Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland
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ABSTRACT |
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The epithelial Na+ channel (ENaC) is regulated via PY motif-WW domain interaction by the mouse (m) ubiquitin-protein ligase mNedd4-2 but not by its close relative mNedd4-1. Whereas mNedd4-1 is composed of one C2, three WW, and one HECT domain, mNedd4-2 comprises four WW domains and one HECT domain. Both proteins have human (h) homologs, hNedd4-1 and hNedd4-2; however, both of them include four WW domains. Therefore, we characterized hNedd4-1 and hNedd4-2 in Xenopus laevis oocytes with respect to ENaC binding and interaction. We found that hNedd4-2 binds to and abrogates ENaC activity, whereas hNedd4-1 does not coimmunoprecipitate with ENaC and has only modest effects on ENaC activity. Structure-function studies revealed that the C2 domain of hNedd4-1 prevents this protein from downregulating ENaC and that WW domains 3 and 4, involved in interaction with ENaC, do not by themselves provide specificity for ENaC recognition. Taken together, our data demonstrate that hNedd4-2 inhibits ENaC, implying that this protein is a modulator of salt homeostasis, whereas hNedd4-1 is not primarily involved in ENaC regulation.
epithelial sodium channel; sodium homeostasis; hypertension; ubiquitination; WW domain; C2 domain
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INTRODUCTION |
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PRECISE REGULATION OF
NA+ homeostasis, extracellular volume, and blood
pressure is an important function of the kidney. Thereby, the
amiloride-sensitive epithelial Na+ channel (ENaC), located
in the apical membrane of tight epithelia in distal regions of the
nephron, constitutes the rate-limiting step of sodium reabsorption and
plays an essential role in sodium handling (14). ENaC is
composed of three homologous subunits (,
, and
) (4,
6) that have similar topology and contain two transmembrane
domains, a cytosolic loop and short cytosolic NH2 and COOH
termini (5, 36, 42). Of interest are proline-rich regions
in the COOH termini of all three subunits that conform to the consensus
sequence of PY motifs (xPPxY, where x is any amino acid, P is proline,
and Y is tyrosine), which have been shown to interact with WW domains
(8). Deletion or mutations of the PY motifs in the COOH
termini of either the
- or
-subunit are the cause of Liddle's
syndrome, an inherited autosomal dominant form of human hypertension
(16, 17, 24, 25, 41, 47). The disease is characterized by
early onset of severe hypertension, salt sensitivity, hypokalemia,
metabolic alkalosis, and low aldosterone and renin plasma
concentrations (30). We have shown that these PY motifs
are the sites interacting with the rat ubiquitin-protein ligase Nedd4
(which we now refer to as rNedd4-1; see below) (44).
Nedd4 is the founding member of the Nedd4/Nedd4-like family of
ubiquitin-protein ligases (Fig. 1; for a
review, see Refs. 20 and 38), proteins that are composed
of a C2 (Ca2+-dependent lipid binding) domain
(34), two to four WW (protein-protein interaction) domains
(45), and the catalytic COOH-terminal HECT (i.e.,
Homologous to E6-AP Carboxy
Terminus) domain (23). The identification of a ubiquitin-protein ligase as a binding partner suggested that the ubiquitin system (21) may play a role
in the regulation of ENaC. Ubiquitination (the modification of proteins with ubiquitin polypeptides) serves to target either intracellular proteins for rapid degradation by the proteasome or plasma membrane proteins for rapid internalization (for comprehensive reviews on
ubiquitination of membrane proteins, see Refs. 3,
22, and 38). We and others have established that
Nedd4 is a regulator of ENaC (1, 11, 12, 15, 18). However,
although we could show that Xenopus laevis Nedd4
(xNedd4) downregulates ENaC when coexpressed in X. laevis oocytes and that this regulation involves the control of
cell surface expression (1), we were not able to
demonstrate this for rat Nedd4 (rNedd4), which prompted us to look for
other mammalian Nedd4 proteins possibly involved in ENaC interaction.
We identified a novel murine Nedd4 protein (mNedd4-2), which is highly
similar to xNedd4 (see Fig. 1) and binds to and suppresses
ENaC activity in X. laevis oocytes (26),
whereas the originally identified Nedd4 proteins from mouse or rat
(referred to as Nedd4-1) do not bind to and regulate ENaC. mNedd4-2 is
composed of four WW domains and one HECT domain and lacks the C2
domain. This is different from mNedd4-1, which contains one C2 domain, three WW domains, and the HECT domain. As can be seen in Fig. 1B, both proteins have homologs in humans [hNedd4-1 and
hNedd4-2a (encoded by the genes KIAA0093 and KIAA0439, respectively)].
There also exists an alternatively spliced transcript of hNedd4-2a, which we refer to as hNedd4-2b (GenBank accession no. AL137469). In
contrast to rat and mouse Nedd4-1, hNedd4-1 contains four WW domains,
as is the case for the hNedd4-2a protein.
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In this report, we have investigated the characteristics of hNedd4-1 and hNedd4-2 in terms of ENaC regulation. Moreover, the structural differences between the mouse and human Nedd4 proteins (presence of C2 domains, number of WW domains) prompted us to study the role of the human Nedd4 domains in providing specificity for ENaC recognition.
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METHODS |
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Plasmids and constructs.
Human -ENaC (hENaC) cDNAs used for functional
studies were a kind gift of Dr. Pascal Barbry (Valbonne, France). Rat
-ENaC (rENaC) constructs, including
Y673A
Y618H,
Y628A used for
functional studies and
Y618H-flag,
W574stop-flag containing a
FLAG epitope used for coimmunoprecipitation experiments, were
kindly provided by Drs. Laurent Schild and Dmitri Firsov (University of
Lausanne, Switzerland). Human cDNAs for hNedd4-1 (gene
name KIAA0093) and hNedd4-2 (gene name KIAA0439) were
obtained from the Kazusa DNA Research Institute. hNedd4-2b
(alternatively spliced hNedd4-2 variant; GenBank accession no.
AL137469) was obtained from the Resource Center/Primary Database
(Berlin, Germany). A HindIII/HindIII
fragment of hNedd4-1 cDNA was cloned into the pSDeasy plasmid
(35) linearized by HindIII. A
SalI/NotI fragment of hNedd4-2a cDNA was cloned
into the pSDeasy plasmid linearized by SalI and
NotI. For the hNedd4-2a+C2 construct, the first amino acid
(Pro) was replaced by Met. For hNedd4-1 without the C2 construct (hNedd4-1-
C2), amino acids 1-146 were deleted, and
L146 was replaced by Met. Amino terminally truncated
mutants (for hNedd4-1-
N: amino acids 1-411 deleted,
Val412 replaced by Met with preceding Kozak
consensus; for hNedd4-2-
N: amino acids 1-478 deleted,
Glu479 replaced by Met with preceding Kozak consensus) were
cloned into the pSDeasy plasmid.
Expression and function of ENaC channels in X. laevis oocytes.
For functional expression studies, linearized hENaC (or rENaC when
indicated) and hNedd4-1/2 constructs were transcribed by using SP6-RNA
polymerase, and 10 ng cRNA encoding ENaC (3.3 ng of each subunit) with
or without indicated amounts of cRNA encoding hNedd4-1/hNedd4-2 or
their mutants were coinjected into X. laevis oocytes.
Electrophysiological measurements were performed 12-24 h after
cRNA injection, using the two-electrode voltage-clamp technique and
measuring the amiloride-sensitive Na+ currents at 100 mV
as described before (40). All values were normalized to
the control value (oocytes only injected with ENaC) in one given batch
of oocytes. Data are presented as means ± SE. The statistical
significance of the differences between the means was estimated by
using a bilateral Student's t-test for unpaired data.
Biochemical analyses.
For Western blot analysis of hNedd4-1/2 expression, oocytes were kept
at 4°C after the electrophysiological measurements were made, pooled,
and lysed (25 µl/oocyte) in Triton X-100 homogenization buffer (20 mM
Tris · HCl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluroide, 10 µg/ml leupeptin, 10 µg/ml peptstatin A, 10 µg/ml aprotinin) at 4°C. After centrifugation at
4°C for 10 min at 20,000 g, the supernatant was recovered
and stored at 80°C. For the coimmunoprecipitation, oocytes
coinjected with cRNA encoding rENaC subunits with a FLAG-epitope on the
extracellular loop (13), and hNedd4-1/2 or their mutants
were incubated overnight in 0.1 mCi/ml [35S]methionine,
and homogenized in 50 mM HEPES, pH 7.4, 83 mM NaCl, 1 mM
MgCl2, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml
pepstatin A, and 10 µg/ml aprotinin. An aliquot was removed for
analysis of the total homogenate. Membranes were prepared by
centrifugation at 4°C for 10 min at 900 g in a microfuge,
followed by centrifugation of the supernatant at 4°C for 20 min at
20,000 g. The recovered membranes were solubilized (25 µl/oocyte) in Triton X-100 homogenization buffer.
Coimmunoprecipitation was performed with anti-FLAG antibody (Kodak) and
protein G-Sepharose (Sigma). The immunoprecipitated material was then
analyzed by separating it on a 7% SDS-PAGE gel, followed by
autoradiography, and the homogenate was analyzed on a 10% SDS-PAGE
gel, followed by Western blotting using an anti-rNedd4-WW2 antibody at
a dilution of 1:1,000 (44).
Antibodies. For Western blot analysis, the following antibodies were used: anti-rNedd4 WW domain 2 (44), anti-rNedd4 HECT domain (46), and an antibody raised against a glutathione S-transferase fusion protein containing amino acids 302-376 of mNedd4-2.
Northern blot analysis.
A multiple-tissue Northern blot (MTN) containing ~2 µg purified
polyA+ RNA/lane from 12 different human tissues was
obtained from Clontech. The blot was prehybridized for 1 h at
65°C in ExpressHyb (Clontech) according to the manufacturer's
directions. A hNedd4-1 EcoRI/EcoRI fragment
(nucleotides 3596-3939), a hNedd4-2
XhoI/XhoI fragment (nucleotides 1-541), or
-actin cDNA (provided by Clontech) was used as a random primed
32P-labeled probe. Hybridization was for 2 h at
65°C. The blot was rinsed twice with 2× standard sodium
citrate/0.1% SDS and twice for 30 min at 55°C with 0.1× standard
sodium citrate/0.1% SDS. The blot was analyzed by autoradiography.
Phylogenetic tree analysis. Protein sequences of Nedd4 and Nedd4-like proteins were analyzed by using the ClustaL_X package (48), which consists of following steps: 1) construction of a distance matrix by pairwise dynamic programming global alignment, followed by approximate conversion of similarity scores to evolutionary distances using the model of Kimura (28); 2) construction of a guide tree by a neighbor-joining clustering algorithm by Saitou and Nei (39); and 3) progressive alignment at nodes in order of decreasing similarity using sequence-sequence, sequence-profile, and profile-profile alignment. The calculated tree was viewed by using the TreeView program.
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RESULTS |
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Existence of two human Nedd4 proteins. We have recently shown that in the mouse there are at least two Nedd4 proteins (mNedd4-1 and mNedd4-2) (26), both of which are expressed in murine epithelial cells derived from the cortical collecting duct and behave differently in terms of ENaC regulation, as mNedd4-2 binds to and regulates ENaC and mNedd4-1 is incompetent in ENaC binding and regulation in X. laevis oocytes (26). As illustrated in Fig. 1A, the Nedd4 proteins are part of the Nedd4/Nedd4-like family of ubiquitin-protein ligases, which are composed of a variable number of WW domains, a HECT domain, and, in most cases, a C2 domain (20, 27, 38). Phylogenetic analysis shows that they cluster into at least five different branches. The branch comprising Rsp5, Pub1, CAB16903, and CAB91803, proteins that are all either yeast or fungal, likely represents the ancestral members of this family. The other branches contain the following proteins: 1) WWP1, WWP2, and AIP4 (Itchy); 2) SMURF; 3) KIAA0322; and 4) the Nedd4 proteins. The latter proteins cluster into two clades, one including hNedd4-1, mNedd4-1, and rNedd4-1 and the other hNedd4-2, mNedd4-2, and xNedd4. This characterizes them as close Nedd4 paralogs, clearly distinct from the other Nedd4-like proteins (Fig. 1A). The branching pattern and the taxonomic composition of the two Nedd4 clades, as well as the identification of a Drosophila melanogaster genomic sequence encoding a protein closely related to both hNedd4-1 (45% identity) and hNedd4-2 (46% identity), suggest a duplication event early in vertebrate evolution before amphibian and mammalian diversification. On the basis of these observations, we speculate that Nedd4-1 and Nedd4-2 orthologs will be found in all mammals.
Differential expression of hNedd4 mRNAs.
As a first step toward the characterization of hNedd4-1 and hNedd4-2,
we determined the tissue expression of hNedd4-2 and hNedd4-1 mRNA by
performing Northern blot analysis on a multiple tissue blot using cDNA
probes for either hNedd4-2 or hNedd4-1 (Fig.
2). We found at least four different
hNedd4-2 mRNA species in the kidney, lung, and placenta of ~10, 5, 4.2, and 4.0 kb (Fig. 2, top). Some of these transcripts,
albeit at a lower abundance, were also observed in brain, heart,
skeletal muscle, colon, liver, and small intestine, whereas no hNedd4-2
mRNAs were detectable in thymus, spleen, and the peripheral blood
leukocytes. Most of these bands were also seen by using a cDNA probe
derived from the 3'-noncoding end (data not shown), suggesting that
they all originate from the same gene and represent alternatively
spliced transcripts. For hNedd4-1 (Fig. 2, middle), there
were two detectable mRNA species of ~8 and 6 kb. The 8-kb message was
only present in skeletal muscle, whereas the 6-kb message was observed
in heart, skeletal muscle, kidney, liver, and placenta (Fig. 2,
middle). Hence, hNedd4-2 and hNedd4-1 have different
expression patterns in the tissues analyzed.
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hNedd4-2 inhibits ENaC activity more efficiently than hNedd4-1.
We have recently demonstrated that, in X. laevis oocytes,
mNedd4-2, but not mNedd4-1, binds to and inhibits ENaC
(26). As outlined above, there are two major differences
in the molecular organization between the two murine proteins: mNedd4-2
contains no C2 domain and four WW domains, as opposed to mNedd4-1,
which has a C2 domain but only three WW domains. The observed
difference in ENaC regulation could therefore be due either to the
presence of an additional WW domain in mNedd4-2 or to the C2 domain in mNedd4-1, which prevents mNedd4-1 from interaction with ENaC. In
humans, both hNedd4-1 and hNedd4-2a contain four WW domains (Fig.
1B). Therefore, we wanted to know whether the findings on the mNedd4 proteins regarding ENaC inhibition are also valid for the
human Nedd4 orthologs and tested the expression of hNedd4-1 and
hNedd4-2 in X. laevis oocytes. Lysates from such oocytes
injected with cRNA encoding either hNedd4-1 or hNedd4-2 were analyzed
by Western blotting by using three different anti-Nedd4 antibodies, demonstrating that both hNedd4-1 and hNedd4-2 were properly expressed (Fig. 3). For subsequent analysis, the
anti-WW2 antibody was used, as it was allowed to follow the expression
of both isoforms. Next, we injected increasing amounts of cRNA encoding
either hNedd4-1 or hNedd4-2 into X. laevis oocytes together
with human ENaC (hENaC) cRNA. After an overnight incubation,
amiloride-sensitive Na+ currents were measured by the
two-electrode voltage-clamp method, an indication of ENaC activity at
the plasma membrane. Oocytes were kept on ice after measurements and
used for biochemical analysis. Figure
4A shows the expression levels
of the two Nedd4 proteins, as determined by Western blotting of cell
lysates with the anti-Nedd4 WW2 antibody. Figure 4B
demonstrates that, despite the strong expression of hNedd4-1, there was
a relatively modest effect on hENaC activity, even at the highest level
of injected cRNA (~30% inhibition of amiloride-sensitive
Na+ currents for 60 ng of hNedd4-1 cRNA injected per
oocyte) (Fig. 4B, ). On the other hand, hNedd4-2a had a
dramatic effect on ENaC activity: even at the lowest concentration of
cRNA injected, at which the protein was hardly detectable by Western
blot techniques, we observed very strong suppression of hENaC currents
(~85% inhibition for 3.7 ng of cRNA injected) (Fig. 4B,
), suggesting that the efficiency of hNedd4-2a in downregulating
ENaC is more than an order of magnitude higher than that of hNedd4-1.
Similar results were obtained with rat ENaC (rENaC; not shown).
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Removal of the C2 domain renders hNedd4-1 more competent for ENaC
regulation.
As mentioned above, all the identified Nedd4-1 proteins contain a C2
domain, whereas mNedd4-2 and possibly hNedd4-2 lack the C2 domain (Fig.
1B), suggesting that the C2 domain may play a role in
substrate specificity and interaction with ENaC. Regarding hNedd4-2a,
we cannot exclude the possibility that the sequence published in
GenBank is incomplete at its 5'-end and that another methionine is
situated further upstream, which would result in the presence of a
complete C2 domain. We therefore expressed in X. laevis
oocytes either a hNedd4-1 mutant lacking the C2 domain (hNedd4-1-C2)
or a hNedd4-2a construct containing a C2 domain (hNedd4-2a+C2),
obtained by adding an artificial methionine further upstream (see
METHODS). Both proteins were properly expressed (Fig.
5A). Figure 5B
shows that hNedd4-1
C2 was much more effective in ENaC suppression
(Fig. 5B,
; ~60% inhibition with 6 ng of cRNA
injected) compared with the wild-type hNedd4-1 form containing the C2
domain (compare with Fig. 4B,
; there was no reduction of
ENaC currents with 10 ng of cRNA injected). On the other hand, addition
of the C2 domain to hNedd4-2a did not significantly change the
characteristics of hNedd4-2a regarding ENaC inhibition (Fig. 5B,
). Hence the presence of a C2 domain in hNedd4-1, but
not in hNedd4-2a, prevents this protein from downregulating the
epithelial Na+ channel in X. laevis oocytes.
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WW domains 3 and 4 are involved in ENaC recognition but are not
sufficient to determine substrate specificity.
We have previously shown that the WW domains 1 and
2 of xNedd4 and mNedd4-2 are less important than
the WW domains 3 and 4 for ENaC inhibition
(26), suggesting that WW domains 3 and
4 provide substrate specificity. To further analyze this
issue, we truncated both hNedd4-1 and hNedd4-2, thereby removing WW
domains 1 and 2 and creating mutants that only
contain WW domains 3 and 4 and the HECT domain
(Fig. 7A, hNedd4-1-N and
hNedd4-2-
N). We also tested the splice variant hNedd4-2b, which
lacks the second WW domain. These constructs were expressed together
with hENaC in oocytes, their expression verified by Western blotting
(Fig. 7A), and the effect on amiloride-sensitive
Na+ currents was investigated. We found that both hNedd4
truncation mutants were efficient in ENaC suppression, whereas the
splice variant (hNedd4-2b) modulated ENaC activity to a lower extent, comparable to the effect of hNedd4-2a (Fig. 7B). The finding
that both truncated hNedd4 proteins inhibit ENaC confirms that the WW
domains 3 and 4 are involved in Nedd4-dependent
ENaC downregulation. This is further corroborated with the splice
variant hNedd4-2b, which lacks the second WW domain and still abrogates
ENaC activity. However, the observation that hNedd4-1-
N and
hNedd4-2-
N inhibit ENaC to a similar extent shows that the
selectivity in ENaC recognition is not given by WW domains 3 and 4 but by more NH2-terminal regions of the
proteins.
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DISCUSSION |
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In this report we show that hNedd4-1 and hNedd4-2a, despite their
structural similarity (64% identity and 74% similarity at the protein
level) and the presence of four highly homologous WW domains in both
proteins, have different characteristics in terms of binding and
regulation of the ENaC. hNedd4-2a, similar to its mouse and X. laevis orthologs (1, 26), strongly suppresses ENaC
activity and coimmunoprecipitates in a PY motif-dependent mode. In
contrast, hNedd4-1 has only modest effects on ENaC, and interaction
between hNedd4-1 and ENaC cannot be detected by coimmunoprecipitation experiments. We cannot exclude, however, that some hNedd4-1 proteins (below the detection limit of our system) bind to ENaC. Our data provide some interesting cues about the structural parameters, which
determine the differences in substrate specificity of the two human
Nedd4 ubiquitin-protein ligases. Clearly, the number of WW domains is
not decisive, as both human proteins contain four WW domains, but it is
possible that either the different arrangement of the WW domains within
the protein or individual affinities of the hNedd4-1 and hNedd4-2 WW
domains toward the PY motifs on ENaC account for the difference in ENaC
inhibition. Our coimmunoprecipitation experiments suggest that the WW
domains 3 and 4 of hNedd4-2 have a higher
affinity toward ENaC than the corresponding WW domains of hNedd4-1. On
the other hand, there is no difference in ENaC downregulation between
the two truncation mutants, and the fact that both hNedd4-1-N and
hNedd4-2-
N (in contrast to full-length hNedd4-1) are binding in vivo
to ENaC demonstrates that the apparent difference in affinity is not
sufficient to explain the specificity in ENaC recognition; other more
NH2-terminal regions are also involved. Indeed, our data
demonstrate that the C2 domain partially accounts for the difference
between hNedd4-1 and hNedd4-2a, because the C2 domain of hNedd4-1
prevents suppression of ENaC by this protein (compare Figs.
4B and 5B). On the other hand, addition of a C2
domain to hNedd4-2 has no effect on ENaC regulation. C2 domains are
protein-lipid and protein-protein interaction domains originally
identified in Ca2+-responsive isoforms of protein kinase C
(10, 29) and later were found in a large number of
proteins such as phospholipase A2 (9),
synaptotagmin (32), rasGAP (49), and
phosphoinositide-specific phospholipase C (43) (for a
review on C2 domains, see Refs. 31 and 37). The C2 domain
in Nedd4 has been shown to be involved in apical targeting of rNedd4
(the rNedd4-1 ortholog) in Madin-Darby canine kidney cells
(34) and to interact with annexin XIIIb (33).
It is therefore possible that the Nedd4-1 C2 domain either interacts
with another endogenous oocyte protein, thereby sequestering it from
ENaC, or that the oocyte is missing an important adapter protein, which
facilitates the interaction with hNedd4-1, or that there is some
sterical hindrance provided by the C2 domain, which prevents proper
interaction of ENaC and hNedd4-1. Our findings are supported by the
ones of Farr et al. (12), who also showed that a hNedd4-1
protein lacking portions of the C2 domain abrogates ENaC activity, when
coexpressed in X. laevis oocytes (12).
Our Northern blot analysis demonstrates that the two hNedd4 proteins are differentially expressed, suggesting that they have distinct cellular functions in different tissues. The pattern of hNedd4-1 expression corresponds to the results reported by Anan et al. (2). hNedd4-2 is strongly expressed in the kidney, which is in agreement with the idea that this protein plays an important role in this tissue. For both hNedd4-1 and hNedd4-2, we detect messages of different sizes. In kidney, four hNedd4-2 messages with sizes of ~10, 5, 4.1, and 4.0 kb were detected. These forms may arise through alternative splicing, which may represent another mechanism of increasing the diversity of Nedd4 proteins or another level of regulation. As described above, the hNedd4-2b mRNA species represents such an alternatively spliced form of hNedd4-2 mRNA, which is derived from human testis and encodes a protein that lacks the second WW domain (Fig. 1B).
The present data establish that human Nedd4-2 is the primary ubiquitin-protein ligase regulating ENaC, an important finding in the understanding of Liddle's syndrome and salt-sensitive hypertension in humans. Our data and a previous report (26) suggest that WW domains 3 and 4 are involved in interaction with ENaC. Hence mutations in these domains could be expected to have an impact on hNedd4-2 dependent ENaC inhibition. The function of hNedd4-1 is less clear. Its effect on ENaC is much weaker than that of hNedd4-2, and, on the basis of the coimmunoprecipitation experiments, no direct interaction can be detected in vivo. However, this does not exclude that this protein may, under certain circumstances, participate in ENaC regulation. It is possible that mutations in humans, which lead to the truncation of a functional C2 domain in hNedd4-1 or change the affinities of the WW domains toward the PY motifs, may interfere with proper ENaC regulation and thereby either yield a pseudohypoaldosteronism type 1 (7) or a Liddle's phenotype (30). To determine, whether hNedd4-1 may interfere with the hNedd4-2 interaction, we have performed coinjection experiments but found no evidence for any kind of competition or mutual interaction between hNedd4-1 and hNedd4-2 (C. Debonneville and O. Staub, unpublished observations). Also, the C2 domain has been shown to be cleaved from Nedd4-1 protein during apoptosis, but no physiological function has been attributed to it (19).
Collectively, the present data show that there are important differences between hNedd4-1 and hNedd4-2a with respect to inhibition of ENaC, despite high similarity and the presence of four closely related WW domains in both proteins. hNedd4-2a is a much stronger inhibitor of ENaC than hNedd4-1. Part of the variation is due to the presence of the C2 domain in hNedd4-1, which prevents this protein from downregulating ENaC. WW domains 3 and 4, which appear to have a higher affinity toward ENaC in hNedd4-2 than in hNedd4-1, are not exclusively involved in mediating substrate specificity, as NH2-terminal regions are also important. In conclusion, we propose that hNedd4-2a is an inhibitor of ENaC and, hence, may be a regulator of Na+ homeostasis and therefore represent a susceptibility gene for arterial hypertension.
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ACKNOWLEDGEMENTS |
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We thank Dr. Pascal Barbry for providing cDNAs encoding human ENaC, the Kazusa DNA Research Institute, Japan, for cDNA encoding human Nedd4-1 (KIAA0093) and human Nedd4-2 (KIAA0439), Sophie Cordonnier for technical assistance, and Drs. Laurent Schild and Dmitri Firsov for providing rENaC mutants. We also thank Drs. B. Rossier, L. Schild, P. Shaw, J.-D. Horisberger, L. Müller, and D. Firsov for critically reading the manuscript.
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FOOTNOTES |
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This work was supported by grants of the Swiss National Science Foundation (31-52178.97), the Leenards Foundation, the Fondation Emma Muschamp, and the Novartis Research Foundation.
Address for reprint requests and other correspondence: O. Staub, Institute of Pharmacology and Toxicology, Univ. of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland (E-mail:olivier.staub{at}ipharm.unil.ch).
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.
Received 2 February 2001; accepted in final form 27 April 2001.
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