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).


CONCLUSIONS
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.
Kuwahara
Michio
M
Department of Nephrology, Tokyo Medical and Dental University Graduate School, Japan. mkuwahara.kid@tmd.ac.jp
Asai
Tomoki
T
Terada
Yoshio
Y
Sasaki
Sei
S
D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D004285:Dogs; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology*; D014162:Transfection
D051399:Aquaporin 2; D000242:Cyclic AMP