Genetic Disorders – Development
Kidney International (2005) 68, 1999–2009; doi:10.1111/j.1523-1755.2005.00654.x
The C-terminal tail of aquaporin-2 determines apical trafficking
MICHIO KUWAHARA, TOMOKI ASAI, YOSHIO TERADA and SEI SASAKI
Department of Nephrology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
Correspondence: Michio Kuwahara M.D, Department of Nephrology, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8519, Japan E-mail: mkuwahara.kid@tmd.ac.jp
Received 5 October 2004; Revised 15 March 2005; Re-revised 13 May 2005; Accepted 3 June 2005.
Abstract
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.
Keywords:
AQP-2, C-terminal tail, PDZ-interacting motif, signal-induced proliferation-associated gene-1
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