Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains.
Mutations that disrupt a PY motif in epithelial Na+ 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 >40 exons. Multiple 5'-exons were identified by 5'-rapid amplification of cDNA ends, and tissue-specific expression of these transcripts was noted by RT-PCR and RNase protection assay. Alternate polyadenylation signal sequences led to varying lengths of the 3'-untranslated region. 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. Coexpression, in Fischer rat thyroid 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 alpha-, beta-, and gamma-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.
Itani
Omar A
OA
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, USA.
Campbell
Jason R
JR
Herrero
Juan
J
Snyder
Peter M
PM
Thomas
Christie P
CP
D020413:3' Untranslated Regions / Q000235:genetics; D017398:Alternative Splicing; D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001692:Biological Transport / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D026723:Polyadenylation / Q000502:physiology; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D011916:Rats, Inbred F344; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000502:physiology
D020413:3' Untranslated Regions; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D012964:Sodium; 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