Hrs controls sorting of the epithelial Na+ channel between endosomal degradation and recycling pathways.
Epithelial Na(+) absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na(+) channel (ENaC) at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling.
Zhou
Ruifeng
R
Department of Internal Medicine, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
Kabra
Rajesh
R
Olson
Diane R
DR
Piper
Robert C
RC
Snyder
Peter M
PM
D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D056929:Liddle Syndrome / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D011916:Rats, Inbred F344; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D010750:Phosphoproteins; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases