From the Hanson Centre for Cancer Research, Institute
of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia and the ¶ Department of Physiology, University of
Sydney, Sydney, NSW 2006, Australia
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ABSTRACT |
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The amiloride-sensitive epithelial sodium channel
(ENaC) plays a critical role in fluid and electrolyte homeostasis and
consists of The amiloride-sensitive epithelial sodium channel
(ENaC)1 plays a critical role
in fluid and electrolyte homeostasis and is widely expressed in
absorptive epithelia such as the renal collecting duct (1, 2), the
colon (1, 2), the lung, (3, 4) and sweat and salivary ducts (1, 2). It
consists of The PY motifs in the carboxyl termini of ENaC subunits are believed to
be necessary for interaction with the WW domains of Nedd4 (20), a
widely expressed ubiquitin-protein ligase (20-24). Nedd4 is believed
to down-regulate Na+ channel activity in response to
increases in intracellular Na+ (17, 25) by ubiquitinating
the channel (16, 25, 26), leading to its endocytosis (16, 18) and
degradation (16, 26).
The detailed mechanisms by which Nedd4 interacts with Na+
channels remain, however, unclear. Nedd4 consists of a
ubiquitin-protein ligase domain, multiple WW domains, and a
Ca2+ and lipid binding domain (20-22). There is now good
evidence that Nedd4 is a functional ubiquitin-protein ligase and that
it ubiquitinates ENaC (16, 24-26). The Ca2+ and lipid
binding domain has been shown to mediate
Ca2+-dependent redistribution of Nedd4 from the
cytoplasm to the cell membrane (27), although the importance of this is
unclear given that intracellular Ca2+ has no role in the
control of Na+ channels by intracellular Na+
(17, 28). Both mouse and rat Nedd4 proteins contain three WW domains,
whereas human Nedd4 has an additional WW domain (20, 22). It has
previously been reported that WW domains 1, 2, and 3 from rat Nedd4 can
bind to the PY motifs of the Expression Plasmids--
The expression construct used to
generate protein containing all three WW domains of mouse Nedd4 fused
to glutathione S-transferase (GST) has been described
previously (25). Single WW domain-GST constructs were generated by
amplifying each WW domain individually by PCR using appropriate
primers2 followed by cloning
into the BamHI/EcoRI sites of pGEX-2TK (Amersham Pharmacia Biotech). WW domain mutations were produced according to a
published protocol (29), PCR amplified, and cloned into the
BamHI/EcoRI sites of pGEX-2TK. The regions
encoding the carboxyl termini of human and mouse Cloning ENaC from Mouse Mandibular Duct Cells--
The regions
encoding the carboxyl termini of the murine Production of GST Fusion Proteins--
Overnight cultures of
Escherichia coli DH5 SDS-Polyacrylamide Gel Electrophoresis and Far-Western
Analysis--
32P-labeled protein probes were produced by
directly labeling the appropriate GST fusion protein using protein
kinase A (New England Biolabs). Glutathione beads containing bound
fusion protein were incubated with protein kinase A and
[ Whole-cell Patch Clamping--
Isolated granular duct cells were
prepared by collagenase digestion of mouse mandibular glands from male
mice as previously described (30). The standard bath solution (pH 7.4)
contained 145 mM NaCl, 5.5 mM KCl, 1 mM CaCl2, 1.2 mM MgCl2,
1.2 mM NaH2PO4, 7.5 mM
HEPES, and 10 mM glucose. The standard (0 Na+)
pipette solution (pH 7.2) contained 150 mM NMDG-glutamate,
1 mM MgCl2, 10 mM HEPES, 5 mM EGTA, and 10 mM glucose. In the 70 mM Na+ pipette solution, Na+ was
adjusted by substitution of Na+-glutamate for
NMDG-glutamate. Standard whole-cell patch clamp techniques were used
(25, 30). After establishing the whole-cell configuration, the bath
solution was replaced with one containing 145 mM
Na+-glutamate, 5 mM NaCl, 1 mM
MgCl2, 10 mM HEPES, 1 mM EGTA, and 10 mM glucose (pH 7.4). The amiloride-sensitive current was
measured as described previously (25, 30). Results are presented as mean ± S.E. Statistical significance was assessed using
Student's unpaired t test. All experiments were performed
at 20-22 °C.
Production of GST-WW Fusion Proteins in E. coli--
To
investigate the specificity of the interaction between the WW domains
of Nedd4 and the subunits of ENaC, a dual approach involving
far-Western blotting and patch clamp analysis was used. To do this, a
variety of WW domain proteins fused to GST were produced (Fig.
1). Murine Nedd4 protein contains three
WW domains located between the amino-terminal Ca2+ and
lipid binding domain and carboxyl-terminal ubiquitin-protein ligase
domain (22). GST fusion proteins containing all three WW domains of
murine Nedd4 with one domain mutated or all three domains in their
wild-type configuration were produced as well as wild-type and mutant
versions of each individual WW domain (Fig. 1). WW domain mutations
were generated by converting the second conserved Trp to Phe and the
conserved Pro to Ala. These mutations were designed to abolish WW
domain binding activity without significantly altering the tertiary
structure. The human Nedd4 protein contains four WW domains of which
domains 1, 2, and 4 correspond to domains 1, 2, and 3, respectively, of
the mouse protein (22). These three domains are highly conserved between mouse and human Nedd4 (22) and are thus expected to have
similar ligand binding specificity. However, WW domain 3 in human Nedd4
is not found in the mouse protein. We therefore generated a GST fusion
protein containing WW domain 3 of the human protein for our binding and
patch clamp assays.
To explore the binding specificity of ENaCs further, individual WW
domains were fused to GST and used for far-Western analysis. As
depicted in Fig. 3B, the
The WW domain 1 protein (Fig. 1, s1) used for binding
studies contained an extra 5 kDa at the carboxyl terminus of the WW domain, whereas s2 and s3 proteins contained only the minimal WW domain
sequence fused to GST (Fig. 1). To examine whether the extra sequence
in s1 altered the binding characteristics of WW domain 1, a shorter
construct containing only WW domain 1 was used in far-Western
experiments. As was the case with s1, this shorter protein did not bind
to any of the ENaC subunits (data not shown).
Interactions between Murine ENaC Subunits and Murine
Nedd4--
The analysis presented in Figs. 2 and 3 was open to
objection that interactions between human ENaC carboxyl termini and
murine Nedd4 may differ from those we would observe if we were to use murine ENaC carboxyl termini. We therefore, using reverse transcriptase PCR, cloned the coding regions of the carboxyl termini of
As none of the mouse or human ENaC subunits showed binding affinity for
WW domain 1 that was immobilized on a nitrocellulose membrane, we used
32P-labeled WW domain 1 to probe immobilized ENaC-GST
fusion proteins. A positive control probe, wt, bound to all mouse and
human ENaC subunits, whereas, WW domain 1 did not exhibit any
significant binding to any of the subunits (data not shown), further
confirming the results shown in Figs. 2 and 3.
All Three WW Domains of Mouse Nedd4 Need to Be Occluded to Inhibit
the Na+ Feedback Loop--
As mentioned in the
introduction, Nedd4 mediates the feedback inhibition of Na+
channel activity produced by an increase in intracellular
Na+. We have previously shown using the whole cell patch
clamp technique that Na+ channel activity of single mouse
mandibular granular duct cells can be inhibited by increasing the
pipette solution Na+ to 70 mM (25, 28, 32), a
value within the physiological range for cytosolic Na+
concentration in exocrine tissues (33, 34). This feedback inhibition by
raised intracellular Na+ can be inhibited by inclusion in
the pipette solution of: (i) an antibody directed against the WW
domains of Nedd4,3 (ii) an
antibody directed against the ubiquitin-protein ligase domain of Nedd4
(25), or (iii) a GST-Nedd4 WW domain fusion protein that presumably
acts as a dominant negative mutant by displacing endogenous Nedd4 from
ENaC subunits (25).
We thus decided to use the ability of this GST-WW domain fusion protein
(wt in Fig. 1) to block Na+ feedback inhibition
of Na+ channels as the basis for an investigation of the
roles of the individual WW domains of Nedd4 in regulating ENaC
activity. We first investigated the concentration-dependence of the
ability of the wt protein to block Na+ feedback inhibition
of Na+ channels in mouse granular duct cells. We found that
the inclusion in the pipette solution of 100 µg/ml GST-wt, but not 50 µg/ml GST-wt, was sufficient to overcome feedback inhibition of
Na+ channels (Fig. 5). In
subsequent studies we thus used a concentration of 100 µg/ml as the
reference concentration of the GST-wt construct.
To test whether all three WW domains of Nedd4 are required to inhibit
the Na+-dependent feedback pathway, GST fusion
proteins containing two intact WW domains and one mutant WW domain (m1,
m2, and m3 in Fig. 1) were examined. We found that none of these
proteins were able to overcome the inhibition of Na+
channel activity produced by inclusion of 70 mM
Na+ in the pipette solution (Fig.
6A). These results suggested
that each of the three WW domains in murine Nedd4 binds to a distinct, noninterchangeable site and that to prevent Na+ feedback
control of Na+ channels it is necessary to occlude all
three sites. We further tested this interpretation by examining whether
the inclusion in the pipette solution of a mixture of individual WW
domains of murine Nedd4 (s1, s2, and s3 in Fig. 1, each at 100 µg/ml) could block Na+ feedback. Indeed, as shown in Fig.
6B, the effect of adding the three individual WW-GST
proteins to the pipette solution was similar to the wt WW-GST fusion
protein containing the three WW domains. Furthermore, omission of any
of these isolated domains from the mixture led to the loss of this
blocking effect despite the adjustment of the concentration of each of
the two remaining WW domain proteins to 150 µg/ml to maintain the
total concentration of GST-WW domain fusion proteins in the pipette
solution at 300 µg/ml (Fig. 6B). Finally, we examined
whether the WW domain unique to human Nedd4 (h3 in Fig. 1)
could replace any of the 3 murine WW domains. We found that
it was unable to do so (Fig. 6C). Inclusion of the h3 domain
(100 µg/ml) in the 0 Na+ pipette solution did not
suppress the amiloride-sensitive Na+ current (data not
shown). Thus we concluded that the additional WW domain in human Nedd4
does not substitute for any of the three WW domains that are present in
both murine and human Nedd4.
This study has shown that to block
Na+-dependent feedback inhibition of epithelial
Na+ channels, distinct binding sites for all three WW
domains of murine Nedd4 must be occluded. It also suggests that,
in vivo, each of these WW domains binds one of these sites
and is unable to bind to the other two. In vitro, the WW
domains of Nedd4 were found to have varying specificity for the PY
motifs of different ENaC subunits. Murine WW domains 2 and 3, and human
WW domain 3 showed varying affinity for all three ENaC subunits,
whereas WW domain 1 failed to bind to either of the three ENaC
subunits. This suggests that WW domain 1 either prefers a different PY
motif to WW domains 2 and 3 or different residues surrounding the PY motif. There is a precedence for WW domains preferring proline-rich motifs other than PPxY; the WW domains of some formin binding proteins
(FBPs) prefer a PPLP motif (35, 36), whereas other FBPs prefer a
proline-, methionine-, and glycine-rich motif (37). The Nedd4 WW domain
1 is much more homologous to Nedd4 WW domains 2 and 3 than to the WW
domains of FBPs, so it is reasonable to expect that WW domain 1 will
bind to a PPxY motif containing protein. Staub et al. (20)
reported that WW domain 1 of rat Nedd4, like WW domains 2 and 3, bound
to the PY motif of both the In light of our binding assays, it may have been reasonable to expect
that WW domains 2 and 3 alone would be capable of exerting a dominant
negative effect on the Na+ feedback pathway as measured by
patch clamp analysis. This was not the case, however, as an intact WW
domain 1 was required, in concert with WW domains 2 and 3, to restore
Na+ channel activity under high Na+ conditions.
We therefore suggest that WW domain 1 must play a role in ENaC
regulation that is not dependent on direct binding of an ENaC subunit
PY motif. We postulate that WW domain 1 binds to an additional, as yet
unidentified, molecule that is activated under high salt conditions,
possibly downstream of G0. This molecule may recruit Nedd4
to the ENaC complex and/or stabilize the ENaC/Nedd4 complex. We
postulate that a stable Nedd4/ENaC complex will comprise one ENaC
tetramer, two molecules of Nedd4, and two molecules of the unknown
recruitment/stabilization factor (Fig.
7). The possibility also exists that a
single molecule of Nedd4 binds to a single ENaC tetramer, leaving two
ENaC PY motifs free. Further experiments are required to explore these
possibilities. In vitro, human WW domain 3 showed affinity
for all three subunits of ENaC, comparable with the binding
characteristics shown by murine WW domains 2 and 3 (Fig. 3), but it was
unable to complement either of these WW domains or WW domain 1 in
vivo. This suggests that, although human WW domain 3 recognizes
the PY motifs of ENaC subunits in vitro, it does not play a
role in the Na+-dependent feedback inhibition
of Na+ channels.
,
, and
subunits. The carboxyl terminus of each
ENaC subunit contains a PPxY motif which is necessary for interaction
with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or
alter the PY motif of either the
or
subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the
ubiquitin-dependent down-regulation of
Na+ channel activity in response to increased
intracellular Na+. In this paper, we demonstrate that WW
domains 2 and 3 bind
-,
-, and
-ENaC with varying degrees of
affinity, whereas WW domain 1 does not bind to any of the subunits. We
further show using whole-cell patch clamp techniques that
Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells
involves binding of the WW domains of Nedd4 to three distinct sites. We
propose that Nedd4-mediated down-regulation of Na+ channels
involves the binding of WW domains 2 and 3 to the Na+
channel and of WW domain 1 to an unknown associated protein.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
subunits which are thought to assume a
tetrameric
2
structure at the membrane (5, 6). The
carboxyl terminus of each ENaC subunit contains a PPxY sequence (the PY
motif), which when mutated or deleted in either the
or
ENaC
subunits leads to Liddle's syndrome, an autosomal dominant form of
hypertension (7-11). Therefore, mutating just one PY motif from a
single subunit of the tetrameric ENaC complex is sufficient to lead to
a disease phenotype. In in vitro systems, identical
mutations to those that cause Liddle's syndrome, have been shown to
increase amiloride-sensitive Na+ current (8, 11-15). This
increase is believed to result from the presence of increased numbers
of active Na+ channels in the cell membrane (12, 16-18),
although an increase in channel open probability may also contribute
(12, 17, 19).
and
ENaC subunits in
vitro (20). It is not known however whether all, or only a subset,
of these three WW domains are required for Nedd4 to regulate
Na+ channel activity. In this study, we have used a
combination of in vitro binding assays and whole cell patch
clamp analysis to investigate the role of the WW domains of Nedd4 in
mediating the feedback inhibition of Na+ channels by raised
intracellular Na+.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
ENaC
subunits were PCR amplified2 and cloned into either
BamHI or BamHI/EcoRI sites of
pGEX-2TK.
,
, and
subunits
of ENaC were cloned from mouse mandibular duct cells, obtained as
described previously (30), by reverse transcriptase PCR. mRNA was
reverse transcribed and then PCR amplified using a set of nested
primers.2 Amplified fragments were cloned into pGEM-T-easy
(Promega) and verified by sequencing.
harboring the appropriate GST
expression plasmid were diluted 1/50, grown for 2 h at 37 °C,
induced with 1 mM isopropyl
-D-thiogalactoside, and grown for an additional 5 h
at 37 °C. Bacterial cell pellets were resuspended in
phosphate-buffered saline, lysed by sonication, and clarified by
centrifugation at 10,000 × g for 10 min.
Glutathione-Sepharose (Amersham Pharmacia Biotech) was incubated with
the cleared lysate for 60 min at room temperature, and then the beads
were washed three times with phosphate-buffered saline. Fusion protein
was eluted with glutathione buffer according to the manufacturer's protocol. Protein concentration was measured using a BCA kit (Pierce).
-32P]ATP in a buffer containing 20 mM
Tris-HCl (pH 7.5), 100 mM NaCl, 12 mM
MgCl2, and 1 mM dithiothreitol for 60 min at
4 °C. Beads were washed five times in phosphate-buffered saline, and
labeled protein was eluted with glutathione buffer. To prepare WW
domain protein filters, approximately 2 µg of each GST fusion protein was resolved on SDS-polyacrylamide electrophoresis gels and transferred to nitrocellulose membrane (Schleicher & Schüll). Membranes were blocked in Hyb75 (31) and then hybridized with either
,
, or
ENaC 32P-labeled protein probes for 4 h at 4 °C in
Hyb75. Membranes were washed three times in Hyb75 and exposed to x-ray film.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the regions of
Nedd4 that were fused to GST for protein production. All three WW
domains of mouse Nedd4 (mNedd4, wt) along with
mutants in either WW domain 1 (m1), WW domain 2 (m2), or WW domain 3 (m3) were fused to GST.
Single WW domains were also fused to GST in wild-type and mutant forms
and are labeled s1, s2, and s3
(wild-type WW domains 1, 2, and 3, respectively) and sm1,
sm2, and sm3 (mutant WW domains 1, 2, and 3, respectively). The third WW domain of human Nedd4 (hNedd4,
h3) was also fused to GST.
,
, and
ENaC Subunits Selectively Interact with Nedd4 WW
Domains--
The PY motifs of
,
, and
subunits of ENaC have
been shown to be required for interaction with Nedd4 WW domains (20), and therefore the intracellular carboxyl terminus of each ENaC subunit
containing the PY motif was cloned into the pGEX-2TK vector and used
for production of 32P-labeled protein probes (Fig.
2). Various GST-WW domain fusion proteins
were immobilized on nitrocellulose membranes by electroblotting following electrophoresis on polyacrylamide gels and probed with 32P-labeled GST-ENaC subunits. As shown in Fig.
2B, the
subunit of ENaC does not bind to the control,
GST (lane 1), but binds with equal strength to the wild type
(wt) protein (lane 2) and the WW domain 1 mutant,
m1 (lane 3), suggesting that it does not bind to
WW domain 1. The
subunit of ENaC shows reduced binding ability to
mutant WW domain 2 (m2) and even less affinity for mutant WW
domain 3 (m3) which suggests that both WW domains 2 and 3 can bind to the
subunit, although the
subunit has a preference
for WW domain 3. The
ENaC subunit has very similar binding
characteristics to the
subunit and binds with similar strength to
wt and m1 but with reduced affinity to m2 and m3 (Fig. 2C).
This suggests that the
subunit can bind to WW domains 2 and 3 but
not to WW domain 1 and may also have a slight preference for WW domain
3. The
ENaC subunit bound efficiently to both wt and m1, once again
suggesting that it does not interact with WW domain 1. The
ENaC
subunit bound weakly to m2 and m3, indicating that it has affinity for
both WW domains 2 and 3.
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Fig. 2.
Varying specificity of
,
, and
ENaC for Nedd4 WW domains. A,
Coomassie Blue stained gel of GST fusion proteins as outlined in Fig. 1
and indicated at top of the gel. Lane 1 contains
GST alone and molecular mass markers in kDa are indicated on the
right hand side of the gel. B-D, far-Western
blots of the above gel probed with 32P-labeled
,
, or
ENaC protein probes, respectively.
ENaC subunit bound to WW domains 2 and 3 with similar affinity, but not
to WW domain 1, confirming the result in Fig. 2B. The
subunit also bound to human WW domain 3, which is not found in mouse
Nedd4. As was the case in Fig. 2, the
subunit showed similar
binding characteristics to the
subunit by binding to murine WW
domains 2 and 3 and human WW domain 3, but not to WW domain 1 (Fig.
3C). The
ENaC subunit displayed a higher affinity for
murine WW domain 2 and human WW domain 3 and reduced affinity for
murine WW domain 3, but like the
and
ENaC subunits, showed no
affinity for WW domain 1 (Fig. 3D).
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Fig. 3.
Specificity of
,
, and
ENaC for individual Nedd4 WW domains.
A, Coomassie Blue stained gel of GST fusion proteins as
outlined in Fig. 1 and indicated on top of the gel.
Molecular mass markers in kDa are indicated on the right hand
side of the gel. B-D, far-Western blots of the above
gel probed with 32P-labeled
,
, or
ENaC protein
probes, respectively.
,
, and
subunits of murine ENaC from granular duct cells of mouse mandibular glands. When used in far-Western analyses, GST fusion proteins generated from all three subunits of murine ENaC carboxyl termini gave results similar to those shown in Figs. 2 and 3 (data not
shown). This is not surprising given the close homologies evident in
and around the PY motifs of human and mouse ENaC subunits (Fig.
4). The PY motifs in the
and
subunits (PPAY and PPNY, respectively) of mouse and human ENaC are
identical, whereas in the
subunit, the sequence differs by a single
conservative change (PPRY in mouse compared with PPKY in human) (Fig.
4).
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Fig. 4.
A comparison of the carboxyl-terminal amino
acid sequences derived from ,
, and
subunits of mouse
and human ENaC. Only the sequences of the 50-60 amino acid
residues at the carboxyl termini of the three ENaC subunits are shown.
The PPxY motifs present in all three subunits are
underlined. The residues identical between the human and
mouse proteins are shown in bold. *, termination
codon.
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Fig. 5.
Dose response curve of inhibition of the
Na+-dependent feedback pathway by
the GST-WW protein. The figure represents the concentration
response relationship for the effects of inclusion in the pipette
solution of the GST-WW fusion protein on the chord conductance of the
amiloride-sensitive current when the pipette contained 70 mM Na+. The broken line indicates
the mean chord conductance when the 0 Na+ pipette solution
was used.
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Fig. 6.
All three WW domains of Nedd4 are required to
inhibit the Na+-dependent feedback
pathway. A, the effects on the chord conductance of the
amiloride-sensitive Na+ current of the inclusion in the 70 mM pipette solution of the control GST-WW fusion protein
(100 µg/ml) and of variants of this fusion protein (100 µg/ml) in
which one WW domain has been mutated, leaving the other two intact
(m1, m2, m3). B, the
effects on the chord conductance of the amiloride-sensitive
Na+ current of the inclusion in the 70 mM
pipette solution of various mixtures of fusion proteins containing
individual WW domains of murine Nedd4 (s1, s2,
s3) in equal concentrations totaling 300 µg/ml.
C, the effects on the chord conductance of the
amiloride-sensitive Na+ current of the inclusion in the 70 mM pipette solution of the h3 construct carrying the WW
domain 3 of human Nedd4 (100 µg/ml) with pairs of constructs of
single WW domains of murine Nedd4 (s1, s2, and
s3, 100 µg/ml each). Details of the constructs used in
this figure are given in Fig. 1.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
ENaC subunits. Because mouse
and rat Nedd4 WW domain 1 sequences are highly homologous (95%
identical), the possibility of different ligand specificities is highly
unlikely. Our data show that, similar to mouse, the human Nedd4 WW
domain 1 does not interact with either of the three ENaC subunits. We
are therefore unsure about the reason for discrepancy between our
results and those reported by Staub et al. (20). It is worth
noting that two other proteins p45/NF-E2 and RNA polymerase II, which
interact with Nedd4 through their PY motifs, also do not bind WW domain
1 (38), suggesting that WW domain 1 of Nedd4 may have very restricted
ligand-binding specificity.
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Fig. 7.
Model for interaction of Nedd4 with
ENaC. In response to high intracellular Na+, an as yet
unidentified factor (X) is activated and binds to Nedd4 via
WW domain 1. This serves to recruit Nedd4 to ENaC and/or stabilize the
binding of Nedd4 WW domains 2 and 3 to the ,
, and
subunits
of ENaC. We postulate that a stable Nedd4/ENaC complex will
comprise one ENaC tetramer, two molecules of Nedd4, and two molecules
of factor X. Once stably bound, Nedd4 can ubiquitinate (u)
ENaC and stimulate its internalization and/or subsequent
degradation.
The finding that all three WW domains of murine Nedd4 must be occluded
to inhibit the Na+ feedback loop suggests that more than
one WW domain/PY motif contact needs to occur for stable complex
formation between Nedd4 and ENaC. This is supported by the molecular
understanding of the hypertensive disorder Liddle's syndrome, where
mutation in a single ENaC subunit ( or
) is sufficient to cause a
disease phenotype (7-11). As ENaC is a tetramer, presumably three
subunits with intact PY motifs will still be present in Liddle's
syndrome patients. This would seem insufficient to form a stable
complex with Nedd4, however, as hypertension results, presumably as a consequence of lack of negative regulation of ENaC by Nedd4.
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ACKNOWLEDGEMENT |
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We are grateful to Prof. M. Welsh for
providing the cDNA clones for human ,
, and
ENaC.
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FOOTNOTES |
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* This work was supported by grants from the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia.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.
§ Supported by a Dawes Scholarship from the Royal Adelaide Hospital.
Fellow of the Medical Foundation of the University of Sydney.
** Wellcome Trust Senior Fellow in Medical Science. To whom correspondence should be addressed: The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, P. O. Box 14, Rundle Mall, Adelaide, SA 5000, Australia. Fax: 61-8-8222-3139; E-mail: sharad.kumar{at}imvs.sa.gov.au.
2 Primer sequences are available upon request.
3 A. Dinudom and D. I. Cook, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ENaC, epithelial sodium channel; PY motif, PPxY sequence; GST, glutathione S-transferase; NMDG, N-methyl-D-glucamine; PCR, polymerase chain reaction; wt, wild type; FBP, formin binding protein.
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