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INTRODUCTION |
Gap junction channels permit the transport of low molecular weight
substances between the cytoplasm of neighboring cells. Each gap
junction channel is made of two hemichannels, which are composed of six
subunits called connexins (1). At least 14 different connexin isoforms
have been identified in the mouse. Different tissues express different
combinations of connexins, which might allow the formation of
heteromeric or heterotypic channels and facilitate the modulation of
gap junction permeability in these cells (1). Alterations in gap
junction permeability affect many biological phenomena including
cellular differentiation and development, metabolic homeostasis, and
electrotonic coupling of excitable tissue (2).
Osteoblastic cells express connexin43
(Cx43)1, Cx45 and Cx46 (3).
Although Cx46 is retained within the exocytic pathway in these cells
(4), Cx43 and Cx45 are expressed on the plasma membrane and form gap
junction channels. Cx43 and Cx45 have different molecular
permeabilities, and changes in the relative expression of Cx43 and Cx45
in bone cells can alter the permeability of gap junctions and the
expression of bone matrix proteins (2, 5). These results suggest that
interactions among different gap junction proteins might alter
cell-cell communication in cell networks thereby modulating the
expression of bone matrix proteins and cell activities. However little
is known about the processes through which different gap junction
proteins interact.
One protein that may be involved in the trafficking or organization of
gap junction proteins is the tight junction-associated protein ZO-1.
Cx43 associates with ZO-1 (6, 7), a member of the membrane-associated
guanylate kinase family of proteins, all of which contain three
distinct amino acid motifs that mediate protein-protein interactions:
up to three PSD95/Dlg/ZO-1 (PDZ) domains, an Src homology 3 domain, and
a guanylate kinase domain (8). Membrane-associated guanylate kinases
bind to the carboxyl termini of membrane proteins and to internal
domains of other membrane-associated guanylate kinases, thereby
organizing these proteins into two-dimensional multi-protein complexes
at cell-cell boundaries (9). Mutational analysis of the interaction
between Cx43 and ZO-1 suggests that the second PDZ domain of ZO-1
interacts with the carboxyl-terminal region of Cx43 (6, 7, 10).
To understand the interactions between Cx43 and Cx45 in osteoblastic
cells, in the current work we sought to identify proteins that interact
with Cx45 and in particular wished to determine whether ZO-1 interacts
with Cx45 as well as with Cx43. We found that ZO-1 could be isolated
with Cx45 from ROS/Cx45 cells using a coimmunoprecipitation assay.
Immunofluorescence studies showed that Cx45 colocalized with ZO-1 and
Cx43 in the transfected cells. We found that in vitro
translated ZO-1 bound to an oligopeptide corresponding to the final 12 amino acids in Cx45, suggesting that ZO-1 recognizes the
carboxyl-terminal of Cx45.
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MATERIALS AND METHODS |
Reagents and Plasmid--
Anti-Cx43 (11) and anti-Cx45 antiserum
(5) were previously characterized. Polyclonal and monoclonal antibodies
directed against ZO-1 were obtained from Zymed Laboratories
Inc. (South San Francisco, CA) whereas the monoclonal antibody
directed against Cx43 was purchased from Chemicon (Temecula, CA).
Unless otherwise noted, reagents were purchased from Sigma.
Myc-tagged ZO-1 (ZO-1myc) in pBluescript was a gift from Dr. Alan
Fanning in the Dr. J. M. Anderson Laboratory (Yale University, New
Haven, CT) (12).
Cell Culture and Transfection--
ROS 17/2.8 cells are an
osteosarcoma cell line that has been shown to express Cx43 and Cx46 but
not Cx45 (13). Our experiments showed that these cells express ZO-1 and
N-cadherin and suggest that they do not express ZO-2 or
occludin (data not shown). ROS cells were cultured in minimum
Eagle's medium containing 10% heat inactivated bovine calf serum
containing 2 mM glutamine, 1 mM sodium
pyruvate, 1% nonessential amino acids, 5 units/ml penicillin, and 5 µg/ml streptomycin. The Cx45-transfected ROS cells had been reported
previously (5, 14). Stably transfected ROS cells were selected and
cultured in the same culture media containing 400 µg/ml G418.
Immunoprecipitation--
The coimmunoprecipitation protocol was
adapted from a procedure used to precipitate ZO-1-associated proteins
(15, 16). Cells were labeled with [35S]methionine (100 µCi/ml) (Amersham Pharmacia Biotech or ICN, Costa Mesa, CA) for
2 h in methionine-depleted minimum Eagle's medium containing 10%
calf serum. The media was removed, and the cells were harvested by
scraping and subsequently were solubilized in mildly dissociating
conditions with an IP buffer containing PBS, 1% Triton X-100, 0.5%
CHAPS, 0.1% SDS, and protease inhibitors (1:100 dilution of a protease
inhibitor mixture (Sigma)). The antigen-antibody complexes were
collected with protein A-Sepharose. In most experiments the
antigen-antibody complexes were washed with IP buffer prior to analysis
by SDS-PAGE and fluorography. In some experiments the antigen-antibody
complexes were collected with protein A-Sepharose and then washed in
RIPA buffer, PBS containing 1% Triton X-100, 0.6% SDS, and protease
inhibitors after they were collected with protein A-Sepharose. This
immunoprecipitated material was then analyzed by SDS-PAGE and
fluorography. In some experiments the cells were not radioactively
labeled, and the immunoprecipitated material was analyzed by immunoblotting.
Immunoblot--
Proteins immunoprecipitated by
anti-Cx45-antibodies were transferred to Immobilon-P membranes. The
membranes were probed with 1:1000 dilution of monoclonal antibody
directed against Cx43 (Chemicon) or ZO-1 (Zymed Laboratories
Inc.). All blots were then probed with the appropriate
peroxidase-conjugated secondary antibody (Roche Molecular
Biochemicals or Jackson Immunoresearch, West Grove, PA)) and
developed with the SuperSignal West Pico chemiluminescence system (Pierce).
Immunofluorescence Microscopy--
ROS cells were cultured on
coverslips to 60-80% confluence and fixed in 50% methanol/50%
acetone for 2 min at room temperature and permeabilized in 1% Triton
X-100/PBS. The cells were blocked with PBS/1% Triton X-100/2% normal
goat serum overnight and then incubated for 1 h in primary
antibody. We used monoclonal anti-ZO-1 (Zymed Laboratories
Inc.) at a 1:1000 dilution, monoclonal anti-Cx43 (Chemicon) at a
1:1000 dilution, or rabbit anti-Cx45 antiserum at a 1:1000 dilution
(alone or in combination) as primary antibodies. The coverslips were
washed and then incubated for an hour in secondary antibody. In these
experiments we used a 1:2000 dilution of CY3-conjugated donkey
anti-mouse IgG (Jackson Immunoresearch) and a 1:2000 dilution of
Alexa-488-conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR). The cells were viewed by epifluorescence on a Nikon Eclipse E600FN microscope using a 60× (numerical aperture 1.4) or a
40× (numerical aperture 1.3) objective, and the images were processed
using Tillvision software (Martinsried, Germany). Confocal images were
collected on a Bio-Rad MRC10-24 microscope with LaserShop software,
and the images were analyzed using Adobe Photoshop software.
ZO-1 Binding Assay--
To mimic the carboxyl-terminal of Cx45,
peptides consisting of amino acids 383-394 of chicken Cx45
(CSKSGDGKNSVWI), which we call Cx45CT, were synthesized
(Research Genetics, Huntsville, AL.) and conjugated to SulfoLink Gel
(Pierce) through the added amino-terminal cysteine. Control peptides
used in this study included the amino acids 373-382 of rat Cx43
(CSRPRPDDLEI), which we call Cx43 CT, and amino acids 367-378 on
chicken Cx45 (CSREKKSKAGSNK), which we call Cx45OC (which is
41.7% identical with amino acids 479-490 in rat occludin), and amino
acids 252-271 of rat Cx43 (CGPLSPSKDCGSPKYAYFNGC), which we
call Cx43PET, were synthesized by Research Genetics and coupled to
SulfoLink Gel (Pierce) through the added amino-terminal cysteine.
Peptides corresponding to amino acids 317-329 on rat Cx40
(CQPKEQPSGASAGH), which we call Cx40I, were synthesized previously and
coupled to SulfoLink Gel through the added amino-terminal cysteine.
ZO-1myc was translated in a TNT T7 quick transcription system (Promega,
Madison, WI) and divided into eight equal portions, which were
incubated with 50 µl of the peptide-coupled SulfoLink Gels, protein
A-Sepharose or protein A-Sepharose and anti-ZO-1 polyclonal antibody.
The gels were extensively washed with RIPA buffer and analyzed by
SDS-PAGE and fluorography. We found that washing the resins with less
stringent buffers, such as IP buffer, produced an unacceptably high
level of nonspecific binding.
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RESULTS |
A 220-kDa Polypeptide Precipitates with Cx45 in ROS/Cx45
Cells--
We identified proteins that associate with Cx45 in ROS/Cx45
transfectants by immunoprecipitation of Cx45-containing complexes in
mild detergent conditions (Fig.
1A). Detergent lysates were made from [35S]methionine-labeled cells solubilized with
IP buffer (PBS containing 1% Triton X-100, 0.5% CHAPS, and 0.1%
SDS). Anti-Cx45 antibodies were added to the lysate, antigen-antibody
complexes were collected with protein A-Sepharose, and the
immunoprecipitated material was extensively washed with IP Buffer or
the more stringent RIPA buffer (PBS containing 1% Triton X-100 and
0.6% SDS) (Fig. 1A) prior to analysis by SDS-PAGE and
fluorography. As shown in Fig. 1A, the immunoprecipitated
material derived from ROS/Cx45 cells revealed a 45-kDa band in both IP
buffer-washed and RIPA buffer-washed samples. However, the IP
buffer-washed samples also contained a 220-kDa band that was not
detected in the RIPA buffer washed precipitates. As expected, nothing
was detected in the IP or RIPA buffer-washed precipitates from the
untransfected ROS cells (Fig. 1A). To confirm that this
220-kDa coprecipitated band occurred in cells expressing endogenous
Cx45 as well as in the ROS/Cx45 transfectant, we isolated Cx45
immunoprecipitates from the osteoblastic cell line UMR 106-01, which
expresses endogenous Cx45, and again detected a band that migrated at
~220 kDa (Fig. 1B). The molecular mass of this polypeptide
is similar to that of ZO-1, a protein that is known to bind to
Cx43.

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Fig. 1.
A 220-kDa protein coprecipitates with
Cx45. A, ROS and ROS/Cx45 cells were labeled with
[35S]methionine and lysed in the IP buffer and Cx45, and
associated proteins were isolated by immunoprecipitation with Cx45
antiserum. Immunoprecipitates were then washed with RIPA buffer or the
less stringent IP buffer, and SDS-PAGE and fluorography were performed.
B, ROS, ROS/Cx45, and UMR cells were labeled with
[35S]methionine and lysed in the IP buffer, and Cx45 and
associated proteins were isolated by immunoprecipitation with Cx45
antiserum. Immunoprecipitates were then washed with IP buffer, and
SDS-PAGE and fluorography were performed.
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ZO-1 and Cx43 Are Present in Cx45 Immunoprecipitates--
We next
asked whether the 220-kDa protein that coisolated with Cx45 was ZO-1.
In the experiments shown in Fig.
2A, ROS cells, ROS/Cx45 cells,
and UMR cells were harvested and solubilized in IP buffer, and the
soluble material was immunoprecipitated with an anti-Cx45 antiserum.
The antigen-antibody complexes were collected with protein A-Sepharose,
and the precipitated material was washed with either IP buffer or RIPA
buffer and analyzed by immunoblotting with a monoclonal antibody
directed against ZO-1. ZO-1 was found in IP buffer-washed anti-Cx45
immunoprecipitates derived from ROS/Cx45 cells but not in the RIPA
buffer-washed immunoprecipitates (Fig. 2A). As expected, the
anti-Cx45 antibody did not precipitate detectable ZO-1 from ROS
cells.

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Fig. 2.
ZO-1 and Cx43 precipitate with Cx45.
A, ROS, ROS/Cx45, and UMR cells were lysed in the IP buffer,
and Cx45 and associated proteins were isolated by immunoprecipitation
with Cx45 antiserum. Immunoprecipitates were then washed with a RIPA
buffer or the less stringent IP buffer, run on SDS-PAGE, and
transferred to Immobilon membrane. The membrane was probed with a
monoclonal antibody against ZO-1 and peroxidase-conjugated secondary
antibodies. B, ROS and ROS/Cx45 cells were lysed in the IP
buffer, and Cx45 and associated proteins were isolated by
immunoprecipitation with Cx45 antiserum. Immunoprecipitates were
isolated in RIPA buffer or the less stringent IP buffer as above. The
membrane was probed with a monoclonal antibody against Cx43 and
peroxidase-conjugated secondary antibodies.
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Because Cx43 is a known binding partner of ZO-1 and our previous data
suggest an interaction between Cx43 and Cx45 in cells expressing both
connexins (14), we asked whether Cx43 was also precipitated by Cx45
antiserum in ROS/Cx45 cells. Proteins immunoprecipitated from
transfected ROS cells were analyzed by immunoblotting with a monoclonal
antibody directed against Cx43, showing that Cx43 was associated with
the anti-Cx45 immunoprecipitate from the ROS/Cx45 cells (Fig.
2B). The anti-Cx45 antiserum did not immunoprecipitate Cx43
from the untransfected ROS cells. Similarly there was no Cx43 in the
RIPA buffer-washed immunoprecipitates derived from either cell line.
ZO-1 Immunoprecipates with Cx43 in ROS and ROS/Cx45 Cells--
To
confirm the interactions identified above, we performed
coimmunoprecipitations from radioactively labeled ROS and ROS/Cx45 cells using anti-Cx43 serum. The immunoprecipitated proteins were then
analyzed by SDS-PAGE and fluorography (Fig.
3A). Anti-Cx43 precipitated
both 45-kDa and 220-kDa radioactively labeled polypeptide proteins from
both cell lines. There were more of both proteins in the ROS/Cx45 cells
than in the ROS cells. Another striking feature of these
immunoprecipitates is that other than the 45-kDa and 220-kDa proteins
there are no other [35S]methionine-labeled proteins that
copurify with Cx43 on these gels.

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Fig. 3.
ZO-1 coprecipitates with Cx43.
A, ROS and ROS/Cx45 cells were labeled with
[35S]methionine and lysed in the IP buffer, and Cx43 and
associated proteins were isolated by immunoprecipitation with anti-Cx43
polyclonal antiserum. Immunoprecipitates were then washed with IP
buffer, and SDS-PAGE and fluorography revealed proteins precipitated by
anti-Cx43 antiserum from the corresponding cell lines. B,
ROS and ROS/Cx45 cells were lysed in the IP buffer, and Cx43 and
associated proteins were isolated by immunoprecipitation with anti-Cx43
antiserum. Immunoprecipitates were then washed with the IP buffer,
separated by SDS-PAGE, and transferred to Immobilon membrane. The
membrane was probed with a monoclonal antibody against ZO-1 and
peroxidase-conjugated secondary antibodies.
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To confirm that the anti-Cx43 antiserum also immunoprecipitated ZO-1,
anti-Cx43 immunoprecipitates that were derived from unlabeled ROS and
ROS/Cx45 were subject to immunoblotting with a monoclonal antibody
directed against ZO-1. ZO-1 was found in anti-Cx43 immunoprecipitates
derived from each cell type (Fig. 3B). The
immunoprecipitates derived from ROS/Cx45 cells had more ZO-1 than the
immunoprecipitates derived from ROS cells. These data confirm that
anti-Cx43 antiserum can isolate ZO-1 in the ROS cells.
Cx45 Colocalizes with ZO-1 and Cx43 in Cx45-transfected ROS
Cells--
Subsequently we examined the localization of Cx45, ZO-1,
and Cx43 in the transfected ROS cells by confocal microscopy of
immunofluorescently stained ROS/Cx45 cells. Cells were fixed,
permeabilized, and stained with a monoclonal antibody directed against
ZO-1 and a polyclonal antiserum against Cx45. These cells were than
examined using laser scanning confocal microscopy. And as seen in Fig.
4, there was little staining for Cx45 in
the ROS cells, whereas the ROS/Cx45 cells exhibited punctate and linear
appositional membrane staining for Cx45. There was plentiful linear
staining for ZO-1 at appositional membranes in both cell lines. Merging
the Cx45 (Alexa-488) and ZO-1 (CY3) micrographs revealed areas where
the two signals colocalized. While plasma membrane Cx45 was associated
with ZO-1, some of the ZO-1 at the plasma membrane was not associated
with Cx45. In contrast there was very little Cx45 staining seen in the
ROS cells. Thus Cx45 at the cell surface colocalized with ZO-1,
consistent with the data above suggesting that these proteins could
associate.

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Fig. 4.
ZO-1 colocalize with Cx45
in ROS/Cx45 transfectants. Cells were fixed, permeabilized, and
stained as above. Confocal immunofluorescence microscopy shows the Cx45
and ZO-1 staining in ROS and ROS/Cx45 cells. In the right
panels, the Cx45 and ZO-1 images were merged to show yellow
areas that were positive for both Cx45 and ZO-1.
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We next confirmed that Cx43 also colocalized with Cx45 in these cells
as was seen previously (14). The Cx45-transfected cells were
simultaneously stained with an antibody directed against Cx43 and Cx45
as seen in Fig. 5. In ROS cells not
expressing Cx45, Cx43 was present at cell-cell boundaries and some
staining in cytoplasmic vesicles, while minimal staining was seen with
the Cx45 antibody. The anti-Cx45 antiserum and the anti-Cx43 antibody stained the ROS/Cx45 cells in discrete spots along the plasma membrane,
and a significant amount of staining was evident in a perinuclear
region as well. The merged image shows that there is substantial
colocalization of Cx43 and Cx45 in the ROS/Cx45 cells. These results
are consistent with the hypothesis that transfected Cx45 associates
with Cx43 in the ROS/Cx45 cells.

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Fig. 5.
Immunolocalization of Cx45 and Cx43 in
transfected ROS cells. ROS cells and transfected ROS cells were
fixed, permeabilized, and stained with rabbit antibodies against Cx45
and a mouse monoclonal antibody against Cx43. Rabbit antibodies were
seen with Alexa-488-conjugated donkey anti-rabbit IgG, and mouse
antibody was revealed with a CY3-conjugated donkey anti-mouse IgG. In
the right panels, the Cx45 and Cx43 images were merged to
show yellow areas that were positive for both Cx45 and
Cx43.
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ZO-1 Binds to the Cx45 Carboxyl-terminal--
ZO-1 binding usually
occurs via interactions with the carboxyl terminus of membrane
proteins. To determine whether ZO-1 binds to the carboxyl-terminal of
Cx45 oligopeptides corresponding to the final 12 amino acids in Cx45
(Cx45CT), the carboxyl-terminal nine amino acids of Cx43 (Cx43CT), and
internal peptides from Cx45 (Cx45OC) and Cx40 (Cx40I) were synthesized
and conjugated to SulfoLink Gel through an added amino-terminal
cysteine. Equal portions of in vitro translated
[35S]methionine-labeled ZO-1myc were incubated with the
peptide-derivatized gel beads, protein A-Sepharose, or anti-ZO-1
antibody and protein-A-Sepharose, and these beads were then extensively
washed with RIPA buffer. As can be seen in the resulting gel (Fig.
6), the Cx45CT peptide and the Cx43CT
peptide bind significant amounts of ZO-1myc. In contrast to this,
protein A-agarose, the Cx40I peptide-coupled agarose, and Cx45OC
peptide-coupled agarose do not bind ZO-1myc. The multitude of bands on
this gel are probably due to incomplete transcription of the relatively
lengthy ZO-1myc mRNA as inclusion of the Sigma protease inhibitor
mixture did not significantly alter the presence of these bands (data
not shown); nonetheless they were recognized by the anti-ZO-1 antibody
(data not shown), and thus they are portions of the ZO-1myc
polypeptide. In a parallel experiment ZO-1myc did not bind to amino
acids 252-271 of rat Cx43 that had been coupled to agarose (data not
shown). All of these results suggest that ZO-1 recognizes the
carboxyl-terminal of Cx45.

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Fig. 6.
In vitro translated ZO-1myc binds to the to
the carboxyl-terminal of Cx45. ZO-1myc was in vitro
translated in the presence of [35S]methionine and then
incubated with protein A-Sepharose, with protein A-Sepharose and
anti-ZO-1 antibody, or agarose conjugated with Cx43CT, Cx40I, Cx45OC,
or Cx45CT peptides. The beads were collected by centrifugation and
extensively washed with RIPA buffer, and the bound peptides were
analyzed by SDS-PAGE and fluorography.
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DISCUSSION |
These studies demonstrate that ZO-1 interacts with Cx45, and that
this Cx45/ZO-1 interaction probably requires recognition of the
carboxyl terminus of Cx45. The primary evidence of this is that Cx45
and ZO-1 can be coprecipitated from lysates of ROS/Cx45 cells.
Furthermore Cx45 and ZO-1 colocalize in transfected ROS cells as seen
by immunofluorescence and confocal microscopy suggesting that these
proteins may be in close proximity to each other at the plasma
membrane. We also found that in vitro translated ZO-1 binds
specifically to the 12 most carboxyl-terminal amino acids in Cx45. The
interaction between the carboxyl terminus of Cx45 and ZO-1 is likely to
occur via binding to a ZO-1 PDZ domain in the same manner as Cx43
carboxyl-terminal.
Work in other laboratories has shown that Cx43 binds to the second PDZ
domain in ZO-1 (6, 7). Both groups isolated the second ZO-1 PDZ domain
in a yeast two-hybrid assay using the carboxyl-terminal of Cx43 as
bait. Giepmans and Moolenar showed that the absolute carboxyl-terminal
of Cx43 is needed to bind ZO-1; masking the carboxyl-terminal with a
Myc epitope or removing the absolute carboxyl-terminal isoleucine
abrogated this interaction (7), which is consistent with a
canonical PDZ domain-carboxyl-terminal interaction. The
carboxyl-terminal sequence of Cx45 (NSVWI) is somewhat different from
Cx43 (DDLEI), which raises the possibility that the Cx43 and Cx45
carboxyl termini might bind to different domains in ZO-1. Indeed the
carboxyl termini of claudins (XXXYV), a known binding
partner for the first PDZ domain in ZO-1, seem to be more similar to
the Cx45 carboxyl-terminal (NSVWI). Additionally the presence of Cx43,
ZO-1, and Cx45 in the same immunoprecipitates suggests that it is
unlikely that Cx43 and Cx45 bind to the same PDZ domain.
It is still unclear what role ZO-1 plays in the life cycle of a
connexin. One hypothesis is that ZO-1 makes a scaffold that temporarily
secures the different connexins in gap junction plaques at the
cell-cell boundary. The amino acid sequences of the carboxyl-terminal of all of connexins, with the exception of Cx32, end in a hydrophobic residue suggesting that they might bind to PDZ domains in ZO-1 (18).
This might indicate that sets of connexins that bind to ZO-1, like Cx45
and Cx43, could be found in the same gap junctions along with ZO-1. In
contrast Cx32 and Cx43 sort to different plasma membrane locales in
thyroid epithelial cells, and only Cx43 colocalized with ZO-1 (19). It
is also possible that interaction with a ZO-1 scaffold stabilizes the
connexin at the gap junction. Recent data from Toyofuku et
al. are consistent with this notion as Cx43 that cannot interact
with ZO-1 (due to mutation or phosphorylation by c-Src) turns over much
more rapidly than Cx43 that can interact with ZO-1 (10). In a
scaffolding model, the different domains of ZO-1 serve as docking
modules for kinases and phosphatases that interact with the different
connexin polypeptides. ZO-1 could then serve a common function in the
life cycle of a number of different connexins by providing a docking
platform for these signaling molecules.