The C-Terminal Tail of Aquaporin-2 Determines Apical Trafficking
|Title:||The C-Terminal Tail of Aquaporin-2 Determines Apical Trafficking|
|Authors:||Kuwahara, Michio; Asai, Tomoki; Terada, Yoshio; Sasaki, Sei|
|Date Published:||November 01, 2005|
BACKGROUND: Aquaporin-2 (AQP-2) proteins are mainly expressed at the apical region of the collecting duct cells. We previously reported three different mutations in the C-terminus of AQP-2 that all-cause autosomal-dominant nephrogenic diabetes insipidus. When one of these mutant AQP-2s was expressed in Madin-Darby canine kidney (MDCK) cells, it was mistargeted to the basolateral membrane, suggesting a critical role of the C-terminal tail in the apical trafficking of AQP-2.
METHODS: Portions of the AQP-2 C-terminal tail (residues 226-271) were mutated by the polymerase chain reaction (PCR) technique and inserted into the pcDNA3.1 vector. Constructs were transfected into MDCK cells to examine the localization of mutated AQP-2 proteins by immunofluorescence microscopy. Cell surface expression was detected by biotinylation assay.
RESULTS: The wild-type AQP-2 was localized at the apical membrane, whereas mutants lacking residues 262-271 (the last 10 amino acids) were predominantly distributed in the endoplasmic reticulum. Deletion mutants of the initial (226-240del) and middle (241-252del) portions of the C-terminal tail were identified at the apical membrane, suggesting that residues 226-252 have no involvement in apical targeting. An AQP-4-AQP-2 chimera in which a portion of the AQP-4 C-terminal tail was replaced by the corresponding site in AQP-2 (residues 256-271) was found at the apical membrane. The sequence of the last 4 amino acids of AQP-2 (G-T-K-A) corresponds to a PDZ-interacting motif. Our investigations identified a mutant of this portion mostly localized to the subapical region. Further, apical expression was found to be significantly decreased in mutants lacking a consensus sequence for cyclic adenosine monophosphate (cAMP)-dependent phosphorylation (residues 253-256).
CONCLUSION: The sequence at 256-271 is sufficient for apical trafficking in AQP-2. The putative PDZ-interacting motif (G-T-K-A, residues 268-271) plays a key role in apical membrane expression. In addition, cAMP-dependent phosphorylation was found to be critical for apical targeting.
You may, however, learn more about this article at the publisher's site.
To return to this page, use your "back" key.
This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)
The aquaporin 2 (AQP2) protein is a chain of 271 amino acid residues that scientists theorize locates partly within the cell membrane, partly outside the cell and partly inside the cell. Both ends of the protein, the N- and C-termini, are located within the cell. Initially, AQP2 resides inside the cell until a chemical sequence initiated by the binding of the hormone arginine vasopressin (AVP) and the vasopressin 2 receptor (V2R) protein signals the AQP2 to travel to the apical (top) section of the cell membrane. When AQP2 can not travel to the cell membrane due to either a defect in the AQP2 or the V2R, it can result in NDI.
Previous research by Kuwahara, et al., indicated that mutations in the C-terminus of the AQP2 result in autosomal NDI. Their findings suggest that the AQP2’s C-terminal tail plays a critical role in AQP2’s ability to travel to the cell membrane from the cell interior.
To refine their understanding of this, Kuwahara, et al., mutated portions of the AQP2’s C-terminal tail, specifically those amino acid residues that were the 226th through the 271st residues in the AQP2. The researchers observed that the AQP2 mutants that were missing the amino acid residues 262 through 271 could not travel to the cell membrane. AQP2 mutants lacking the portion of the C-terminus made of residues 226-252 could travel to the apical section of the cell membrane.
Experimentation with aquaporin proteins made up of two different AQP – AQP2 and AQP4 – wherein the AQP4 contained AQP2 residues 256 -271 in the C-terminus showed that this new protein was able to travel to the apical membrane.