Parathyroid hormone inhibits renal phosphate transport by phosphorylation of serine 77 of sodium-hydrogen exchanger regulatory factor–1
Edward J. Weinman, Rajat S. Biswas, Quihong Peng, Lily Shen, Christina L. Turner, Xiaofei E, Deborah Steplock, Shirish Shenolikar, Rochelle Cunningham
Edward J. Weinman, Rajat S. Biswas, Quihong Peng, Lily Shen, Christina L. Turner, Xiaofei E, Deborah Steplock, Shirish Shenolikar, Rochelle Cunningham
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Research Article

Parathyroid hormone inhibits renal phosphate transport by phosphorylation of serine 77 of sodium-hydrogen exchanger regulatory factor–1

Abstract

Parathyroid hormone (PTH), via activation of PKC and/or protein kinase A, inhibits renal proximal tubular phosphate reabsorption by facilitating the internalization of the major sodium-dependent phosphate transporter, Npt2a. Herein, we explore the hypothesis that the effect of PTH is mediated by phosphorylation of serine 77 (S77) of the first PDZ domain of the Npt2a-binding protein sodium-hydrogen exchanger regulatory factor–1 (NHERF-1). Using recombinant polypeptides representing PDZ I, S77 of NHERF-1 is phosphorylated by PKC but not PKA. When expressed in primate kidney epithelial cells (BSC-1 cells), however, activation of either protein kinase phosphorylates S77, suggesting that the phosphorylation of PDZ I by PKC and PKA proceeds by different biochemical pathways. PTH and other activators of PKC and PKA dissociate NHERF-1/Npt2a complexes, as assayed using quantitative coimmunoprecipitation, confocal microscopy, and sucrose density gradient ultracentrifugation in mice. Murine NHERF-1–/– renal proximal tubule cells infected with adenovirus-GFP-NHERF-1 containing an S77A mutation showed significantly increased phosphate transport compared with a phosphomimetic S77D mutation and were resistant to the inhibitory effect of PTH compared with cells infected with wild-type NHERF-1. These results indicate that PTH-mediated inhibition of renal phosphate transport involves phosphorylation of S77 of the NHERF-1 PDZ I domain and the dissociation of NHERF-1/Npt2a complexes.

Authors

Edward J. Weinman, Rajat S. Biswas, Quihong Peng, Lily Shen, Christina L. Turner, Xiaofei E, Deborah Steplock, Shirish Shenolikar, Rochelle Cunningham

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Figure 6

The effect of PTH on Npt2a/NHERF-1 complexes in mouse brush border membranes.

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The effect of PTH on Npt2a/NHERF-1 complexes in mouse brush border membr...
(A) Representative Western immunoblots of Npt2a and NHERF-1 in BBM harvested from control wild-type mice (–PTH) and from mice treated with an intraperitoneal injection of PTH 45 minutes prior to harvest (+PTH). (B) Representative experiment on BBM harvested from control wild-type mice and from mice treated with an intraperitoneal injection of PTH 45 minutes prior to harvest. BBMs were treated with Triton X-100 and resolved by ultracentrifugation using a sucrose density gradient, and the fractions were immunoblotted for Npt2a, NHERF-1, ezrin, and clathrin. (C) Summary of the sucrose density gradient ultracentrifugation of wild-type mouse BBM in the absence (solid line) or after the intraperitoneal injection of PTH (dashed line). Npt2a is shown in the top panel and NHERF-1 in the bottom panel. Results are expressed as percent of total (mean ± SEM); n = 4.