1 Institute of Pharmacology and Toxicology, University of Lausanne, 1005
Lausanne, Switzerland
2 Department of Pediatrics, University Hospitals, 1211 Geneva, Switzerland
3 Department of Physiology, University of Geneva, 1211 Geneva, Switzerland
Authors for correspondence (e-mails:
olivier.staub{at}ipharm.unil.ch;
marc.chanson{at}hcuge.ch)
Accepted 17 February 2003
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Summary |
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Key words: Connexins, Gap junctional communication, Trafficking, Degradation, Endocytosis
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Introduction |
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Unlike most membrane proteins, gap junctions are dynamic structures with
half-lives ranging from 1.5 to 5 hours
(Darrow et al., 1995;
Laird et al., 1991
;
Musil et al., 1990a
). The life
cycle of connexins involves the noncovalent oligomerisation of subunits into
connexons, the translocation of assembled connexons to the cell surface,
intercellular pairing of connexons and channel clustering into paracrystallin
arrays referred to as gap junction plaques (reviewed in
(Kumar and Gilula, 1996
). The
retrieval of gap junction plaques from the cell surface has been proposed to
entail the endocytosis of partial or complete junctional plaques as a double
membrane annular junction that is subsequently degraded or possibly reutilised
(Gaietta et al., 2002
;
Jordan et al., 2001
;
Larsen et al., 1979
;
Naus et al., 1993
). Hence,
processes involving turnover and degradation, as well as remodelling, may
provide important mechanisms to regulate intercellular communication under
normal or pathological conditions
(Beardslee et al., 1998
;
Laird, 1996
;
Luke and Saffitz, 1991
;
Traub et al., 1983
).
Connexin43 (Cx43), the most studied gap junction protein so far, undergoes
several types of post-translational modifications, including phosphorylation
and ubiquitination. Numerous studies have established that the conversion of
unphosphorylated Cx43 to slower migrating species on SDS-polyacrylamide gel is
caused by phosphorylation of Cx43, an event that facilitates gap junction
channel formation and gating (Kwak et al.,
1995; Laird et al.,
1991
; Lampe, 1994
;
Moreno et al., 1994
;
Musil et al., 1990b
). The
phosphorylation state of Cx43 has also been proposed to control Cx43
degradation in rat mammary tumour cells and in intact rat heart
(Beardslee et al., 1998
;
Laird et al., 1995
). The
degradation of Cx43 has been shown to occur by the lysosomal
(Laing et al., 1997
;
Larsen and Hai, 1978
;
Musil et al., 2000
;
Naus et al., 1993
;
Vaughan and Lasater, 1990
) and
the ubiquitin-proteasomal (Laing et al.,
1997
; Musil et al.,
2000
; Laing and Beyer,
1995
; Rutz and Hulser,
2001
) pathways, the relative contribution of which appears to be
largely cell-type specific. It is likely that some of the proteasomal
degradation occurs at the level of the endoplasmic reticulum (ER), as a
quality control step, to remove poorly folded or oligomerised connexin
polypeptides (Musil et al.,
2000
; VanSlyke et al.,
2000
).
Despite the large body of knowledge on the rapid turnover and degradation
of Cx43, and the proteolytic systems involved, virtually nothing is known
about the signals and motifs that control sorting to the lysosome or promote
degradation by the ubiquitin-proteasome system. Cx43 does not have an amino
terminus (basic or hydrophobic, bulky amino acids) that would be recognised by
the N-end rule (Varshavsky,
1992). Many rapidly degrading proteins contain PEST sequences
(rich in proline, glutamic acid, serine and threonine), which have been
suggested to be signals for rapid degradation
(Rogers et al., 1986
). Indeed,
low consensus PEST sequences were described for Cx43
(Darrow et al., 1995
;
Laird et al., 1991
); however,
their role in Cx43 turnover has not been shown experimentally. Interestingly,
Cx43 contains a proline-rich motif in its C-terminus, which conforms to the
consensus of a PY motif (xPPxY, P=proline, Y=tyrosine, x=amino acid)
(Fig. 1A). Such PY motifs have
been shown to act as ligands for WW domain-containing proteins
(Chen and Sudol, 1995
). More
importantly, several different ion channels interact with members of the
Nedd4/Nedd4-like family of ubiquitin-protein ligases, via PY motif/WW domain
interactions, leading to their ubiquitination-dependent downregulation at the
plasma membrane (Abriel et al.,
2000
; Abriel et al.,
1999
; Schwake et al.,
2001
; Staub et al.,
1996
). Hence, it is possible that this motif directs the
ubiquitination of Cx43 and plays a role in its targeting for endocytosis and
destruction. Overlapping the PY-motif, however, is a tyrosine-based sorting
signal conforming to the consensus Yxx
(where Y is a tyrosine, x is any
amino acid and
is an amino acid with a bulky hydrophobic side chain).
Signals of this nature, contained in the cytosolic domains of many plasma
membrane proteins, are also known to mediate internalisation and lysosomal
targeting for degradation. In addition, some Yxx
motifs can direct
traffic within the endosomal and late secretory pathways
(Bonifacino and Dell'Angelica,
1999
; Kirchhausen et al.,
1997
; Owen and Evans,
1998
). The specificity of these processes is believed to be
achieved through the interaction of these signals with alternative adapter
complex molecules that associate with different protein-sorting machineries
(Bonifacino and Dell'Angelica,
1999
).
|
To better understand the molecular mechanisms that control connexin retrieval from the plasma membrane and degradation, we studied the possible contribution of the PY-motif and its overlapping tyrosine-based motif on Cx43 degradation. To this end, we studied the effects of amino acid substitutions within this region on protein stability and sensitivity to proteasome and lysosome inhibitors, as well as on functional expression. We report here that the tyrosine-based sorting signal is a primary element in this region controlling Cx43 turnover.
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Materials and Methods |
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Cell culture and transfection
SKHep1 cells were purchased from the American Type Culture Collection
(Rockville, MD) and maintained in Dulbecco's Modified Eagle's Medium (DMEM),
supplemented with 10% FCS and 0.6% penicillin/streptomycin (Invitrogen) in an
atmosphere of humidified air/5% CO2 at 37°C. For the
development of the stable cell lines Cx43-WT (wildtype) and Cx43-Y286A,
plasmids were transfected into SKHep1 cells using the Effectene transfection
reagent (Qiagen, Hilden, Germany), and transformants were selected for
neomycin resistance using 400 µg/ml of G418. Likewise, transient
transfections were carried out using the Effectene transfection kit, and
plasmids were allowed to express Cx43 for 48 hours before SDS-PAGE and western
blot analysis.
Western blot analysis
SKHep1 cells, stably or transiently transfected with Cx43 constructs, were
washed twice in cold PBS, lysed in radioimmunoprecipitation (RIPA) buffer pH
8.0 (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS. 0.5%
deoxycholate, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10
µg/ml aprotinin) and the cells harvested on ice by scraping. After
centrifugation at 4°C for 5 minutes at 20,000 g, the
supernatants were recovered and samples denatured by heating at 95°C for 5
minutes in protein sample buffer (295 mM sucrose, 2% SDS, 2.5 mM EDTA, 62.5 mM
Tris-Cl pH 8.8, 0.05% bromophenol blue, 26 mM dithiothreitol (DTT)).
Dephosphorylation experiments were performed by treating the cellular lysates
with 50 units of calf intestinal phosphatase (New England Biolabs, Beverly,
MA) for 3 hours at 37°C before denaturing in protein sample buffer.
Cellular lysates were then electrophoresed on a 10% polyacrylamide gel
(SDS-PAGE), before being transferred onto a nitrocellulose membrane. Membranes
were then probed with a polyclonal antibody directed against the C-terminus of
Cx43 (Zymed, San Francisco, CA) or ß-Actin (Sigma). For experiments with
protease inhibitors, the stable cell lines Cx43-WT and Cx43-Y286A were treated
for 3 hours with either lactacystin (10 µM), leupeptin (10 µM) or
NH4Cl (10 mM) before cell harvesting and western blotting.
Quantitation of recognised levels was performed on fluorograms, using a
molecular imager FX (Biorad, Hercules, CA), and the results were normalised to
the controls and expressed as mean±s.e.m. Statistical analyses were
performed using the unpaired two-tailed Student's t test.
Pulse-chase analysis
Cx43-WT and Cx43-Y286A cells were grown to 80% confluency and then starved
in depletion medium (DMEM without methionine) for 30 minutes at 37°C.
Cells were then labelled for 60 minutes in depletion medium containing 0.1
mCi/ml [35S]-methionine. After labelling, cells were placed on ice
and washed three times in ice-cold wash medium (DMEM, 10% FCS, 0.6%
penicillin/streptomycin, 10 mM methionine) before being chased, for various
periods of time, in pre-warmed wash medium (alone or supplemented with either
10 µM lactacystin or 10 mM NH4Cl). At the end of the chase,
cells were transferred onto ice, washed three times with ice-cold PBS, then
lysed in RIPA buffer. Cells were harvested by scraping, centrifuged for 5
minutes at 20,000 g (4°C) and the supernatants
immunoprecipitated overnight (4°C with rotation) using an anti-Cx43
antibody (Zymed) together with protein A sepharose beads. After
immunoprecipitation, the beads were washed four times in RIPA buffer and then
the immunoprecipitated proteins eluted with protein sample buffer and boiling
for 5 minutes at 95°C. Radiolabelled proteins were then visualised by
SDS-PAGE and autoradiography. Quantitation of [35S]-labelled Cx43
was performed on autoradiographs using a molecular imager FX (Biorad). The
results were normalised to the control (t=0) and expressed as
mean±s.e.m. Mono-exponential curves for each independent experiment
were fitted through the values measured at each time point according to the
formula y=100*exp(kt), using Kaleidagraph v. 3.52 (Synergy Software,
Reading, PA). The decay rate constants (k), which are representative of the
rates of protein degradation, were determined for each curve and the mean
values calculated. For statistical analyses, two-way ANOVA was performed using
Prism (GraphPad, San Diego, CA).
Immunofluorescence microscopy
For immunofluorescent labelling, cell lines were cultured on glass
coverslips and incubated for 3 hours in new medium alone (control) or
supplemented with 10 µM lactacystin, 10 mM NH4Cl or 2 µg/ml
Brefeldin A before fixation for 2-3 minutes in methanol at 20°C.
The coverslips were then rinsed in PBS and incubated successively with 0.2%
Triton X-100 for 60 minutes, 0.5 M NH4Cl for 15 minutes and PBS
supplemented with 2% bovine serum albumin for an additional 30 minutes. Cells
were then rinsed and incubated overnight with polyclonal antibodies (diluted
1:30) against Cx43 (Alpha Diagnostics, San Antonio, TX). After washing in PBS,
the coverslips were incubated with secondary antibodies, conjugated to FITC
for 3 hours and then examined using fluorescent microscopy. Images were
acquired with a high-sensitivity CCD Visicam (Visitron systems GmbH, Germany)
camera connected to a personal computer. Images were captured using the
software Metafluor 4.01 (Universal Imaging, West Chester, PA) and processed
using Adobe Photoshop 5.5 (Adobe Systems Inc.).
Cell-coupling measurements
Dye coupling studies were performed on subconfluent monolayers of cells
incubated in a solution (external solution) containing (in mM): 136 NaCl, 4
KCl, 1 CaCl2, 1 MgCl2, and 2.5 glucose, and was buffered
to pH 7.4 with 10 mM HEPES-NaOH. Single cells were impaled with
microelectrodes backfilled with a 4% lucifer yellow solution prepared in 150
mM LiCl (buffered to pH 7.2 with 10 mM Hepes). The fluorescent tracer was
allowed to fill the cells by simple diffusion for 3 minutes. After the
injection period, the electrode was removed and the number of fluorescent
cells was counted. Cells were visualised using epifluorescent illumination
provided by a 100 W mercury lamp and the appropriate set of filters. The
results were expressed as mean±s.e.m. To examine the effects of
proteasome/lysosome inhibitors on dye coupling, subconfluent monolayers of
cells were incubated for 3 hours in the external solution supplemented with
either 10 µM lactacystin, 10 mM NH4Cl or 2 µg/ml Brefeldin
A.
For electrical coupling studies, the dual whole-cell patch-clamp approach was applied to pairs of cells incubated in the external solution. Both cells of a pair were voltage clamped at a common holding potential of 0 mV. To measure gap junctional currents (Ij), transjunctional potential differences (Vj) were elicited by changing the holding potential of one member of a cell pair. Ij was defined as the current recorded in the cell kept at a 0 mV. Junctional conductance (gj) was then calculated by gj=Ij/Vj and the results displayed as a scattered plot displaying the individual gj values, including the mean±s.e.m. Series resistance was not compensated for and was less than 2% of the combined junctional and cell input resistance. Patch electrodes were filled with a pCa 7 solution containing (in mM): 138 KCl, 1 NaCl, 2.9 CaCl2, 5.5 EGTA, 2 MgCl2, and buffered to pH 7.2 with 10 mM HEPES-KOH. Statistical analyses were performed using the two-tailed Student's t test for unpaired data.
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Results |
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|
The increased expression level of Cx43-Y286A could be due to differences either in the rate of biosynthesis or in the degradation of mutant Cx43, as compared with Cx43-WT. To discriminate between these two possibilities, we developed stable SKHep1 cell lines expressing either Cx43-wildtype or Cx43-Y286A, which was representative of the tyrosine sorting mutations, and examined the turnover of these proteins by pulse-chase analysis. Cells were metabolically labelled for 60 minutes in the presence of [35S]-methionine and then chased for 0, 2, 4 and 6 hours with an excess of unlabelled methionine. Cx43 was then immunoprecipitated and the amount of radiolabelled protein analysed by SDS-PAGE and fluorography. The results, depicted in Fig. 3A, revealed that the degradation of Cx43, harbouring the Y286A mutation, was considerably slower than that of the wild-type Cx43. The two proteins had comparable translation rates as judged by the similar amounts of [35S]-methionine incorporation at time 0. Using this approach, however, Cx43 was predominantly detected as a single band which, according to its predicted molecular weight, corresponds to the NP-form. This suggests that either this assay is not sensitive enough to detect the less-abundant P forms or, alternatively, that immunoprecipitation could not isolate the phosphorylated species. To ensure that the disappearance of the NP form in the pulse-chase experiments represented degradation and not maturation into the phosphorylated forms, we also performed similar experiments in the presence of calf intestinal phosphatases so as to analyse the entire pool of Cx43, and found identical results (data not shown). The [35S]-labelled Cx43 species were quantified in four different experiments, and measured values were subjected to ANOVA statistical analysis. As shown in Fig. 3B, the degradation of Cx43-Y286A was significantly slower (P<0.0001) compared with that of Cx43-WT, with the half-life of Cx43 being increased from approximately 2 to 6 hours. The calculated rate of degradation for Cx43-WT (k=0.34 h1) was reduced 2.72-fold on mutation of Cx43-Y286A (k=0.125 h1). This approximates the increased steady-state abundance of Cx43-Y286A determined in our transient transfection assays (Fig. 2B; 3.5-fold), which can be expected if the accumulation rate is similar between wild-type and mutant protein. Collectively, these data show that the increase in the steady-state levels brought about by the substitution of tyrosine 286 with alanine results from a decrease in the rate of degradation of the mutant protein, suggesting that this amino acid plays an important role in Cx43 turnover.
|
Effects of proteasomal and lysosomal inhibitors on connexin
turnover
Having established that the tyrosine 286 was involved in Cx43 stability, we
next investigated the underlying cellular mechanism. As Cx43 has been shown to
be degraded by both the proteasome and the lysosome
(Beardslee et al., 1998;
Laing and Beyer, 1995
;
Laing et al., 1997
;
Musil et al., 2000
), we used
pharmacological agents to inhibit the function of these intracellular
compartments and measured Cx43 protein levels by western blot analysis
(Fig. 4A). Note that because of
the high connexin levels in Cx43-Y286A-expressing cells, reduced fluorography
has been used on this blot for comparative analysis with the wildtype.
Quantitative analysis of Cx43 expression with these treatments is shown in
Fig. 4B. Treatment of
Cx43-WT-expressing cells with the proteasomal inhibitor lactacystin
(Fenteany et al., 1995
)
resulted in an approximate 3-fold increase only in the phosphorylated forms of
Cx43 (Fig. 4B; right panel,
grey columns). A similar effect could be observed using MG-132
(Palombella et al., 1994
), an
alternative proteasomal inhibitor (data not shown). By contrast, treatment of
wild-type cells with two different lysosomal inhibitors, namely leupeptin and
NH4Cl (Hart et al.,
1983
), resulted in an increase in the nonphosphorylated form
compared with the P1 and P2 forms, in the order of 1.5- and 2-fold,
respectively (Fig. 4B; left
panel, grey columns). These results are in agreement with the notion that both
proteolytic pathways control the steady-state levels of Cx43.
|
In contrast to wild-type Cx43, Cx43-Y286A-expressing cells showed a very
different response to proteasomal and lysosomal inhibitors
(Fig. 4A). As previously shown,
the ratio of P:NP forms of Cx43-WT (1:5.6) was already increased in untreated
cells compared with Cx43-Y286A cells (1:2.6), and quantification further
revealed that treatment with lactacystin had a much less prominent effect on
the accumulation of the P1 and P2 forms
(Fig. 4B; right panel, black
columns). Most strikingly though, leupeptin and NH4Cl no longer had
an effect on the steady-state levels of unphosphorylated Cx43
(Fig. 4B; left panel, black
columns). To further confirm this result, pulse-chase experiments were
performed in the presence of NH4Cl. As shown in
Fig. 5A,B, treatment with
NH4Cl markedly reduced Cx43-WT turnover but had only a minor effect
on the more stable mutant Cx43. Quantitative analysis of Cx43 expression
levels revealed that the degradation rate of Cx43-WT (k=0.34
h1) was significantly reduced (fivefold) in the
presence of NH4Cl (k=0.07h1), as indicated by
ANOVA analysis (P<0.0001). But NH4Cl treatment had a
much weaker effect on the tyrosine mutant, as seen by the calculated
degradation rates (control k=0.12 h1, NH4Cl
k=0.074 h1). In contrast to NH4Cl, lactacystin
did not affect the turnover of either Cx43-WT or Cx43-Y286A (data not shown;
recall that only the NP form of Cx43 can be detected in the pulse-chase
experiments). Taken together, these results show that the differences in Cx43
steady-state levels reflect a differential degradation of the wild-type and
Y286A connexin primarily by the lysosome. Inhibition of the proteasome appears
to affect mostly the ratio between NP and P forms and, to a much lesser
extent, the total pool of Cx43.
|
Mutation of tyrosine 286 increases gap junctional staining
As a next step, we sought to examine the localisation of the wild-type and
Y286A Cx43 within cells. Immunofluorescence localisation studies were
therefore performed on both cell lines using an anti-Cx43 polyclonal antibody.
Modest Cx43 immunoreactivity was detected in cells transfected with wild-type
Cx43 (Fig. 6A; control), both
intracellularly and at appositional membranes (indicated by arrows). The
staining was strikingly stronger in the mutant cell line, which displayed
larger and more abundant gap junctional plaques, as well as vesicular
intracellular structures (Fig.
6B; control). We then examined the effects of proteasomal and
lysosomal inhibitors on Cx43 localisation. Lactacystin, which specifically
increased the phosphorylated forms of Cx43
(Fig. 2), markedly increased
the gap junctional staining of Cx43-WT-expressing cells without any
appreciable changes in intracellular labelling
(Fig. 6A; lactacystin). This is
in agreement with the notion that conversion of Cx43 to the P1 and P2 forms
occurs after transport to the cell surface
(Laird et al., 1995;
Musil and Goodenough, 1991
;
Nagy et al., 1997
). By
contrast, lactacystin had no effect on the Cx43 labelling in the Y286A cell
line (Fig. 6B).
|
Treatment of Cx43-WT-expressing cells with NH4Cl gave a very
different response than lactacystin. As can be seen in
Fig. 6A, NH4Cl,
consistent with its inhibitory effect on lysosomal degradation, markedly
increased Cx43 immunoreactivity within intracellular vesicles, which was
probably due to the accumulation of undigested Cx43 in lysosomes. Moreover,
the appositional staining was virtually abolished, suggesting that
NH4Cl may have some additional effects such as impairing the
delivery or the recycling of connexins to the plasma membrane. In sharp
contrast, NH4Cl had no effect on Cx43 staining in the Cx43-Y286A
mutant, with strong staining still clearly evident at cell-cell interfaces
(Fig. 5B; NH4Cl).
The failure of NH4Cl to disrupt the Cx43-Y286A-containing plaques
may suggest that endocytosis had been affected in the connexin mutant. To
further investigate this hypothesis, we used Brefeldin A (BFA), which prevents
the delivery of newly synthesised proteins to the cell surface
(Lippincott-Schwartz et al.,
1991), to follow the fate of the Cx43 pool localised at the plasma
membrane. In cells expressing wild-type Cx43, treatment with BFA resulted in a
loss of Cx43 immunoreactivity at appositional membranes while increasing the
intracellular fluorescence. By contrast, in the Y286A mutant, large
gap-junctional plaques were still clearly visible in the presence of BFA
(Fig. 6B; BFA).
Tyrosine 286 affects Cx43 gap junctional communication
To investigate whether mutation within Cx43's tyrosine-based motif was also
associated with a change in gap junctional communication (GJC), intracellular
injections of lucifer yellow (LY) were carried out on both SKHep1 stable cell
clones. As shown in Fig. 7A (control), both cell clones transferred LY to a similar extent, indicating
that Cx43-Y286A can form functional gap junction channels. This was further
confirmed by measuring junctional conductance in pairs of cells by the dual
patch-clamp approach. No significant difference in electrical coupling between
the wild-type and mutant cell clones was detected
(Fig. 7B; P=0.064). We
cannot exclude, however, the possibility that Cx43-Y286A has a lower
permeability for LY and/or a reduced single-channel conductance that is
compensated for by a larger number of channels at the cell surface.
|
Exposure of Cx43-WT and Cx43-Y286A cells to lactacystin did not significantly change the extent of dye coupling (Fig. 7A; lactacystin). By contrast, treatment with NH4Cl had a marked effect on GJC. According to the immunohistochemistry, such a treatment should result in nearly complete loss of junctional staining in the wild-type, preventing LY transfer, while having little effect on the mutant. Indeed, under this condition, GJC in wild-type cells was significantly decreased compared with Y286A-cells, which remained LY competent (Fig. 7; NH4Cl). Likewise, treatment with BFA had a marked effect on the ability of Cx43-WT cells to transfer LY, whereas diffusion of the fluorescent tracer remained efficient in cells expressing mutant Cx43 (Fig. 7; BFA).
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Discussion |
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Our data show that the tyrosine residue at position 286 (Y286) is a crucial
amino acid involved in Cx43 turnover. Pulse-chase analysis in stably
transfected SKHep1 cells indicates that substitution of Y286 with alanine
increases the half-life of Cx43 from approximately 2 to 6 hours. Consistent
with this, the steady-state levels of Cx43-Y286A is also elevated by a factor
of 3.5, which can be expected if one assumes equal biosynthesis rates of
wild-type and mutant Cx43, which appears to be the case
(Fig. 3A; compare time 0 of
Cx43-WT with Cx43-Y286A). As outlined above, Y286 is part of two putative
protein-protein interaction motifs (Fig.
1A): first, a PY-motif (Chen
and Sudol, 1995
), known to interact with WW domain-containing
proteins such as the Nedd4/Nedd4-like family of ubiquitin-protein ligases
(Rotin et al., 2000
), and
second, a tyrosine-based sorting signal (consensus: Yxx
, where
is a
hydrophobic amino acid), which is part of a family of degenerate motifs
involved in the targeting of many transmembrane proteins to different cell
compartments (Bonifacino and Dell'Angelica,
1999
). Our mutational analysis of the region around Y286 and the
subsequent transfection experiments in SKHep1 cells supports the notion that
it is primarily the tyrosine motif that determines the stability of Cx43.
Mutation of valine 289 to aspartate (V289D) in the tyrosine motif had the same
drastic effect on the stability of Cx43 as Y286A, whereas mutation of proline
283 to leucine (P283L), which is part of the PY motif, increased Cx43
stability to a much weaker extent. Importantly, Y286A, being part of the two
putative motifs, showed no additive effect on the stability (compared with
V289D), as was the case for a double mutant (P283L/V289D) (data not shown).
This therefore suggests that the PY motif plays only a limited, if any, role
in the control of Cx43 turnover. It is possible, considering the steric
properties of proline residues, that the P283L substitution has affected the
functionality of the tyrosine-based sorting motif, providing an explanation
for the comparably small increase in stability of this mutant. Indeed, it has
been proposed recently that residues upstream of the crucial tyrosine may be
important for the binding of effector molecules to tyrosine sorting motifs
(Owen et al., 2001
).
Alternatively, we cannot exclude that the PY motif and the tyrosine motif act
on the same pathway. The mutagenesis results further revealed that the
substitution mutant Y286F behaves in a similar manner to that of the Y286A
construct. This indicates that it is the tyrosine itself that is of crucial
importance to Cx43 turnover and not its aromatic nature. Interestingly,
tyrosine-based sorting signals are remarkably similar to the
phospho-tyrosine-based motifs that direct SH2 domain binding
(Pawson, 1995
;
Songyang et al., 1993
). On
this basis, we cannot exclude an involvement of tyrosine phosphorylation in
this effect, especially as phosphorylation of such sorting signals has been
shown to control protein localisation by regulating their interaction with the
transport machinery (Bradshaw et al.,
1997
; Schaefer et al.,
2002
; Shiratori et al.,
1997
; Stephens and Banting,
1997
). There is no evidence, however, that Y286 is a site of Cx43
phosphorylation.
Tyrosine-based sorting signals have been shown to interact with the medium
chain (mu) subunits of the adaptor complexes (AP), which are components of the
machinery involved in either clathrin-dependent or -independent formation of
membrane-bound transport intermediates (e.g. coated vesicles)
(Bonifacino and Dell'Angelica,
1999; Hirst et al.,
1999
; Hirst and Robinson,
1998
; Simpson et al.,
1996
). Interestingly, annular gap junctions (e.g. internalised gap
junctions) have been proposed to be clathrin coated
(Larsen et al., 1979
).
Moreover, Cx43 has been shown to colocalise with clathrin
(Huang et al., 1996
), and has
been found in close proximity to clathrin-coated pits within the plasma
membrane (Naus et al., 1993
),
suggesting the possible involvement of a clathrin-mediated pathway in Cx43
trafficking. Therefore, it will be interesting to examine if the YKLV motif
exerts its effects by interacting with one of the different adaptor
complexes.
Several lines of evidence suggest that Y286 regulates the stability of Cx43
by controlling targeting of Cx43 for lysosomal degradation. Inhibitors of
endosomal/lysosomal degradation, such as NH4Cl and leupeptin,
slowed down dramatically the degradation of Cx43-WT. This was accompanied by
increased levels of the NP form of Cx43-WT
(Fig. 4) and by an
intracellular accumulation of the protein in vesicles that probably represent
endosomes/lysosomes (Fig. 6).
This is consistent with previous reports implicating the lysosomal system in
the degradation of Cx43 (Berthoud, 2000;
Laing and Beyer, 1995;
Laing et al., 1997
;
Musil et al., 2000
;
Naus et al., 1993
). By
contrast, NH4Cl (Fig.
4) and leupeptin (not shown) displayed only a marginal effect on
the degradation rate and did not affect the steady-state levels of Cx43-Y286A,
which remained elevated in all conditions
(Fig. 4). The fact that
NH4Cl can still repress marginally the decay of the Y-mutant
(Fig. 5) suggests that
NH4Cl has some small effects on the degradation mechanisms of Cx43
that are independent of Y286A. Brefeldin A, an inhibitor of transport from the
ER to the plasma membrane, affected the cell-surface location and GJC of
Cx43-WT, but not of Cx43-Y286A, indicating that mutation of tyrosine 286 may
affect the retrieval of Cx43 from the plasma membrane. Our data further show
that in transfected SKHep1 cells, the proteasome inhibitors lactacystin and
MG-132 have only minor effects on overall stability
(Fig. 4), but that they
increase the Cx43 P forms in Cx43-WT-expressing cells, and, to a lesser
extent, in Cx43-Y286A cells. Consistent with this observation, they also
increase the staining of Cx43-WT at appositional membranes
(Fig. 5). Possibly, proteasome
inhibitors may inhibit ER-dependent degradation (ERAD), as described
previously for Cx32 and Cx43 (VanSlyke et
al., 2000
; VanSlyke and Musil,
2002
), which could lead to an increased export of Cx43 to the cell
surface. Alternatively, they may interfere with a direct role of the
proteasome in the internalisation of Cx43, as has been described for the
growth hormone receptor (van Kerkhof et
al., 2000
).
Surprisingly, the Y286A mutation increased the level of Cx43
immunoreactivity at cell-cell membrane contacts without affecting the extent
of dye coupling. Dual patch-clamp analysis of pairs of Cx43-WT- and
Cx43-Y286A-expressing cells confirmed that the two cell clones do not differ
in terms of junctional conductance values. One explanation may be that the
expression levels achieved in cells transfected with a CMV-driven Cx43
construct are high enough to cause maximal dye coupling that is already seen
in Cx43-WT cells. Alternatively, the Cx43-Y286 mutant protein may be less
efficient than Cx43-WT in transferring LY, which is compensated for by a
larger number of channels at the cell surface. In our SKHep1 clones, no
specific information could be obtained on the biophysical properties of the
mutant Cx43 due to the high level of Cx45 channel activity in these cells
(Moreno et al., 1995).
Possibly, co-expression of Cx43 and Cx45 may form heteromeric channels with
novel biophysical properties that may alter the normal behaviour of individual
connexin components (Martinez et al.,
2002
). Despite these possibilities, mutation of Y286 prevented the
decrease in GJC by inhibitors of the endosomal/lysosomal degradation pathways
that was normally observed in cells expressing wild-type Cx43.
In conclusion, our data show that a tyrosine-based sorting signal present
in the C-terminus of Cx43 controls turnover by targeting the protein for
lysosomal degradation, thereby regulating the strength of gap junctional
communication. The existence of such putative sequences in other connexin
genes may suggest a common mechanism for the sorting of some members of the
gap junction family. However, tyrosine-based signals are not the only
recognised sequences to direct endocytosis and sorting of transmembrane
proteins (Hu et al., 2001;
Johnson and Kornfeld, 1992
;
Letourneur and Klausner, 1992
;
Stroh et al., 1999
). Thus, the
existence of several mechanisms for the sorting and degradation of gap
junction channels made of distinct connexins may play important roles in
various pathophysiological situations to maintain and/or modulate specific
connexin expression and function at the junctional membrane.
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Acknowledgments |
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Footnotes |
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References |
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