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.
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ABSTRACT: Inhibition of the renal brush border membrane (BBM) Na/H exchanger by cAMP-dependent protein kinase, PKA, requires participation of a recently cloned regulatory cofactor, Na/H exchanger-regulatory factor (NHE-RF). As deduced from the cDNA of this 358-amino acid protein, amino acids 11-101 and amino acids 150-241 of the NHE-RF protein share 74% overall homology suggesting duplication of these PDZ containing domains. The serine residues at amino acid position 289 and 340 are considered to be the most likely sites for PKA mediated phosphorylation. To study the structure- function relation between NHE-RF and PKA mediated inhibition of the rabbit BBM Na/H exchanger, the effect of recombinant proteins representing full-length NHE-RF as well as truncated and mutant forms of NHE-RF were determined using a reconstitution assay. The reconstitution assay employed a fraction of rabbit BBM proteins that contains Na/H exchanger activity that is not regulated by PKA. NHE-RF in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a significant left shift in the dose-response relation such that 10(-12) M NHE-RF inhibited Na/H exchange transport by 30% in the presence but not in the absence of PKA. A recombinant polypeptide representing amino acids 1-151 (Domain I) did not affect Na/H exchange transport in the presence or absence of PKA. A polypeptide representing amino acids 149-358 (Domain II) in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a left shift in the dose-response relation. 10(-12) M of Domain II polypeptide inhibited transport by 18% in the presence but not in the absence of PKA. Mutation of serine residues 287, 289, and 290 to alanine did not affect the inhibitory effect in the absence of PKA but abolished the left shift in the dose-response relation elicited by PKA. Mutation of serine residues 339 and 340 to alanine were without effect on PKA dependent regulation of Na/H exchange transport. These studies indicate that NHE-RF inhibits basal rabbit renal BBM Na/H exchange activity-an effect which is augmented by PKA. The amino acid sequences in the polypeptide containing only the NH2-terminal PDZ domain of NHE-RF have no intrinsic activity as an inhibitor but appears to be required for the full-length NHE-RF to express its full inhibitory effect on the BBM Na/H exchanger. One or more of the serine residues at positions 287, 289, and/or 290 represent the critical PKA phosphorylation site(s) on the NHE-RF protein that mediates the physiologic effect of cAMP on the renal BBM Na/H exchanger.Journal of Clinical Investigation 06/1998; 101(10):2199-206. · 15.39 Impact Factor
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ABSTRACT: The proximal tubular brush border membrane type IIa Na/P(i)-cotransporter is an important element in overall phosphate (Pi) homeostasis. Its regulation is tightly associated with membrane retrieval/reinsertion mechanisms. Specific molecular domains are involved in its internalization (predicted third intracellular loop) and in its apical expression (carboxy-terminus). Regulation and apical expression require a correct ('proximal tubular') cellular context and interaction with specific cellular proteins (scaffolding). Basic cotransport function is via a 3 Na+ to 1 P(i)-coupling ratio, also including the possibility of a Na+-leak, and is strongly affected by changes in pH. This function can be assigned to monomeric transporter molecules. The predicted first intracellular and third extracellular loops contribute important functional characteristics. It is suggested that they may form "re-entrant loops" and thereby a "permeation pore." Sequences in this region determine also pH-sensitivity and affinities in P(i)- and in Na+-interaction, respectively.Kidney International 09/2002; 62(2):375-82. · 6.61 Impact Factor
Article: Parathyroid hormone treatment induces dissociation of type IIa Na+-P(i) cotransporter-Na+/H+ exchanger regulatory factor-1 complexes.[show abstract] [hide abstract]
ABSTRACT: The type IIa Na+-P(i) cotransporter (NaP(i)-IIa) and the Na+/H+ exchanger regulatory factor-1 (NHERF1) colocalize in the apical membrane of proximal tubular cells. Both proteins interact in vitro. Herein the interaction between NaP(i)-IIa and NHERF1 is further documented on the basis of coimmunoprecipitation and co-pull-down assays. NaP(i)-IIa is endocytosed and degraded in lysosomes upon parathyroid hormone (PTH) treatment. To investigate the effect of PTH on the NaP(i)-IIa-NHERF1 association, we first compared the localization of both proteins after PTH treatment. In mouse proximal tubules and OK cells, NaP(i)-IIa was removed from the apical membrane after hormonal treatment; however, NHERF1 remained at the membrane. Moreover, PTH treatment led to degradation of NaP(i)-IIa without changes in the amount of NHERF1. The effect of PTH on the NaP(i)-IIa-NHERF1 interaction was further studied using coimmunoprecipitation. PTH treatment reduced the amount of NaP(i)-IIa coimmunoprecipitated with NHERF antibodies. PTH-induced internalization of NaP(i)-IIa requires PKA and PKC; therefore, we next analyzed whether PTH induces changes in the phosphorylation state of either partner. NHERF1 was constitutively phosphorylated. Moreover, in mouse kidney slices, PTH induced an increase in NHERF1 phosphorylation; independent activation of PKA or PKC also resulted in increased phosphorylation of NHERF1 in kidney slices. However, NaP(i)-IIa was not phosphorylated either basally or after exposure to PTH. Our study supports an interaction between NHERF1 and NaP(i)-IIa on the basis of their brush-border membrane colocalization and in vitro coimmunoprecipitation/co-pull-down assays. Furthermore, PTH weakens this interaction as evidenced by different in situ and in vivo behavior. The PTH effect takes place in the presence of increased phosphorylation of NHERF1.AJP Cell Physiology 08/2005; 289(1):C159-67. · 3.54 Impact Factor