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 466-471 (August 2001) DOI: 10.1046/j.1523-1755.2001.060002466.x Copyright © 2001 International Society of Nephrology Terms and Conditions

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 2001 60, 466-471DOI: (10.1046/j.1523-1755.2001.060002466.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

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 2001 60, 466-471DOI: (10.1046/j.1523-1755.2001.060002466.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

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 2001 60, 466-471DOI: (10.1046/j.1523-1755.2001.060002466.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

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 2001 60, 466-471DOI: (10.1046/j.1523-1755.2001.060002466.x) Copyright © 2001 International Society of Nephrology Terms and Conditions