Distinct characteristics of two human Nedd4 proteins with respect to epithelial Na+ channel regulation

Elena Kamynina, Caroline Tauxe, and Olivier Staub

Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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 (alpha , beta , and gamma ) (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 beta - or gamma -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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Fig. 1.   The Nedd4/Nedd4-like family of ubiquitin-protein ligases. A: phylogenetic tree analysis of Nedd4 and Nedd4-like proteins were done using the ClustaL_X package and viewed by TreeView (as described in METHODS). The scale bar represents 10 estimated changes/ 100 sites. Accession nos. of compared sequences are the following: AAB48949 (rNedd4-1); BAA12803 (mNedd4-1); BAA07655 (hNedd4-1); BAA23711 (hNedd4-2a); AAK00809 (mNedd4-2); CAA03915 (xNedd4); AAF49328 (dNedd4); AAF08298 (SMURF-1); AAD52564 (xSMURF-1); AAF57824 (dLack); CAB91803 (CAB91803); CAA68867 (Pub1); AAC03223 (Rsp5); CAB16903 (CAB16903); AAF60858 (AAF60858); AAD38975 (dSOD); AAC51325 (hWWP2); AAC51324 (hWWP1); AAB99764 (mItchy); and AAC04845 (hAIP4), where m is mouse and h is human. B: schematic view of the Nedd4 proteins. Accession no. of hNedd4-2b is AL137469.

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Plasmids and constructs. Human alpha beta gamma -ENaC (hENaC) cDNAs used for functional studies were a kind gift of Dr. Pascal Barbry (Valbonne, France). Rat alpha beta gamma -ENaC (rENaC) constructs, including alpha Y673Abeta Y618H,gamma Y628A used for functional studies and beta Y618H-flag, gamma 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-Delta C2), amino acids 1-146 were deleted, and L146 was replaced by Met. Amino terminally truncated mutants (for hNedd4-1-Delta N: amino acids 1-411 deleted, Val412 replaced by Met with preceding Kozak consensus; for hNedd4-2-Delta 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 beta -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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ABSTRACT
INTRODUCTION
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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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Fig. 2.   Expression of hNedd4-2 and hNedd4-1 in various tissues. A human multiple tissue Northern blot (MTN, Clontech) containing 2 µg of poly (A)+ RNA from the indicated tissues was hybridized with a probe derived from either hNedd4-2a (top), hNedd4-1 (middle), or beta -actin (bottom) as described in METHODS. sk., Skeletal; sm., small; periph. bl. leuk., peripheral blood leukocyte.

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, open circle ), 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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Fig. 3.   Both hNedd4-1 and hNedd4-2 can be properly expressed in Xenopus laevis oocytes. X. laevis oocytes were injected with either 10 ng of hNedd4-1 or hNedd4-2a and lysed after an overnight incubation. The proteins expressed in the lysate were analyzed by SDS-PAGE/Western by using either an anti-rat Nedd4 WW2 domain (left), anti-rat Nedd4 HECT domain (specific for Nedd4-1; middle), or an anti-mouse Nedd4-2 antibody. The anti-rat Nedd4 WW2 domain antibody crossreacts with other proteins, likely proteins containing WW domains as well. n.i., Noninjected oocytes.



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Fig. 4.   hNedd4-2 efficiently inhibits human epithelial sodium channel (hENaC) activity in a dose-dependent manner, whereas hNedd4-1 has only a minor effect. A: immunoblot analysis showing expression of hNedd4-1 or hNedd4-2a with increasing amounts of injected hNedd4 cRNA (ng/oocyte indicated). B: corresponding normalized amiloride-sensitive Na+ currents (INa). The measured currents were normalized to control oocytes (i.e., 0 ng of hNedd4-1 or hNedd4-2a cRNA). , hNedd4-1; open circle , hNedd4-2a (n = 40 oocytes from 4 frogs).

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-Delta 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 hNedd4-1Delta 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, open circle ). 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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Fig. 5.   Effect of removal of C2 domain from hNedd4-1 or addition of C2 domain to hNedd4-2a on regulation of ENaC. A: dose-dependent expression of hNedd4-1-Delta C2 without C2 or hNedd4-2+C2, as analyzed by Western blotting, using an anti-Nedd4 antibody. Amount of injected hNedd4 cRNA per oocyte is indicated. B: corresponding amiloride-sensitive INa normalized to control. , hNedd4-1-Delta C2 without C2; open circle , hNedd4-2a+C2 (n = 20 oocytes from 2 frogs).

hNedd4-1 and hNedd4-2 have only minor effects on ENaC lacking all PY motifs. We have previously shown that suppression of ENaC activity by either mNedd4-2 or its ortholog xNedd4 depends on the presence of intact PY motifs within ENaC. To confirm these findings with hNedd4, we coexpressed the hNedd4 constructs together with a rENaC channel in which the tyrosine of each PY motif, shown to be essential for the interaction with mNedd4-2 (26), was mutated (rENaCDelta PY). We found that when 10 ng of hNedd4 cRNA were injected, neither the hNedd4-2a proteins (with and without the C2 domain) nor hNedd4-1Delta C2, had a significant inhibitory effect, whereas wild-type hNedd4-1 containing the C2 domain reduces ENaCDelta PY activity to ~75% (Fig. 6), indicating that the previously observed reduction in wild-type ENaC activity by wild-type hNedd4-1 (see Fig. 4B) was not mediated by the PY motifs.


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Fig. 6.   Effect of various Nedd4 constructs on ENaC channels lacking intact PY motifs. Oocytes were injected with cRNA encoding alpha Y673Abeta Y618Hgamma Y628A together with 10 ng of cRNA encoding the hNedd4 proteins as indicated. Amiloride-sensitive INa are normalized to control cells (injected only with the rENaC mutants; n = 20 oocytes from 2 frogs). *P < 0.05.

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-Delta N and hNedd4-2-Delta 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-Delta N and hNedd4-2-Delta 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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Fig. 7.   Effect of truncated Nedd4 constructs and of a splice variant (hNedd4-2b) on ENaC activity. A: dose-dependent expression of hNedd4-1-Delta N, hNedd4-2-Delta N, and hNedd4-2b as analyzed by immunoblotting of cell lysates. The amount of injected Nedd4 cRNA per oocyte is indicated. B: corresponding amiloride-sensitive INa normalized to the control (n = 20 oocytes from 2 frogs).

hNedd4-2, but not hNedd4-1, binds to ENaC in a PY motif-dependent manner. To determine how the different hNedd4 forms interact with ENaC, we expressed rENaC subunits tagged with a FLAG epitope on beta - and gamma -rENaC in X. laevis oocytes (see METHODS), together with the hNedd4 constructs. Oocytes were labeled overnight with [35S]methionine, membrane fractions were solubilized, and the FLAG-tagged rENaC subunits were immunoprecipitated with anti-FLAG antibodies. The precipitated proteins were analyzed by SDS-PAGE and autoradiography (Fig. 8). As can be seen in Fig. 8 (top), all three ENaC subunits (arrowheads) were efficiently immunoprecipitated. When either hNedd4-2a (with or without the C2 domain) (Fig. 8, top, lanes 4 and 5) or the truncated forms of hNedd4-1 and hNedd4-2 (lanes 6 and 7) were expressed with rENaC, additional faint bands were coimmunoprecipitated (Fig. 8, top, *). Whereas we could identify the coimmunoprecipitated bands in lanes 6 and 7 as hNedd4-1-Delta N and hNedd4-2-Delta N, respectively, by Western blotting with anti-Nedd4 antibodies (data not shown), this was not possible for hNedd4-2a and hNedd4-2a+C2 (lanes 4 and 5), most likely because the coimmunoprecipitated material was below the detection level. However, these proteins migrated at the expected size (compared with the corresponding proteins in the cellular lysate identified by Western blotting using anti-Nedd4 proteins, Fig. 8, bottom). In contrast, when hNedd4-1 (with or without C2 domain) was expressed, no additional bands were visible (Fig. 8, top, lanes 2 and 3), although these proteins were strongly expressed (Fig. 8, bottom, lanes 2 and 3). Regarding the two truncated hNedd4 constructs, we note that hNedd4-2-Delta N precipitated more efficiently than hNedd4-1-Delta N (Fig. 8, top, lane 6 vs. lane 7), which may suggest that the WW domains 3 and 4 of hNedd4-2 have a higher affinity toward the ENaC PY motifs. When rENaC subunits lacking functional PY motifs were expressed, no proteins coimmunoprecipitated with the channel subunits (Fig. 8, middle), indicating that the above observed interactions depended on the presence of a complete PY motif. Surprisingly, both truncated mutants, in contrast to the other hNedd4 proteins tested, affected the total cellular pool of ENaC (Fig. 8, top, lanes 6 and 7), thereby reducing the levels of alpha - and beta -ENaC considerably. This result is probably due to the direct interaction of these hNedd4 truncation mutants with ENaC and not to some general toxic or competitive effect on protein biosynthesis, as channel proteins devoid of functional PY motifs were not similarly reduced (Fig. 8, middle).


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Fig. 8.   In vivo interaction of the various Nedd4 mutants with ENaC, as revealed by immunoprecipitation. Oocytes were injected with cRNA encoding flagged rENaC, either alone (lane 1) or together with hNedd4 constructs as indicated. Oocytes were labeled overnight with [35S] methionine and homogenized; a membrane fraction was solubilized, and immunoprecipitations were performed with anti-FLAG antibody. The immunoprecipitated proteins were analyzed by autoradiography (top and middle) or Western blotting with anti-Nedd4 antibody (bottom) Top: wild-type ENaC. Middle: same as in top but with alpha Y673Abeta Y618H-flaggamma W574stop-flag rENaC. Bottom: cell lysates analyzed by Western blotting using an anti-Nedd4 antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-Delta N and hNedd4-2-Delta 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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