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1 Medicine, University of Louisville, Louisville, Kentucky, United States
2 Medicine, Veterans Affairs, Baltimore, Maryland, United States; Medicine, University of Maryland, Baltimore, Maryland, United States
3 Medicine, University of Maryland, Baltimore, Maryland, United States
4 Biology, University of Memphis, Memphis, Tennessee, United States
5 Molecular,Cellular and Craniofacial Biology, University of Louisville, Louisville, Kentucky, United States
6 Biochemistry, University of Louisville, Louisville, Kentucky, United States
7 Medicine, Veterans Affairs, Louisville, Kentucky, United States; Medicine, University of Louisville, Louisville, Kentucky, United States
* To whom correspondence should be addressed. E-mail: e.lederer{at}louisville.edu.
Several lines of evidence show that sodium hydrogen exchanger regulatory factor (NHERF-1) regulates the expression and activity of the type IIa sodium phosphate transporter (Npt2a) in renal proximal tubule. We have previously demonstrated that expression of a C-terminal ezrin binding domain deficient NHERF-1 in opossum kidney (OK) cells decreased expression of Npt2a in apical membranes but did not affect response to PTH. We hypothesized that NHERF-1 regulates apical membrane expression of Npt2a in renal proximal tubule cells. To address the 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 the OK-WT cells. Transient transfection of GFP-tagged Npt2a cDNA into OK-H cells resulted in aberrant localization of an Npt2a fragment to the cytosol, not the apical membrane. OK-H cells also exhibited a marked decrease in the expression of Npt2a mRNA. As demonstrated by luciferase assay, Npt2a promoter activity was significantly decreased in OK-H cells compared to OK-WT cells. Transfection of OK-H cells with human NHERF-1 restored Npt2a expression both at the protein and mRNA level and regulation by PTH. Expression of NHERF-1 constructs with mutations in the PDZ domains or the ezrin binding domain in OK-H cells suggested that the PDZ-2 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.
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