Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus.
The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763-772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-type AQP2 is predicted to be a 271-amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330-333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2.
Kuwahara
M
M
Department of Homeostasis Medicine and Nephrology, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8519, Japan. mkuwahara.kid@tmd.ac.jp
Iwai
K
K
Ooeda
T
T
Igarashi
T
T
Ogawa
E
E
Katsushima
Y
Y
Shinbo
I
I
Uchida
S
S
Terada
Y
Y
Arthus
M F
MF
Lonergan
M
M
Fujiwara
T M
TM
Bichet
D G
DG
Marumo
F
F
Sasaki
S
S
D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D018500:Diabetes Insipidus, Nephrogenic / Q000235:genetics*; D005260:Female; D005799:Genes, Dominant / Q000235:genetics*; D006801:Humans; D007223:Infant; D007564:Japan; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism; D010375:Pedigree; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
C496023:AQP2 protein, human; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins