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1 Department of Physiology, University of Iowa, Iowa City, IA, USA; Department of Internal Medicine, University of Iowa, Iowa City, IA, USA
2 Department of Internal Medicine, University of Iowa, Iowa City, IA, USA
3 Axcell Biosciences, Newton, PA, USA
4 Department of Internal Medicine, University of Iowa, Iowa City, IA, USA; Graduate Program in Molecular Biology, University of Iowa College of Medicine; the Veterans Affairs Medical Center, Iowa City, IA, USA
5 Department of Internal Medicine, University of Iowa, Iowa City, IA, USA; Department of Physiology, University of Iowa, Iowa City, IA, USA
* To whom correspondence should be addressed. E-mail: christie-thomas{at}uiowa.edu.
Mutations that disrupt a PY motif in epithelial sodium channel (ENaC) subunits increase surface expression of Na+ channels in the collecting duct resulting in greater Na+ reabsorption. Recently Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4-2 (hNedd4-2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach. The gene is present as a single copy, spans at least 400 kb and contains over 40 exons. Multiple 5' exons were identified by 5' RACE and tissue-specific expression of these transcripts was noted by RT-PCR and ribonuclease protection assay. Alternate polyadenylation signal sequences lead to varying lengths of the 3' UTR. Alternate splicing events within internal exons were also noted. Open reading frame analysis indicates that hNedd4-2 encode multiple protein variants with and without a C2 domain, and with a variable number of WW domains. Co-expression, in FRT epithelia, of ENaC and Nedd4-2 cDNAs, leads to a significant reduction in amiloride-sensitive currents, confirming a role in Na+ transport regulation. In vitro binding studies demonstrated that individual PY motifs of 
,
and 
ENaC have strong affinity for WW domains 3 and 4 but not 1 and 2. These studies indicate that alternate transcripts of Nedd4-2 may interact with ENaC differently. Understanding the function of variant proteins will increase our knowledge of the role of hNedd4-2 in the regulation of ENaC and define protein domains important for Nedd4-2 function.
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