Novel regulatory function for NHERF-1 in Npt2a transcription.
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.
Khundmiri
Syed Jalal
SJ
Department of Medicine, Univ. of Louisville, Kidney Disease Program, 570 S. Preston St, Suite 102, Louisville, KY 40202, USA.
Ahmad
Aamir
A
Bennett
Ryan Everett
RE
Weinman
Edward J
EJ
Steplock
Deborah
D
Cole
Judith
J
Baumann
Patrick D
PD
Lewis
John
J
Singh
Saurabh
S
Clark
Barbara J
BJ
Lederer
Eleanor D
ED
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
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