1. Liddle's syndrome: A novel mouse Nedd4 isoform regulates the activity of the epithelial Na+ channel 
Elena Kamynina, Christophe Debonneville, Robert P. Hirt, Olivier Staub 
Kidney International 
Volume 60, Issue 2, Pages (August 2001)
DOI: /j x
Copyright © 2001 International Society of Nephrology Terms and Conditions

2. Figure 1
Nedd4 isoforms are part of the Nedd4/Nedd4-like protein family. (A) Schematic representation of the proteins of the Nedd4/Nedd4-like protein family. ?, The presence of a C2 domain in hNedd4-2 is uncertain. The sequence of Drosophila Nedd4 predicts a signal sequence at the N terminus. The accession numbers of the protein sequences are indicated to the left. (B) Phylogenetic analyses of Nedd4 and Nedd4-like proteins. To rationalize the sequence divergence between the different Nedd4 proteins (Nedd4-1/2) within a phylogenetic framework, we aligned their protein sequences with more distantly related Nedd4-like proteins19. Three hundred sixty-three unambiguously aligned positions were used for tree inference with distance, parsimony, and maximum likelihood methods25,26. All methods recovered a clade composed of the Nedd4 paralogues (Nedd4-1 and Nedd4-2) with maximum bootstrap support values (100%). The split between Nedd4-1 and Nedd4-2 sequences and the basal position of the Drosophila Nedd4 (dNedd4) sequence were also supported with maximum/high support values. The shown tree (and bootstrap values) corresponds to the ML analysis. It was rooted on the h-KIAA0322 sequence based on the sequence features of the compared sequences (overall sequence length, similarity, and domain composition). Hence, the Nedd4 sequences were considered to have a uniquely shared common ancestor (they are monophyletic). The scale bar represents 10 estimated changes per 100 sites.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

3. Figure 2
Mouse Nedd4-2 (mNedd4-2, but not mNedd4-1, regulates rat epithelial sodium channel (rENaC) by binding to the channel complex in theXenopusoocytes. (A) Oocytes were injected with cRNA encoding αβγrENaC, either alone (rENaC/H2O) or with cRNA for mNedd4-1 (rENaC/mNedd4-1), its catalytically inactive mutant (rENaC/mNedd4-1CS), mNedd4-2 (rENaC/mNedd4-2), or the mNedd4-2 mutant (rENaC/mNedd4-2CS), or with an amino terminally truncated form of mNedd4-2 (rENaC/mNedd4-2ΔN), comprising WW domain 3 and 4 and the HECT domain), and amiloride-sensitive Na+ currents (INa) were measured by the two-electrode voltage-clamp method. The measured currents are normalized to control oocytes. (B) Oocytes were injected with cRNA encoding either nonflagged (lanes 1 through 3) or flagged (lanes 4 through 6) rENaC, either alone (lanes 1 and 4) or together with mNedd4-1 (lane 2 and 5) or mNedd4-2 (lanes 3 and 6). Oocytes were labeled overnight with [35S]-methionine and homogenized. A membrane fraction was solubilized, and immunoprecipitation was performed with anti-FLAG antibody. The immunoprecipitated proteins were analyzed by autoradiography (top panel) or Western blotting with anti-Nedd4 antibody (middle panel). Aliquots from the homogenate analyzed by immunoblotting show that mNedd4-1 and mNedd4-2 were properly expressed (bottom panel) (from21, with permission of the FASEB J).
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

4. Figure 2
Mouse Nedd4-2 (mNedd4-2, but not mNedd4-1, regulates rat epithelial sodium channel (rENaC) by binding to the channel complex in theXenopusoocytes. (A) Oocytes were injected with cRNA encoding αβγrENaC, either alone (rENaC/H2O) or with cRNA for mNedd4-1 (rENaC/mNedd4-1), its catalytically inactive mutant (rENaC/mNedd4-1CS), mNedd4-2 (rENaC/mNedd4-2), or the mNedd4-2 mutant (rENaC/mNedd4-2CS), or with an amino terminally truncated form of mNedd4-2 (rENaC/mNedd4-2ΔN), comprising WW domain 3 and 4 and the HECT domain), and amiloride-sensitive Na+ currents (INa) were measured by the two-electrode voltage-clamp method. The measured currents are normalized to control oocytes. (B) Oocytes were injected with cRNA encoding either nonflagged (lanes 1 through 3) or flagged (lanes 4 through 6) rENaC, either alone (lanes 1 and 4) or together with mNedd4-1 (lane 2 and 5) or mNedd4-2 (lanes 3 and 6). Oocytes were labeled overnight with [35S]-methionine and homogenized. A membrane fraction was solubilized, and immunoprecipitation was performed with anti-FLAG antibody. The immunoprecipitated proteins were analyzed by autoradiography (top panel) or Western blotting with anti-Nedd4 antibody (middle panel). Aliquots from the homogenate analyzed by immunoblotting show that mNedd4-1 and mNedd4-2 were properly expressed (bottom panel) (from21, with permission of the FASEB J).
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

5. Figure 3
Schematic diagram of the hypothesized involvement of Nedd4-2, but not Nedd4-1, in ENaC down-regulation via ubiquitination. Nedd4-2, but not Nedd4-1, binds through its WW domains 3 and 4 to ENaC, likely at an intracellular location (1) Upon translocation to the plasma membrane (2), Nedd4-2 ubiquitinates ENaC (3), leading to the dissociation of Nedd4-2 and the internalization of ENaC (4). After deubiquitination (5), ENaC is either degraded by the lysosomes (6) or is eventually recycled to the plasma membrane (1).
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions