title: Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus.
abstract: 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.
authors.affiliation: Department of Homeostasis Medicine and Nephrology, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8519, Japan. mkuwahara.kid@tmd.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Kuwahara
authors.forename: K
authors.initials: K
authors.lastname: Iwai
authors.forename: T
authors.initials: T
authors.lastname: Ooeda
authors.forename: T
authors.initials: T
authors.lastname: Igarashi
authors.forename: E
authors.initials: E
authors.lastname: Ogawa
authors.forename: Y
authors.initials: Y
authors.lastname: Katsushima
authors.forename: I
authors.initials: I
authors.lastname: Shinbo
authors.forename: S
authors.initials: S
authors.lastname: Uchida
authors.forename: Y
authors.initials: Y
authors.lastname: Terada
authors.forename: M F
authors.initials: MF
authors.lastname: Arthus
authors.forename: M
authors.initials: M
authors.lastname: Lonergan
authors.forename: T M
authors.initials: TM
authors.lastname: Fujiwara
authors.forename: D G
authors.initials: DG
authors.lastname: Bichet
authors.forename: F
authors.initials: F
authors.lastname: Marumo
authors.forename: S
authors.initials: S
authors.lastname: Sasaki
mesh_terms: 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
chemical_list: C496023:AQP2 protein, human; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins