The C-terminal tail of aquaporin-2 determines apical trafficking
| Autor: | Michio Kuwahara, Sei Sasaki, Tomoki Asai, Yoshio Terada |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Molecular Sequence Data
Mutant Aquaporin Biology Kidney Transfection PDZ-interacting motif signal-induced proliferation-associated gene-1 Cell Line Dogs Cyclic AMP Consensus sequence Animals Humans Amino Acid Sequence Phosphorylation Epithelial polarity chemistry.chemical_classification Aquaporin 2 Endoplasmic reticulum Cell Polarity Apical membrane Protein Structure Tertiary Amino acid Cell biology Protein Transport Biochemistry chemistry Mutagenesis AQP-2 Nephrology C-terminal tail |
| Zdroj: | Kidney International. 68:1999-2009 |
| ISSN: | 0085-2538 |
| Popis: | The C-terminal tail of aquaporin-2 determines apical trafficking . 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. |
| Databáze: | OpenAIRE |
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