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. IntroductionParathyroid hormone (PTH) increases the urinary excretion of phosphate by facilitating the retrieval and internalization of Npt2a, the major sodium-dependent phosphate transporter found in the apical membrane of the cells of the renal proximal convoluted tubule (1-3). The precise physiologic and biochemical pathways relating activation of the PTH receptor to the endocytosis of Npt2a, however, are not known. An insight into this process was provided by the observations that Npt2a binds to the PDZ domain adaptor protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) and that NHERF-1 -/-mice demonstrate phosphaturia and mistargeting of Npt2a (4, 5). Subsequent experiments demonstrated that NHERF-1 functions as a membrane retention signal for Npt2a and that sodium-dependent phosphate transport in renal proximal tubule cells from NHERF-1 mice was resistant to the inhibitory effect of PTH (3, 6, 7). NHERF-1 -/-cells were also resistant to the inhibitory effect of activators of PKC and PKA, the 2 major signaling pathways of the PTH1 receptor, indicating that the resistance to PTH derived

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

“…Ezrin, by virtue of its protein kinase A anchoring capacity, positions the PKA holoenzyme in proximity to the Npt2a-NHERF1-ezrin ternary complex. Upon PTH stimulation and the concomitant production of cAMP, anchored PKA phosphorylates NHERF1 on target serines (38,45). This cascade of events induces the dissociation of Npt2a from the ternary complex, resulting in Npt2a internalization and degradation.…”

“…We analyzed peptide fragments harboring Ser 77 located in PDZ1, which has been associated with PTHinduced NHERF1 phosphorylation (38), and the linker region between PDZ2 and the EBD, which contains a serine-rich cluster (Fig. 1A), including Ser 290 , which is constitutively phosphor- ylated (39) and has been implicated in PKC-mediated phosphorylation (40,41).…”

Effects of phorbol ester‐sensitive PKC (c/nPKC) activation on the production of adiponectin in 3T3‐L1 adipocytes

“…Our previous study (13) showed that activation of atypical PKC (aPKC) led to NF-jB activation and a decrease of PPARc expression. Considering the fact that various hormones, including insulin, angiotensin II, glucagons, parathyroid hormone, endothelin-1, thrombin, and vitamin D3, and high glucose concentration, activate c/nPKC (14)(15)(16)(17)(18)(19), evaluating the role of c/nPKC on gene expression in adipocyte may provide a new understanding of the regulation of insulin sensitivity.…”

Phosphate transporters of the SLC20 and SLC34 families

“…Neither short term up-or down-regulation seems to involve changes on mRNA levels, suggesting a posttranscriptional control. The nature of this posttranscriptional mechanism has been clarified in the case of the PTH-induced downregulation of NaPi-IIa: PTH administration leads to phosphorylation of NHERF1, an adaptor protein that contributes to stabilize NaPi-IIa at the proximal BBM; phosphorylation of NHERF1 decreases its affinity for NaPi-IIa, resulting in a reduced stability and therefore removal of the cotransporter from the apical membrane (Deliot et al, 2005;Weinman et al, 2007). Whether or not similar protein-protein interaction mechanisms contribute to stabilize the membrane expression of NaPi-IIc and /or NaPi-IIb is unknown.…”