Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains

Omar A Itani; Jason R Campbell; Juan Herrero; Peter M Snyder; Christie P Thomas

doi:10.1152/ajprenal.00203.2003

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Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains

Journal article

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Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains

Omar A Itani, Jason R Campbell, Juan Herrero, Peter M Snyder and Christie P Thomas

American journal of physiology. Renal physiology, Vol.285(5), pp.F916-F929

11/2003

DOI: 10.1152/ajprenal.00203.2003

PMID: 12876068

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Abstract

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.

Phosphorylation

Alternative Splicing

Humans

Rats, Inbred F344

3' Untranslated Regions - genetics

Molecular Sequence Data

Sodium Channels - physiology

Amino Acid Sequence - genetics

Sodium - metabolism

Ubiquitin-Protein Ligases - physiology

Genetic Variation

Epithelial Sodium Channels

Nedd4 Ubiquitin Protein Ligases

Transcription, Genetic

Polyadenylation - physiology

Promoter Regions, Genetic

Base Sequence - genetics

Endosomal Sorting Complexes Required for Transport

Rats

Amino Acid Motifs - physiology

Animals

Biological Transport - physiology

Sodium Channels - genetics

Ubiquitin-Protein Ligases - genetics

Protein Structure, Tertiary - physiology

Details

Title: Subtitle: Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains
Creators: Omar A Itani - Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, USA
Jason R Campbell
Juan Herrero
Peter M Snyder
Christie P Thomas
Resource Type: Journal article
Publication Details: American journal of physiology. Renal physiology, Vol.285(5), pp.F916-F929
DOI: 10.1152/ajprenal.00203.2003
PMID: 12876068
NLM abbreviation: Am J Physiol Renal Physiol
ISSN: 1931-857X
eISSN: 1522-1466
Publisher: United States
Grant note: R01 DK054348 / NIDDK NIH HHS DK-54348 / NIDDK NIH HHS
Language: English
Date published: 11/2003
Academic Unit: Molecular Physiology and Biophysics; Stead Family Department of Pediatrics; Cardiovascular Medicine; Obstetrics and Gynecology; Nephrology; Medicine Administration; Internal Medicine
Record Identifier: 9983986370502771

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