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Molecular cloning of Nedd4 from Xenopus laevis
1Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-511, USA.
Abstract:
The tryptophan-bounded WW domains ofNedd4 bind to the proline-tyrosine (PY) motifs contained in the C-terminal cytoplasmic region of the beta and gamma subunits of the rat amiloride-sensitive sodium channel (ENaC). In patients with Liddle's syndrome, the PY motif is mutated and the channel remains constitutively activated leading to sodium retention and hypertension. Although the function ofNedd4 is unknown, it contains a highly conserved ubiquitin protein ligase domain that may attach ubiquitin to ENaC, targeting it for degradation or it may modulate ENaC activity through another undetermined pathway. Xenopus laevis-derived cells, such as oocytes and the A6 kidney cell line, are important models currently used for the study of ENaC regulation. We describe the X. laevis homologue of Nedd4 (xNedd4). A partial clone, approximately 2.6 Kb, was isolated from an aldosterone-treated A6 cell cDNA library. Further 5' sequence, approximately 1.2 Kb, was obtained using a modified 5' rapid amplification of cDNA (RACE) protocol and cDNA from untreated A6 cells as the substrate. The identity and similarity of xNedd4 with human Nedd4 are approximately 63 and 71%, respectively. xNedd4 contains the C2, ubiquitin protein ligase, and 4 WW domains previously described for Nedd4 from other species.
Insights
Researchers identified a Xenopus laevis homologue of Nedd4 (xNedd4), crucial for regulating the amiloride-sensitive sodium channel (ENaC). This finding aids in understanding Liddle's syndrome and hypertension.
Area of Science:
- Molecular Biology
- Cell Biology
- Physiology
Background:
- Nedd4 binds to proline-tyrosine (PY) motifs on the amiloride-sensitive sodium channel (ENaC) subunits.
- Mutations in the PY motif cause Liddle's syndrome, characterized by sodium retention and hypertension.
- Nedd4's function is unclear but it possesses a ubiquitin protein ligase domain, suggesting a role in ENaC regulation.
Purpose of the Study:
- To identify and characterize the Xenopus laevis homologue of Nedd4 (xNedd4).
- To investigate the potential role of xNedd4 in regulating ENaC activity in a relevant model system.
Main Methods:
- Isolation of a partial xNedd4 clone from an aldosterone-treated A6 cell cDNA library.
- 5' rapid amplification of cDNA (RACE) to obtain further sequence from untreated A6 cells.
- Sequence analysis to determine identity and similarity to human Nedd4.
Main Results:
- A partial clone of xNedd4 (2.6 Kb) was isolated, with an additional 1.2 Kb obtained via 5' RACE.
- xNedd4 shares approximately 63% identity and 71% similarity with human Nedd4.
- xNedd4 possesses conserved domains: C2, ubiquitin protein ligase, and 4 WW domains.
Conclusions:
- The identification of xNedd4 provides a new tool for studying ENaC regulation in Xenopus laevis models.
- xNedd4 is likely involved in modulating ENaC activity, similar to its mammalian counterpart.
- Further research can elucidate the specific mechanisms by which xNedd4 regulates ENaC and its implications for hypertension.
