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.
Edward J. Weinman, Rajat S. Biswas, Quihong Peng, Lily Shen, Christina L. Turner, Xiaofei E, Deborah Steplock, Shirish Shenolikar, Rochelle Cunningham
Representative immunoblots using an anti–NHERF-1 antibody of whole cell lysates (WH) and BBM fractions of BSC-1 cells transfected with His-tagged wild-type NHERF-1, His-NHERF-1 with a 30-aa C-terminal truncation (ΔERM), His-NHERF-1 with the PDZ II sequence PNGYGF mutated to HNGAGA (PDZ II mutant), or His-NHERF-1 with the PDZ I sequence HNGYGF mutated to PNGAGA (PDZ I mutant).