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1.
J Biol Chem ; 286(43): 37216-21, 2011 Oct 28.
Article in English | MEDLINE | ID: mdl-21908609

ABSTRACT

Fibroblast growth factor-23 (FGF-23) inhibits sodium-dependent phosphate transport in brush border membrane vesicles derived from hormone-treated kidney slices of the mouse and in mouse proximal tubule cells by processes involving mitogen-activated protein kinase (MAPK) but not protein kinase A (PKA) or protein kinase C (PKC). By contrast, phosphate transport in brush border membrane vesicles and proximal tubule cells from sodium-hydrogen exchanger regulatory factor-1 (NHERF-1)-null mice were resistant to the inhibitory effect of FGF-23 (10(-9) m). Infection of NHERF-1-null proximal tubule cells with wild-type adenovirus-GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of FGF-23. Infection with adenovirus-GFP-NHERF-1 containing a S77A or T95D mutation also increased basal phosphate transport, but the cells remained resistant to FGF-23 (10(-9) m). Low concentrations of FGF-23 (10(-13) m) and PTH (10(-11) m) individually did not inhibit phosphate transport or activate PKA, PKC, or MAPK. When combined, however, these hormones markedly inhibited phosphate transport associated with activation of PKC and PKA but not MAPK. These studies indicate that FGF-23 inhibits phosphate transport in the mouse kidney by processes that involve the scaffold protein NHERF-1. In addition, FGF-23 synergizes with PTH to inhibit phosphate transport by facilitating the activation of the PTH signal transduction pathway.


Subject(s)
Fibroblast Growth Factors/metabolism , Kidney Tubules, Proximal/metabolism , Parathyroid Hormone/metabolism , Phosphates/metabolism , Phosphoproteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Adenoviridae , Amino Acid Substitution , Animals , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblast Growth Factor-23 , Fibroblast Growth Factors/agonists , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/pharmacology , Ion Transport/drug effects , Ion Transport/physiology , Mice , Mice, Knockout , Mutation, Missense , Parathyroid Hormone/agonists , Parathyroid Hormone/genetics , Parathyroid Hormone/pharmacology , Phosphoproteins/genetics , Protein Kinase C/genetics , Protein Kinase C/metabolism , Sodium-Hydrogen Exchangers/genetics , Transduction, Genetic
2.
Am J Physiol Renal Physiol ; 300(5): F1123-9, 2011 May.
Article in English | MEDLINE | ID: mdl-21325500

ABSTRACT

The current experiments explore the role of dopamine in facilitating the acute increase in renal phosphate excretion in response to a high-phosphate diet. Compared with a low-phosphate (0.1%) diet for 24 h, mice fed a high-phosphate (1.2%) diet had significantly higher rates of phosphate excretion in the urine associated with a two- to threefold increase in the dopamine content of the kidney and in the urinary excretion of dopamine. Animals fed a high-phosphate diet had a significant increase in the abundance and activity of renal DOPA (l-dihydroxyphenylalanine) decarboxylase and significant reductions in renalase, monoamine oxidase A, and monoamine oxidase B. The activity of protein kinase A and protein kinase C, markers of activation of renal dopamine receptors, were significantly higher in animals fed a high-phosphate vs. a low-phosphate diet. Treatment of rats with carbidopa, an inhibitor of DOPA decarboxylase, impaired adaptation to a high-phosphate diet. These experiments indicate that the rapid adaptation to a high-phosphate diet involves alterations in key enzymes involved in dopamine synthesis and degradation, resulting in increased renal dopamine content and activation of the signaling cascade used by dopamine to inhibit the renal tubular reabsorption of phosphate.


Subject(s)
Dopamine/metabolism , Kidney/metabolism , Phosphorus, Dietary/metabolism , Adaptation, Physiological , Analysis of Variance , Animals , Aromatic Amino Acid Decarboxylase Inhibitors , Carbidopa/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Dopa Decarboxylase/metabolism , Dopamine/urine , Enzyme Inhibitors/pharmacology , Kidney/drug effects , Male , Mice , Mice, Inbred C57BL , Monoamine Oxidase/metabolism , Phosphorus, Dietary/administration & dosage , Phosphorus, Dietary/urine , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction , Time Factors , Up-Regulation
3.
Am J Physiol Renal Physiol ; 300(1): F231-5, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21048030

ABSTRACT

Parathyroid hormone (PTH) inhibits the reabsorption of phosphate in the renal proximal tubule by disrupting the binding of the sodium-dependent phosphate transporter 2A (Npt2a) to the adapter protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), a process initiated by activation of protein kinase C (PKC). To gain additional insights into the dynamic sequence of events, the time course of these responses was studied in living opossum kidney (OK) cells. Using a FRET-based biosensor, we found that PTH activated intracellular PKC within seconds to minutes. In cells expressing GFP-Npt2a and mCherry-NHERF, PTH did not affect the relative abundance of NHERF-1 but there was a significant and time-dependent decrease in the Npt2a/NHERF-1 ratio. The half-time to maximal dissociation was 15 to 20 min. By contrast, PTH had no effect on the fluorescence ratio for GFP-ezrin compared with mCherry-NHERF-1 at the apical surface. These experiments establish that PTH treatment of proximal tubule OK cells leads to rapid activation of PKC with the subsequent dissociation of Npt2a/NHERF-1 complexes. The association of NHERF-1 with Ezrin and their localization at the apical membrane, however, was unperturbed by PTH, thereby enabling the rapid recruitment and membrane reinsertion of Npt2a and other NHERF-1 targets on termination of the hormone response.


Subject(s)
Kidney/metabolism , Multiprotein Complexes/metabolism , Parathyroid Hormone/pharmacology , Phosphoproteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism , Animals , Cell Line , Cytoskeletal Proteins/metabolism , Female , Multiprotein Complexes/drug effects , Opossums
4.
Urol Res ; 38(4): 257-62, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20632170

ABSTRACT

Eukaryotic cells coordinate specific responses to hormones and growth factors by spatial and temporal organization of "signaling components." Through the formation of multiprotein complexes, cells are able to generate "signaling components" that transduce hormone signals through proteins, such as PSD-95/Dlg/ZO-1(PDZ)-containing proteins that associate by stable and dynamic interactions. The PDZ homology domain is a common protein interaction domain in eukaryotes and with greater than 500 PDZ domains identified, it is the most abundant protein interaction domain in eukaryotic cells. The NHERF (sodium hydrogen exchanger regulatory factor) proteins are PDZ domain-containing proteins that play an important role in maintaining and regulating cell function. NHERF-1 was initially identified as a brush border membrane-associated phosphoprotein essential for the cAMP/PKA-induced inhibition of the sodium hydrogen exchanger isoform 3 (NHE3). Mouse, rabbit and human renal proximal tubules also express NHERF-2 (E3KARP), a structurally related protein, which in model cell systems also binds NHE3 and mediates its inhibition by cAMP. PDZK1 (NHERF-3) and IKEPP (NHERF-4) were later identified and found to have similar homology domains, leading to their recent reclassification. Although studies have revealed similar binding partners and overlapping functions for the NHERF proteins, it is clear that there is a significant amount of specificity between them. This review focuses primarily on NHERF-1, as the prototypical PDZ protein and will give a brief summary of its role in phosphate transport and the development of some forms of nephrolithiasis.


Subject(s)
Kidney Tubules, Proximal/metabolism , Phosphoproteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Animals , Humans , Kidney Tubules, Proximal/ultrastructure , Mice , Microscopy, Confocal , PDZ Domains/physiology , Phosphates/metabolism , Protein Binding , Rabbits , Signal Transduction
5.
J Biol Chem ; 285(33): 25134-8, 2010 Aug 13.
Article in English | MEDLINE | ID: mdl-20571032

ABSTRACT

The phosphorylation of the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) plays a key role in the regulation of renal phosphate transport by parathyroid hormone (PTH) and dopamine. Ser(77) in the first PDZ domain of NHERF-1 is a downstream target of both hormones. The current experiments explore the role of Thr(95), another phosphate acceptor site in the PDZ I domain, on hormone-mediated regulation of phosphate transport in the proximal tubule of the kidney. The substitution of alanine for threonine at position 95 (T95A) significantly decreased the rate and extent of in vitro phosphorylation of Ser(77) by PKC. In NHERF-1-null proximal tubule cells, neither PTH nor dopamine inhibited sodium-dependent phosphate transport. Infection of the cells with adenovirus expressing full-length WT GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of both PTH and dopamine. Infection with full-length NHERF-1 containing a T95A mutation, however, increased basal phosphate transport but not the responsiveness to either hormone. As determined by surface plasmon resonance, the substitution of serine for aspartic acid (S77D) in the PDZ I domain decreased the binding affinity to the sodium-dependent phosphate transporter 2a (Npt2a) as compared with WT PDZ I, but a T95D mutation had no effect on binding. Finally, cellular studies indicated that both PTH and dopamine treatment increased the phosphorylation of Thr(95). These studies indicate a remarkable cooperativity between the phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the hormonal regulation of renal phosphate transport. The phosphorylation of Thr(95) facilitates the phosphorylation of Ser(77). This, in turn, results in the dissociation of NHERF-1 from Npt2a and a decrease in phosphate transport in renal proximal tubule cells.


Subject(s)
Biological Transport/drug effects , Dopamine/pharmacology , Parathyroid Hormone/pharmacology , Phosphates/metabolism , Phosphoproteins/metabolism , Serine/metabolism , Sodium-Hydrogen Exchangers/metabolism , Threonine/metabolism , Animals , Blotting, Western , Cell Line , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Humans , Mice , Phosphoproteins/genetics , Phosphorylation/drug effects , Protein Binding/drug effects , Sodium-Hydrogen Exchangers/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism
6.
J Biol Chem ; 285(18): 13454-60, 2010 Apr 30.
Article in English | MEDLINE | ID: mdl-20200151

ABSTRACT

Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in (32)P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.


Subject(s)
Dopamine Agents/pharmacology , Dopamine/pharmacology , Kidney Tubules, Proximal/metabolism , Phosphates/metabolism , Phosphoproteins/metabolism , Signal Transduction/drug effects , Sodium-Hydrogen Exchangers/metabolism , Sodium/metabolism , Adenoviridae , Animals , Ion Transport/drug effects , Ion Transport/genetics , Mice , Mice, Knockout , Opossums , Phosphoproteins/genetics , Phosphorylation , Protein Kinase C/genetics , Protein Kinase C/metabolism , Receptors, Dopamine D1/genetics , Receptors, Dopamine D1/metabolism , Signal Transduction/genetics , Sodium-Hydrogen Exchangers/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIa/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism
7.
Am J Physiol Renal Physiol ; 297(6): F1560-5, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19794105

ABSTRACT

Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was approximately 17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.


Subject(s)
Fluorescence Recovery After Photobleaching , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Parathyroid Hormone/pharmacology , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Down-Regulation , Kidney Tubules, Proximal/cytology , Opossums , PDZ Domains , Phosphoproteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Time Factors , Up-Regulation
8.
Am J Physiol Renal Physiol ; 296(2): F355-61, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18987113

ABSTRACT

The present experiments were designed to detail factors regulating phosphate transport in cultured mouse proximal tubule cells by determining the response to parathyroid hormone (PTH), dopamine, and second messenger agonists and inhibitors. Both PTH and dopamine inhibited phosphate transport by over 30%. The inhibitory effect of PTH was completely abolished in the presence of chelerythrine, a PKC inhibitor, but not by Rp-cAMP, a PKA inhibitor. By contrast, both chelerythrine and Rp-cAMP blocked the inhibitory effect of dopamine. Chelerythrine inhibited PTH-mediated cAMP accumulation but also blocked the inhibitory effect of 8-bromo-cAMP on phosphate transport. On the other hand, Rp-cAMP had no effect on the ability of DOG, a PKC activator, to inhibit phosphate transport. PD98059, an inhibitor of MAPK, had no effect on PTH- or dopamine-mediated inhibition of sodium-phosphate cotransport. Finally, compared with 8-bromo-cAMP, 8-pCPT-2'-O-Me-cAMP, an activator of EPAC, had no effect on phosphate transport. These results outline significant differences in the signaling pathways utilized by PTH and dopamine to inhibit renal phosphate transport. Our results also suggest that activation of MAPK is not critically involved in PTH- or dopamine-mediated inhibition of phosphate transport in mouse renal proximal tubule cells in culture.


Subject(s)
Dopamine/metabolism , Kidney Tubules, Proximal/metabolism , Parathyroid Hormone/metabolism , Phosphates/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cells, Cultured , Cyclic AMP/metabolism , Enzyme Activators , Enzyme Inhibitors , Male , Mice , Mice, Inbred C57BL , Protein Serine-Threonine Kinases/antagonists & inhibitors , Signal Transduction
9.
Am J Physiol Renal Physiol ; 295(6): F1658-65, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18799551

ABSTRACT

There is increasing evidence that mammalian urinary tract epithelial cells utilize membrane channels and transporters to transport solutes across their apical (luminal) and basalateral membranes to modify solute concentrations in both cell and urine. This study investigates the expression, localization, and regulation of the ROMK (K(ir) 1.1) potassium channels in rat and dog ureter and bladder tissues. Immunoblots of homogenates of whole ureter, whole bladder, bladder epithelial cells, and bladder smooth muscle tissues in both rat and dog identified approximately 45- to 50-kDa bands characteristic of ROMK in all tissues. RT-PCR identified ROMK mRNA in these same tissues in both animal species. ROMK protein localized by immunocytochemistry was strongly expressed in the apical membranes of the large umbrella cells lining the bladder lumen and to a lesser extent in the cytoplasm of epithelial cells and smooth muscle cells in the rat bladder. ROMK protein and mRNA were also discovered in cardiac, striated, and smooth muscle in diverse organs. There was no difference in immunoblot expression of ROMK abundance in bladder homogenates (whole bladder, epithelial cell, or muscle cell) or ureteral homogenates between groups of rats fed high- or low-potassium diets. Although the functional role of ROMK in urinary tract epithelia and smooth muscle is unknown, ROMK may participate in the regulation of epithelial and smooth muscle cell volume and osmolality, in the dissipation of potassium leaked or diffused from urine across the epithelial cell apical membranes or tight junctions, and in net or bidirectional potassium transport across urinary tract epithelia.


Subject(s)
Muscle, Smooth/physiology , Potassium Channels, Inwardly Rectifying/physiology , Animals , Epithelial Cells/physiology , Female , Gastrointestinal Tract/physiology , Heart/physiology , Potassium/metabolism , Potassium Channels, Inwardly Rectifying/genetics , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Ureter/physiology , Urinary Bladder/physiology , Urinary Tract/metabolism
10.
Am J Physiol Renal Physiol ; 294(4): F840-9, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18216150

ABSTRACT

Several lines of evidence show that sodium/hydrogen exchanger regulatory factor 1 (NHERF-1) regulates the expression and activity of the type IIa sodium-dependent phosphate transporter (Npt2a) in renal proximal tubules. We have previously demonstrated that expression of a COOH-terminal ezrin binding domain-deficient NHERF-1 in opossum kidney (OK) cells decreased expression of Npt2a in apical membranes but did not affect responses to parathyroid hormone. We hypothesized that NHERF-1 regulates apical membrane expression of Npt2a in renal proximal tubule cells. To address this hypothesis, we compared regulation of Npt2a expression and function in NHERF-deficient OK cells (OK-H) and wild-type cells (OK-WT). In OK-H cells, phosphate uptake and expression of Npt2a protein in apical membranes were significantly lower than in OK-WT cells. Transient transfection of green fluorescent protein-tagged Npt2a cDNA into OK-H cells resulted in aberrant localization of an Npt2a fragment to the cytosol but not to the apical membrane. OK-H cells also exhibited a marked decrease in Npt2a mRNA expression. As demonstrated by luciferase assay, Npt2a promoter activity was significantly decreased in OK-H cells compared with that shown in OK-WT cells. Transfection of OK-H cells with human NHERF-1 restored Npt2a expression at both the protein and mRNA levels and regulation by parathyroid hormone. Expression of NHERF-1 constructs with mutations in the PDZ domains or the ezrin binding domain in OK-H cells suggested that the PDZ2 domain is critical for apical translocation of Npt2a and for expression at the mRNA level. Our data demonstrate for the first time that NHERF-1 regulates Npt2a transcription and membrane insertion.


Subject(s)
Phosphoproteins/physiology , Sodium-Hydrogen Exchangers/physiology , Sodium-Phosphate Cotransporter Proteins, Type IIa/genetics , Animals , Cell Line , DNA Primers , DNA, Complementary/genetics , Genes, Reporter , Kidney , Opossums , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sodium-Phosphate Cotransporter Proteins, Type IIa/deficiency , Transfection
11.
J Clin Invest ; 117(11): 3412-20, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17975671

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.


Subject(s)
Kidney/metabolism , Parathyroid Hormone/metabolism , Phosphates/metabolism , Phosphoproteins/metabolism , Serine/metabolism , Sodium-Hydrogen Exchangers/metabolism , Animals , Biological Transport/physiology , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation , Enzyme Inhibitors/metabolism , Kidney/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , PDZ Domains , Phosphoproteins/genetics , Phosphorylation , Protein Kinase C/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Second Messenger Systems/physiology , Sodium/metabolism , Sodium-Hydrogen Exchangers/genetics
12.
J Biol Chem ; 282(46): 33879-33887, 2007 Nov 16.
Article in English | MEDLINE | ID: mdl-17895247

ABSTRACT

NHERF-1 (Na(+)-H(+) exchanger regulatory factor 1, also known as EBP50 ezrin-binding protein of 50 kDa) is a phosphoprotein that assembles multiprotein complexes via two PDZ domains and a C-terminal ezrin-binding domain. Current work utilized metabolic labeling in cultured cells expressing wild type GFP-NHERF-1 to define the physiological importance of NHERF-1 phosphorylation. Treatment of cells with phosphatase inhibitors calyculin A and okadaic acid enhanced NHERF-1 phosphorylation and inhibited its dimerization. Eliminating C-terminal serines abolished the modulation of NHERF-1 dimerization by phosphatase inhibitors and identified the phosphorylation of the PDZ1 domain that attenuated its binding to physiological targets, including beta(2)-adrenergic receptor, platelet-derived growth factor receptor, cystic fibrosis transmembrane conductance regulator, and sodium-phosphate cotransporter type IIa. The major covalent modification of PDZ1 was mapped to serine 77. Confocal microscopy of cultured cells suggested key roles for PDZ1 and ERM-binding domain in localizing NHERF-1 at the cell surface. The substitution S77A eliminated PDZ1 phosphorylation and increased NHERF-1 localization at the cell periphery. In contrast, S77D reduced NHERF-1 colocalization with cortical actin cytoskeleton. These data suggested that serine 77 phosphorylation played key role in modulating NHERF-1 association with plasma membrane targets and identified a novel mechanism by which PDZ1 phosphorylation may transduce hormonal signals to regulate the function of membrane proteins in epithelial tissues.


Subject(s)
Phosphoproteins/chemistry , Sodium-Hydrogen Exchangers/chemistry , Amino Acid Sequence , Animals , COS Cells , Cell Membrane/metabolism , Chlorocebus aethiops , Dimerization , Humans , Marine Toxins , Mice , Molecular Sequence Data , NIH 3T3 Cells , Okadaic Acid/chemistry , Oxazoles/chemistry , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Receptors, Adrenergic, beta-2/metabolism , Sequence Homology, Amino Acid , Sodium-Hydrogen Exchangers/metabolism
13.
J Biol Chem ; 282(34): 25141-51, 2007 Aug 24.
Article in English | MEDLINE | ID: mdl-17580307

ABSTRACT

The multi-PDZ domain containing protein Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) binds to Na(+)/H(+) exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1(-/-) mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1(-/-) ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1(-/-) mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2'O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1(-/-) mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.


Subject(s)
Ileum/metabolism , Kidney Tubules/metabolism , Phosphoproteins/physiology , Sodium-Hydrogen Exchangers/physiology , Animals , Cyclic AMP/metabolism , Guanine Nucleotide Exchange Factors/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Isoforms , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolism , Tissue Distribution
14.
J Am Soc Nephrol ; 18(5): 1419-25, 2007 May.
Article in English | MEDLINE | ID: mdl-17409311

ABSTRACT

Sodium-hydrogen exchanger regulatory factor-1-deficient (NHERF-1(-/-)) mice demonstrate increases in the urinary excretion of phosphate, calcium, and uric acid associated with interstitial deposition of calcium in the papilla of the kidney. These studies examine the role of NHERF-1 in the tubular reabsorption of uric acid and regulation of mouse urate transporter 1 (mURAT1), a newly described transporter that is responsible for the renal tubular reabsorption of uric acid. In primary cultures of mouse renal proximal tubule cells, uric acid uptake was significantly lower in NHERF-1(-/-) cells compared with wild-type cells over a large range of uric acid concentrations in the media. Western immunoblotting revealed a 56 +/- 6% decrease in the brush border membrane (BBM) expression of mURAT1 in NHERF-1(-/-) compared with wild-type control kidneys (P < 0.05). Confocal microscopy confirmed the reduced apical membrane expression of mURAT1 in NHERF-1(-/-) kidneys and demonstrated mislocalization of mURAT1 to intracellular vesicular structures. Para-aminohippurate significantly inhibited uric acid uptake in wild-type cells (41 +/- 2%) compared with NHERF-1(-/-) cells (8.2 +/- 3%). Infection of NHERF-1(-/-) cells with adenovirus-green fluorescence protein-NHERF-1 resulted in significantly higher rates of uric acid transport (15.4 +/- 1.1 pmol/microg protein per 30 min) compared with null cells that were infected with control adenovirus-green fluorescence protein (7.9 +/- 0.3) and restoration of the inhibitory effect of para-aminohippurate (% inhibition 34 +/- 4%). These findings indicate that NHERF-1 exerts a significant effect on the renal tubular reabsorption of uric acid in the mouse by modulating the BBM abundance of mURAT1 and possibly other BBM uric acid transporters.


Subject(s)
Kidney Tubules, Proximal/metabolism , Membrane Proteins/metabolism , Organic Anion Transporters/metabolism , Phosphoproteins/metabolism , Phosphoproteins/physiology , Sodium-Hydrogen Exchangers/metabolism , Sodium-Hydrogen Exchangers/physiology , Uric Acid/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microvilli/metabolism , Phosphoproteins/genetics , Protein Binding , Sodium-Hydrogen Exchangers/genetics , Transfection
15.
Am J Physiol Renal Physiol ; 291(4): F896-901, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16705152

ABSTRACT

Sodium-dependent phosphate transport in NHERF-1(-/-) proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1(-/-) cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.


Subject(s)
Parathyroid Hormone/pharmacology , Phosphoproteins/deficiency , Phosphoproteins/genetics , Sodium-Hydrogen Exchangers/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism , Sodium-Phosphate Cotransporter Proteins/metabolism , Adenoviridae , Animals , Culture Media , Genetic Vectors , Mice , Mice, Knockout , Phosphates/pharmacology , Phosphoproteins/metabolism , Recombinant Proteins/metabolism , Sodium-Hydrogen Exchangers/metabolism
16.
Am J Physiol Renal Physiol ; 290(4): F838-43, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16249272

ABSTRACT

NHERF-1 binds numerous renal protein targets, including the proximal tubule transporters Na(+)/H(+) exchanger 3 (NHE3) and Na(+)-phosphate cotransporter 2a (Npt2a). Young NHERF-1(-/-) male mice display defective targeting of Npt2a to apical membranes in the renal proximal tubule and manifest hypophosphatemia and increased urinary excretion of phosphate. The present studies describe the changes in the urinary excretion of phosphate, calcium, uric acid, and sodium in male and female wild-type and NHERF-1 null mice over a time period from 12 to 54 wk of age. Young male and female NHERF-1(-/-) mice demonstrated increased urinary excretion of phosphate and urine phosphate/creatinine ratios. There was an age-related decline in the phosphate/creatinine ratio in mutant mice such that there were no differences between wild-type and NHERF-1(-/-) by 24 to 30 wk of age despite the continued presence of hypophosphatemia. Male and female NHERF-1 null mice also demonstrate increased urine calcium/creatinine and uric acid/creatinine ratios compared with wild-type controls. These studies indicate defects in the renal tubule transport of phosphate, calcium, and uric acid in NHERF-1(-/-) male and female mice that could account for the increased deposition of calcium in the papilla of null mice.


Subject(s)
Calcium/urine , Kidney Calculi/physiopathology , Kidney Tubules/physiology , Phosphates/urine , Phosphoproteins/genetics , Phosphoproteins/physiology , Uric Acid/urine , Age Factors , Animals , Creatinine/urine , Female , Kidney Medulla/chemistry , Kinetics , Male , Mice , Mice, Knockout , Sodium-Hydrogen Exchangers
17.
J Am Soc Nephrol ; 16(9): 2598-607, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16000700

ABSTRACT

It was demonstrated that expression of murine sodium hydrogen exchanger regulatory factor (NHERF-1) lacking the ezrin-binding domain blocks parathyroid hormone (PTH) regulation of Na+,K+-ATPase in opossum kidney (OK) cells. The hypothesis that the NHERF-1 PDZ domains contribute to PTH regulation of Na+,K+-ATPase was tested by comparison of PTH regulation of Na+,K+-ATPase in wild-type OK (OK-WT) cells, NHERF-deficient OKH cells, OK-WT transfected with siRNA for NHERF (NHERF siRNA OK-WT), and OKH cells that were stably transfected with full-length NHERF-1 or constructs with mutated PDZ domains. OKH cells and NHERF siRNA OK-WT showed decreased expression of NHERF-1 but equivalent expression of ezrin and Na+,K+-ATPase alpha1 subunit when compared with OK-WT cells. PTH decreased Na+,K+-ATPase activity and stimulated phosphorylation of the Na+,K+-ATPase alpha1 in OK-WT cells but not in NHERF-deficient cells. Rubidium (86Rb) uptake was equivalent in OK-WT, OKH, and OKH cells that were transfected with all but the double PDZ domain mutants. PTH decreased 86Rb uptake significantly in OK-WT but not in OKH cells. PTH also significantly inhibited 86Rb uptake in OKH cells that were transfected with full-length NHERF-1 or NHERF-1 with mutated PDZ 2 but not in OKH cells that were transfected with mutated PDZ 1. Transfection with NHERF expressing both mutated PDZ domains resulted in diminished basal 86Rb uptake that was not inhibited further by PTH. PTH stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation in OK-WT but not in NHERF-deficient cells. Transfection of OKH cells with NHERF constructs that contained an intact PDZ1 domain restored PTH-stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation. These results demonstrate that NHERF-1 is necessary for PTH-mediated inhibition of Na+,K+-ATPase activity and that the inhibition is mediated through the PDZ1, not PDZ2, domain.


Subject(s)
Parathyroid Hormone/pharmacology , Phosphoproteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Base Sequence , Cell Line , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , MAP Kinase Signaling System/drug effects , Mice , Opossums , Phosphoproteins/chemistry , Phosphoproteins/deficiency , Phosphoproteins/genetics , Protein Kinase C-alpha/metabolism , Protein Structure, Tertiary , Protein Subunits , RNA, Small Interfering/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sodium-Hydrogen Exchangers/chemistry , Sodium-Hydrogen Exchangers/genetics , Sodium-Potassium-Exchanging ATPase/chemistry , Transfection
18.
Am J Physiol Renal Physiol ; 289(4): F933-8, 2005 Oct.
Article in English | MEDLINE | ID: mdl-15942053

ABSTRACT

The present experiments using primary cultures from renal proximal tubule cells examine two aspects of the regulation of sodium-dependent phosphate transport and membrane sodium-dependent phosphate transporter (Npt2a) expression by parathyroid hormone (PTH). Sodium-dependent phosphate transport in proximal tubule cells from wild-type mice grown in normal-phosphate media averaged 4.4 +/- 0.5 nmol.mg protein(-1).10 min(-1) and was inhibited by 30.5 +/- 8.6% by PTH (10(-7) M). This was associated with a 32.7 +/- 5.2% decrease in Npt2a expression in the plasma membrane. Proximal tubule cells from Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1)(-/-) mice had a lower rate of phosphate transport compared with wild-type cells and a significantly reduced inhibitory response to PTH. Wild-type cells incubated in low-phosphate media for 24 h had a higher rate of phosphate transport compared with wild-type cells grown in normal-phosphate media but a significantly blunted inhibitory response to PTH. These data indicate a role for NHERF-1 in mediating the membrane retrieval of Npt2a and the subsequent inhibition of phosphate transport in renal proximal tubules. These studies also suggest that there is a blunted phosphaturic effect of PTH in cells adapted to low-phosphate media.


Subject(s)
Kidney Tubules, Proximal/metabolism , Parathyroid Hormone/physiology , Phosphates/metabolism , Phosphoproteins/physiology , Sodium/physiology , Animals , Biological Transport, Active/physiology , Blotting, Western , Cell Membrane/drug effects , Cell Membrane/metabolism , Culture Media , Cyclic AMP/biosynthesis , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinases/biosynthesis , Phosphates/deficiency , Phosphoproteins/genetics , Protein Kinase C/biosynthesis , Sodium-Hydrogen Exchangers
19.
J Biol Chem ; 279(36): 37815-21, 2004 Sep 03.
Article in English | MEDLINE | ID: mdl-15218020

ABSTRACT

The present experiments using primary cultures of renal proximal tubule cells derived from wild-type and NHERF-1 knockout animals examines the regulation of NHE3 by phenylthiohydantoin (PTH) and the regulation of phosphate transport in response to alterations in the media content of phosphate. Forskolin (34.8 +/- 6.2%) and PTH (29.7 +/- 1.8%) inhibited NHE3 activity in wild-type proximal tubule cells but neither forskolin (-3.2 +/- 3.3%) nor PTH (-16.6 +/- 8.1%) inhibited NHE3 activity in NHERF-1(-/-) cells. Using adenovirus-mediated gene transfer, expression of NHERF-1 in NHERF-1(-/-) proximal tubule cells restored the inhibitory response to forskolin (28.2 +/- 3.0%) and PTH (33.2 +/- 3.9%). Compared with high phosphate media, incubation of wild-type cells in low phosphate media resulted in a 36.0 +/- 6.3% higher rate of sodium-dependent phosphate transport and a significant increase in the abundance of Npt2a and PDZK1. NHERF-1(-/-) cells, on the other hand, had lower rates of sodium-dependent phosphate uptake and low phosphate media did not stimulate phosphate transport. Npt2a expression was not affected by the phosphate content of the media in NHERF-1 null cells although low phosphate media up-regulated PDZK1 abundance. Primary cultures of mice proximal tubule cells retain selected regulatory pathways observed in intact kidneys. NHERF-1(-/-) proximal tubule cells demonstrate defective regulation of NHE3 by PTH and indicate that reintroduction of NHERF-1 repairs this defect. NHERF-1(-/-) cells also do not adapt to alterations in the phosphate content of the media indicating that the defect resides within the cells of the proximal tubule and is not dependent on systemic factors.


Subject(s)
Adaptation, Physiological , Kidney Tubules, Proximal/metabolism , Parathyroid Hormone/physiology , Phosphates/metabolism , Sodium-Hydrogen Exchangers/metabolism , Animals , Cells, Cultured , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Sodium-Hydrogen Exchanger 3
20.
J Biol Chem ; 279(24): 25002-9, 2004 Jun 11.
Article in English | MEDLINE | ID: mdl-15070904

ABSTRACT

We previously showed that Na(+)/H(+)-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) co-immunoprecipitated with the human kappa opioid receptor (hKOR) and that its overexpression blocked the kappa agonist U50,488H-induced hKOR down-regulation by enhancing recycling. Here, we show that glutathione S-transferase (GST)-hKOR C-tail interacted with purified NHERF-1/EBP50, whereas GST or GST-C-tails of micro or delta opioid receptors did not. GST-hKOR C-tail, but not GST, bound HA-NHERF-1/EBP50 transfected into Chinese hamster ovary cells and endogenous NHERF-1/EBP50 in opossum kidney proximal tubule epithelial cells (OK cells). The PDZ domain I, but not II, of NHERF-1/EBP50 was involved in the interaction. Association of NHERF-1/EBP50 with hKOR C-tail enhanced oligomerization of NHERF-1/EBP50. NHERF-1/EBP50 was previously shown to regulate Na(+)/H(+)-exchanger 3 (NHE3) activities in OK cells. We found stimulation of OK cells with U50,488H significantly enhanced Na(+)/H(+) exchange, which was blocked by naloxone but not by pertussis toxin pretreatment, indicating it is mediated by KORs but independent of G(i)/G(o) proteins. In OKH cells, a subclone of OK cells expressing a much lower level of NHERF-1/EBP50, U50,488H had no effect on Na(+)/H(+) exchange, although it enhanced p44/42 mitogen-activated protein kinase phosphorylation via G(i)/G(o) proteins similar to that in OK cells. Stable transfection of NHERF-1/EBP50 into OKH cells restored the stimulatory effect of U50,488H upon Na(+)/H(+) exchange. Thus, NHERF-1/EBP50 binds directly to KOR, and this association plays an important role in accelerating Na(+)/H(+) exchange. We hypothesize that binding of the KOR to NHERF-1/EBP50 facilitates oligomerization of NHERF-1/EBP50, leading to stimulation of NHE3. This study provides the first direct evidence that a G protein-coupled receptor through association with NHERF-1/EBP-50 stimulates NHE3.


Subject(s)
Carrier Proteins/physiology , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Phosphoproteins/physiology , Receptors, Opioid, kappa/physiology , Sodium-Hydrogen Exchangers/metabolism , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Animals , CHO Cells , Carrier Proteins/chemistry , Cell Line , Cricetinae , Humans , Mitogen-Activated Protein Kinases/metabolism , Naloxone/pharmacology , Opossums , Pertussis Toxin/pharmacology , Phosphoproteins/chemistry , Phosphorylation , Receptors, Opioid, kappa/chemistry , Sodium-Hydrogen Exchanger 3
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