title: Novel regulatory function for NHERF-1 in Npt2a transcription.
abstract: Several lines of evidence show that sodium/hydrogen exchanger regulatory factor 1 (NHERF-1) regulates the expression and activity of the type IIa sodium-dependent phosphate transporter (Npt2a) in renal proximal tubules. We have previously demonstrated that expression of a COOH-terminal ezrin binding domain-deficient NHERF-1 in opossum kidney (OK) cells decreased expression of Npt2a in apical membranes but did not affect responses to parathyroid hormone. We hypothesized that NHERF-1 regulates apical membrane expression of Npt2a in renal proximal tubule cells. To address this hypothesis, we compared regulation of Npt2a expression and function in NHERF-deficient OK cells (OK-H) and wild-type cells (OK-WT). In OK-H cells, phosphate uptake and expression of Npt2a protein in apical membranes were significantly lower than in OK-WT cells. Transient transfection of green fluorescent protein-tagged Npt2a cDNA into OK-H cells resulted in aberrant localization of an Npt2a fragment to the cytosol but not to the apical membrane. OK-H cells also exhibited a marked decrease in Npt2a mRNA expression. As demonstrated by luciferase assay, Npt2a promoter activity was significantly decreased in OK-H cells compared with that shown in OK-WT cells. Transfection of OK-H cells with human NHERF-1 restored Npt2a expression at both the protein and mRNA levels and regulation by parathyroid hormone. Expression of NHERF-1 constructs with mutations in the PDZ domains or the ezrin binding domain in OK-H cells suggested that the PDZ2 domain is critical for apical translocation of Npt2a and for expression at the mRNA level. Our data demonstrate for the first time that NHERF-1 regulates Npt2a transcription and membrane insertion.
authors.affiliation: Department of Medicine, Univ. of Louisville, Kidney Disease Program, 570 S. Preston St, Suite 102, Louisville, KY 40202, USA.
authors.forename: Syed Jalal
authors.initials: SJ
authors.lastname: Khundmiri
authors.forename: Aamir
authors.initials: A
authors.lastname: Ahmad
authors.forename: Ryan Everett
authors.initials: RE
authors.lastname: Bennett
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Weinman
authors.forename: Deborah
authors.initials: D
authors.lastname: Steplock
authors.forename: Judith
authors.initials: J
authors.lastname: Cole
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Baumann
authors.forename: John
authors.initials: J
authors.lastname: Lewis
authors.forename: Saurabh
authors.initials: S
authors.lastname: Singh
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Clark
authors.forename: Eleanor D
authors.initials: ED
authors.lastname: Lederer
mesh_terms: D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D017930:Genes, Reporter; D007668:Kidney; D009893:Opossums; D010750:Phosphoproteins / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology*; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000172:deficiency / Q000235:genetics*; D014162:Transfection
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D010750:Phosphoproteins; D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; C093376:sodium-hydrogen exchanger regulatory factor