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PLoS One
2012 Jan 01;74:e34764. doi: 10.1371/journal.pone.0034764.
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A new human NHERF1 mutation decreases renal phosphate transporter NPT2a expression by a PTH-independent mechanism.
Courbebaisse M
,
Leroy C
,
Bakouh N
,
Salaün C
,
Beck L
,
Grandchamp B
,
Planelles G
,
Hall RA
,
Friedlander G
,
Prié D
.
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The 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.We 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.We 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.Our 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.
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22506049
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Figure 1. Relationship between serum PTH concentration and the capacity of kidney to reabsorb phosphate normalized for the glomerular filtration rate (TmP/GFR) in 112 control subjects (diamonds).The patient with the mutation in NHERF1 gene is represented by a circle.
Figure 2. Localization of the mutation.A: Sequence analysis of genomic DNA from the patient with NHERF1 mutation. The upper panel shows the portion of the DNA sequence from the patient with NHERF1 mutation. The sequence can be compared with that obtained in a subject with no mutation (lower panel). The nucleotide and the amino acid sequences (above and below the horizontal line respectively) are indicated. A vertical arrow points the mutation. The patient was heterozygous for the mutation. Conservation of the NHERF1 protein sequence surrounding the mutation site in various species. The amino acid modified by the mutation is highlighted. B: Alignment of human NHERF1 amino acid sequence around the site of mutation with homologous sequences of NHERF1 in various species. C: Alignment of human NHERF1 amino acid sequence around the site of mutation with homologous sequences of NHERF2 in various species.
Figure 3. Effects of NHERF1 mutations on cAMP production, PKC activity and phosphate uptake in response to PTH stimulation.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. The mutant E68A has been identified in the patient reported in the present study, the mutants R153Q and E225K have been characterized in a previous study (9). A: Cells were stimulated with PTH 10−8 M. We present the increase in cAMP synthesis above the values measured in the absence of PTH. Results are means ± SD of four independent experiments. The groups were compared with the use of Kruskall-Wallis test (overall P = 0.001, * p<0.05 compared to condition in the absence of NHERF1). B: PKC activity in cells stimulated with 10−5 M or 10−7 M of PTH. For each PTH concentration used, PTH induced PKC activity did not differ in OK cells expressing wild type NHERF1 or the E68A mutant. Results are means ± SD of three independent experiments. C: The inhibitory effect of PTH on phosphate uptake was determined at two concentrations of PTH (10−5 M or 10−7 M). For each PTH concentration, the magnitude of the PTH-induced inhibition on phosphate uptake did not differ between OK cells expressing wild type or E68A NHERF1. Results are means ± SD of three independent experiments.
Figure 4. Measurement of phosphate transport in cells expressing the wild type or the E68A mutant NHERF1 together with the sodium phosphate cotransporter NPT2a.Panel A: Phosphate-induced current recorded in Xenopus oocytes injected with water or with either cRNA of NPT2a alone or in association with the cRNA of the wild type (WT) NHERF1 or the E68A mutant. Results are means ± SD, n = 9. Overall comparison was performed with the use of the Kruskall-Wallis test (P<0.0001) then the group expressing NPT2a alone was compared with other groups with the use of the Mann-Whitney test (p<0.01). Panel B: Sodium-dependent phosphate uptake was measured in Hela cells transfected with empty plasmids or with the plasmid containing the cDNA of NPT2a alone or together with the cDNA of the wild type or the NHERF1 mutant. Results are means ± SD, n = 6. Overall comparison was performed with the use of the Kruskall-Wallis test (P<0.0001) then the group expressing NPT2a alone was compared with the other groups with the use of the Mann-Whitney test (* p<0.01 vs NPT2a+WT NHERF1, # p <0.001 vs NPT2a alone).
Figure 5. Co-immunoprecipitation of NHERF1 with NPT2a in Hela cells.Hela cells were transfected with a plasmid containing the cDNA of NPT2a tagged with GFP at its N- extremity. Cells were also transfected with a plasmid containing the wild type cDNA of NHERF1 or one mutant (E68A or R153Q or E225K) tagged with Flag. Upper panel: Flag-tagged-NHERF1 protein was immunoprecipitated by using an anti-Flag antibody and the immunoprecipitates were probed with anti-GFP antibodies to detect GFP-tagged-NPT2a on Western blot. Middle panel: The total expression of GFP-tagged-NPT2a was analyzed in the cell lysates by anti-GFP Western Blot. Lower panel: Wild type and mutant Flag-NHERF1 was immunoprecipitated and probed by using anti-Flag antibodies to compare Flag-NHERF1 immunoprecipitation in the different conditions.
Figure 6. Effect of mutant NHERF1 on the cell surface expression of NPT2a assessed by biotinylation experiments.Hela cells were transfected with GFP-tagged-NPT2a alone or together with a plasmid containing the cDNA of the wild type NHERF1 or the E68A mutant. NPT2a proteins labelled with sulfo-NHS-SS-biotin were revealed by using anti-GFP antibodies. Labeled proteins were isolated with avidin-agarose beads according to manufacturer's manual (Pierce). Proteins eluted from the beads and total cell lysate were loaded on a SDS gel. Western blot was probed with anti-GFP antibodies. Na+ K+ APTase was used as a loading control and detected with specific antibodies. The higher panel shows a western blot representative of four experiments. Lower panel: Statistical analyses of the quantification of the western blot (n = 4). Results are presented as mean ±sd. Quantification of the surface expression of NPT2a: Anova p = 0.038. Post Hoc test: Tukey-Kramer multiple comparisons test (#: p<0.05; * p<0.01).
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