of receptors, ion channels, and signaling proteins, anchoring them at their target sites (26-32). A C-terminal (T/S)XV consensus motif in the target protein has been identified as one of the interacting sites for the PDZ domain of PSD-95/SAP90 (26-28, 32). However, several studies, including this one, have shown that the interacting sites of the PDZ domain proteins are not restricted to the (T/S)XV consensus motif (29-31, 33). X-ray crystallography analysis indicated that all PDZ domains may share certain structural elements (25). Nevertheless, binding studies with oriented peptide libraries revealed that different PDZ domains display preferences for distinct targets (34). In this regard, it is noteworthy that the various PDZ domains of multivalent proteins are specialized for distinct functions by associating with different target proteins (32, 33). In this way, the various components of signaling cascades are organized into distinct physical and functional units by multivalent PDZ domain proteins, such as ZO-1, which assemble their target proteins at specialized sites and in close proximity.
The dominant-negative assay using the deletion construct of ZO-1
provides an insight into the functional role of ZO-1 in gap junction
formation. Assembly of gap junctions is a multistage process (35, 36).
The first step is the synthesis of connexin. Next, connexons are formed
by the oligomerization of six connexin monomers during their transport
from the endoplasmic reticulum to the Golgi apparatus; this step is
thought to be critical for gap junction formation. Finally, a connexon
in the plasma membrane of one cell docks with a connexon in an opposing
plasma membrane to produce intercellular channels. In this study,
overexpression of the N-terminal domain of ZO-1, which lacked the
ability to localize at cell-cell interfaces, disrupted the transport of
connexin-43-FLAG to the target site. The precise reason why the
N-terminal domain of ZO-1 is not localized at cell-cell interfaces
remains unknown; perhaps it lacks the ability to interact with
cytoskeletal proteins such as -spectrin. Whatever the reason,
overexpression of the N-terminal domain of ZO-1 inhibits the linkage
between endogenous ZO-1 and connexin-43 apparently by dominantly
forming complexes with connexin-43 in cytoplasmic structures. We
therefore conclude that the transport of connexin-43 to regions of
cell-cell interface is achieved by translocation of associated ZO-1
into close proximity with the cell surface. Furthermore, the cell
adhesion molecule cadherin has also been shown to participate in gap
junction formation (37, 38). Taken together with the findings of a
recent study showing that translocation of ZO-1 to the cell surface is
regulated by cadherins (39), it seems likely that the subcellular
targeting of connexin-43 may be regulated by a cadherin-mediated
signaling pathway.
In conclusion, we showed that ZO-1 functions as an adapter for the transport of connexin-43 in cardiac myocytes. Although occludin has been demonstrated to be another target protein of ZO-1 at the tight junction of epithelial cells (13), it is not yet known how the specificity of ZO-1 for different target proteins is determined in different cell types. The identification of all ZO-1 target proteins as well as the corresponding PDZ domains should provide an experimentally tractable system with which to define the structural basis for the interactions between ZO-1 and its target proteins in vivo.
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ACKNOWLEDGEMENT |
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We are indebted to Dr. Noriyuki Yamada for help in the electrophysiological analysis.
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FOOTNOTES |
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* This work was supported in part by the Japanese Ministry of Education, Science, and Culture.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.
To whom correspondence should be addressed. Tel.: 81-6-879-3273;
Fax: 81-6-879-3279; E-mail: toyofuku{at}mr-path.med.osaka-u.ac.jp.
1 The abbreviations used are: PCR, polymerase chain reaction; GST, glutathione S-transferase; GFP, green fluorescent protein.
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REFERENCES |
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