title: Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria.
abstract: Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.
authors.affiliation: Department of Molecular Nutrition, University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: Sakiko
authors.initials: S
authors.lastname: Haito-Sugino
authors.forename: Mikiko
authors.initials: M
authors.lastname: Ito
authors.forename: Akiko
authors.initials: A
authors.lastname: Ohi
authors.forename: Yuji
authors.initials: Y
authors.lastname: Shiozaki
authors.forename: Natsumi
authors.initials: N
authors.lastname: Kangawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishiyama
authors.forename: Fumito
authors.initials: F
authors.lastname: Aranami
authors.forename: Shohei
authors.initials: S
authors.lastname: Sasaki
authors.forename: Ayaka
authors.initials: A
authors.lastname: Mori
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Kido
authors.forename: Sawako
authors.initials: S
authors.lastname: Tatsumi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Segawa
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Miyamoto
mesh_terms: D000818:Animals; D015153:Blotting, Western; D053098:Familial Hypophosphatemic Rickets / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D053565:Hypercalciuria / Q000235:genetics* / Q000378:metabolism*; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D055550:Protein Stability; D050615:Sodium-Phosphate Cotransporter Proteins, Type IIc / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014982:Xenopus laevis
chemical_list: C493562:SLC34A3 protein, human; D050615:Sodium-Phosphate Cotransporter Proteins, Type IIc