A New Human NHERF1 Mutation Decreases Renal Phosphate Transporter NPT2a Expression by a PTH-Independent Mechanism

Marie Courbebaisse,Gérard Friedlander,Christine Leroy,Laurent Beck,Dominique Prié,Christine Salaün,Gabrielle Planelles,Randy A Hall,Naziha Bakouh,Bernard Grandchamp,Michael Butterworth

doi:10.1371/journal.pone.0034764

Abstract

BackgroundThe sodium-hydrogen exchanger regulatory factor 1 (NHERF1) binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH) receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule.MethodsWe compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production.ResultsWe identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A) located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a.ConclusionsOur results indicate that the PDZ1 domain is critical for NHERF1- NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.

Highlights

The maintenance of normal serum phosphate concentration is critical for many biological processes including bone mineralization and cell function
In patients with impaired renal phosphate reabsorption, mutations in the NHERF1 protein that induced an increase in parathyroid hormone (PTH)-induced cyclic adenosine monophosphate (cAMP) production [9]
CAMP accumulation, PKC activity and phosphate uptake inhibition by PTH were measured in opossum kidney (OK) cells transfected with a plasmid alone or containing either the wild type cDNA of NHERF1 or cDNA from mutant NHERF1

Summary

Methods

Objectives We described the consequences of a previously unidentified mutation in the PDZ1 domain of NHRF1 gene. Sequencing was performed on genomic DNA obtained from patients who were referred to our clinical department for renal assessment before initiating potentially nephrotoxic treatment for psoriasis, or before heminephrectomy for living kidney donation, or for examination of renal lithiasis or bone demineralization. In these subjects we measured serum phosphate and PTH concentrations and calculated the TmP/GFR value using Walton and Bijvoet nomogram [13]. Biotinylation and isolation of cell surface proteins were performed in HeLa cells transfected with pEF1-B empty plasmid, NHERF1 wt or E68A mutant and NPT2a-peGFP, using Cell Surface Protein Isolation Kit (Pierce) according to the manufacturer’s instructions.

Results

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