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 sucrose density gradient ultracentrifugation experiment of the distribution of Npt2a (top) and NHERF-1 (bottom).