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ABSTRACT: Control of phosphate (P(i)) homeostasis is essential for many biologic functions and inappropriate low levels of P(i) in plasma have been suggested to associate with several pathological states, including renal stone formation and stone recurrence. P(i) homeostasis is achieved mainly by adjusting the renal reabsorption of P(i) to the body's requirements. This task is performed to a major extent by the Na/Pi cotransporter NaPi-IIa that is specifically expressed in the brush border membrane of renal proximal tubules. While the presence of tight junctions in epithelial cells prevents the diffusion and mixing of the apical and basolateral components, the location of a protein within a particular membrane subdomain (i.e., the presence of NaPi-IIa at the tip of the apical microvilli) often requires its association with scaffolding elements which directly or indirectly connect the protein with the underlying cellular cytoskeleton. NaPi-IIa interacts with the four members of the Na(+)/H(+) exchanger regulatory factor family as well as with the GABA(A)-receptor associated protein . Here we will discuss the most relevant findings regarding the role of these proteins on the expression and regulation of the cotransporter, as well as the impact that their absence has in P(i) homeostasis.
Urological Research 08/2010; 38(4):271-6. · 1.23 Impact Factor
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I Alexandru Bobulescu,
Henry Quiñones, Serge M Gisler,
Francesca Di Sole,
Ming-Chang Hu,
Mingjun Shi,
Jianning Zhang,
Daniel G Fuster,
Nancy Wright,
Marc Mumby,
Orson W Moe
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ABSTRACT: Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56δ (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, coimmunoprecipitation, blot overlay, and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small T antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition.
AJP Renal Physiology 02/2010; 298(5):F1205-13. · 4.42 Impact Factor
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ABSTRACT: Renal reabsorption of inorganic phosphate (P(i)) is mainly mediated by the Na(+)-dependent P(i)-cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study GABA(A) receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S-transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36-68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. GABARAP(-/-) mice have reduced urinary excretion of P(i), higher Na(+)-dependent (32)P(i) uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with GABARAP(+/+) mice. The expression of Na(+)/H(+) exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in GABARAP(-/-) mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary P(i) content.
American journal of physiology. Renal physiology 03/2009; 296(5):F1118-28. · 3.68 Impact Factor
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Paola Capuano,
Desa Bacic,
Marcel Roos, Serge M Gisler,
Gerti Stange,
Jürg Biber,
Brigitte Kaissling,
Edward J Weinman,
Shirish Shenolikar,
Carsten A Wagner,
Heini Murer
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ABSTRACT: Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na(+)-phosphate cotransporter (NaP(i)-IIa) in the brush border membrane (BBM). The activity and localization of NaP(i)-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP(i)-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na(+)/H(+)-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP(i)-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP(i)-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1-34) led to internalization of NaP(i)-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3-34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP(i)-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP(i)-IIa and, specifically, couples the apical PTHR to PLC.
AJP Cell Physiology 03/2007; 292(2):C927-34. · 3.54 Impact Factor
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ABSTRACT: The type IIa Na(+)/P(i) cotransporter (NaPi-IIa) plays a key role in the reabsorption of inorganic phosphate (P(i)) in the renal proximal tubule. The rat NaPi-IIa isoform is a protein of 637 residues for which different algorithms predict 8-12 transmembrane domains (TMDs). Epitope tagging experiments demonstrated that both the N and the C termini of NaPi-IIa are located intracellularly. Site-directed mutagenesis revealed two N-glycosylation sites in a large putative extracellular loop. Results from structure-function studies suggested the assembly of two similar opposed regions that possibly constitute part of the substrate translocation pathway for one phosphate ion together with three sodium ions. Apart from these topological aspects, other structural features of NaPi-IIa are not known. In this study, we have addressed the topology of NaPi-IIa using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NaPi-IIa TMDs. Based on the results of in vitro transcription/translation analyses, we propose a model of NaPi-IIa comprising 12 TMDs, with both N and C termini orientated intracellularly and a large hydrophilic extracellular loop between the fifth and sixth TMDs. The proposed model is in good agreement with the prediction of the NaPi-IIa structure obtained by the hidden Markov algorithm HMMTOP.
Journal of Membrane Biology 02/2006; 212(1):41-9. · 1.81 Impact Factor
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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
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ABSTRACT: In adults, the extent of renal reabsorption of P(i) and consequently the extent of urinary excretion of phosphate are to a large extent determined by the abundance of the Na-P(i) cotransporter NaPi-IIa (SLC34A1). Localization of this cotransporter is restricted to the apical membrane of proximal tubular cells, and its abundance is controlled by a number of factors and pathophysiological conditions. To guarantee a proper apical localization and specific regulated endocytosis of NaPi-IIa, an orchestrated pattern of protein interactions has to be envisaged. Attempts to screen for such interacting proteins resulted in the identification of a PDZ domain containing proteins. The purpose of this review is to discuss the roles of these PDZ proteins in proximal tubular Na-P(i) cotransport.
American journal of physiology. Renal physiology 12/2004; 287(5):F871-5. · 3.68 Impact Factor
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ABSTRACT: PDZ proteins are major structural components of protein assembly. This review covers the implications of these proteins in the regulation of transport systems expressed in renal proximal tubules.
In the last few years, many reports have highlighted the implication of PDZ proteins in two aspects of proximal tubule physiology, namely the generation and maintenance of epithelial polarity and the formation of regulatory complexes that provide spatial and molecular specificity to the intracellular signalling.
PDZ-mediated interactions are involved in a wide range of cellular functions, from cell division to cell polarity to intracellular signalling. Consistent with this functional spectrum, ablation of PDZ protein genes generates a wide panel of pathological phenotypes, some of which link directly to human syndromes. In proximal tubules, PDZ proteins are thought to play a major role in epithelial polarity and transport regulation.
Current Opinion in Nephrology and Hypertension 10/2004; 13(5):569-74. · 4.33 Impact Factor
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ABSTRACT: Adenosine is an autacoid that regulates renal Na(+) transport. Activation of adenosine A(1) receptor (A(1)R) by N(6)-cyclopentidyladenosine (CPA) inhibits the Na(+)/H(+) exchanger 3 (NHE3) via phospholipase C/Ca(2+)/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A(1)R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation. CPA (10(-6) M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3(C terminus); prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3(C terminus) (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3(delta552)) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3(462-552)) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3(Full-length) interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHPNHE3 interaction is regulated by A(1)R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.
Journal of Biological Chemistry 01/2004; 279(4):2962-74. · 4.77 Impact Factor
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Serge M Gisler,
Sandra Pribanic,
Desa Bacic,
Patrik Forrer,
Andrea Gantenbein,
Luc A Sabourin,
Akira Tsuji,
Zhuo-Shen Zhao,
Edward Manser,
Jürg Biber,
Heini Murer
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ABSTRACT: In proximal tubular cells, PDZK1 (NaPi-Cap1) has been implicated in apical expression of the Na+-dependent phosphate cotransporter (NaPi-IIa) via interaction with its C-terminus. PDZK1 represents a multidomain protein consisting of four PDZ domains and thus is believed to have a broader specificity besides NaPi-IIa.
We subjected single PDZ domains derived from PDZK1 either to yeast two-hybrid screens or yeast trap assays. Different pull-down assays and blot overlays were applied to corroborate the PDZK1-mediated interactions in vitro. Co-localization of interacting proteins with PDZK1 in proximal tubular cells was assessed by immunohistochemistry.
In the yeast screens, the most abundant candidate protein to interact with PDZK1 was the membrane-associated protein of 17 kD (MAP17). Besides MAP17, C-terminal parts of following transporters were also identified: NaPi-IIa, solute carrier SLC17A1 (NaPi-I), Na+/H+ exchanger (NHE-3), organic cation transporter (OCTN1), chloride-formate exchanger (CFEX), and urate-anion exchanger (URAT1). In addition, other regulatory factors were found among the clones, such as a protein kinase A (PKA)-anchoring protein (D-AKAP2) and N+/H+ exchanger regulator factor (NHERF-1). All interactions of itemized proteins with PDZK1 were affirmed by in vitro techniques. Apart from PDZK1, strong in vitro interactions of NHERF-1 were also observed with the solute transporters (excluding MAP17) and D-AKAP2. All identified proteins were immunolocalized in proximal tubular cells, wherein all membrane proteins co-localized with PDZK1 in brush borders.
We hypothesize that PDZK1 and NHERF-1 establish an extended network beneath the apical membrane to which membrane proteins and regulatory components are anchored.
Kidney International 12/2003; 64(5):1733-45. · 6.61 Impact Factor
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ABSTRACT: PDZK1, a multiple PDZ protein, was recently found to interact with the type IIa Na/Pi cotransporter (NaPi-IIa) in renal proximal tubular cells. In a preceding study, yeast two-hybrid screens using single PDZ domains of PDZK1 as baits were performed. Among the identified proteins, a C-terminal fragment of the dual-specific A-kinase anchoring protein 2 (D-AKAP2) was obtained by screening PDZ domain 4.
After its molecular cloning by means of RACE, the renal expression of D-AKAP2 was analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Protein interactions were characterized by overlays, pull-downs, and immunoprecipitations from transfected opossum kidney (OK) cells.
Based on 5'-RACE and PDZK1 overlays of mouse kidney cortex separated by two-dimensional electrophoresis, it was suggested that the renal isoform of D-AKAP2 in mouse comprises 372 amino acids and exists as a protein of >40 kD. Immunohistochemistry and real-time PCR localized D-AKAP2 only to the subapical pole of proximal tubular cells in mouse kidney. In pull-down experiments, D-AKAP2 tightly bound PDZK1 as well as N+/H+ exchanger regulator factor (NHERF-1), but the latter with an apparent fourfold lower affinity. Similarly, His-tagged D-AKAP2 specifically retained PDZK1 from mouse kidney cortex homogenate. In addition, myc-tagged D-AKAP2 and HA-tagged PDZK1 co-immunoprecipitated from transfected OK cells.
We conclude that D-AKAP2 anchors protein kinase A (PKA) to PDZK1 and to a lesser extent to NHERF-1. Since PDZK1 and NHERF-1 both sequester NaPi-IIa to the apical membrane, D-AKAP2 may play an important role in the parathyroid hormone (PTH)-mediated regulation of NaPi-IIa by compartmentalization of PKA.
Kidney International 11/2003; 64(5):1746-54. · 6.61 Impact Factor
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Serge M Gisler,
Sandra Pribanic,
Desa Bacic,
Patrik Forrer,
Andrea Gantenbein,
Luc A Sabourin,
Akira Tsuji,
Zhuo-Shen Zhao,
Edward Manser,
J|[uuml]|rg Biber,
Heini Murer
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ABSTRACT: PDZK1: I. A major scaffolder in brush borders of proximal tubular cells.Background In proximal tubular cells, PDZK1 (NaPi-Cap1) has been implicated in apical expression of the Na+-dependent phosphate cotransporter (NaPi-IIa) via interaction with its C-terminus. PDZK1 represents a multidomain protein consisting of four PDZ domains and thus is believed to have a broader specificity besides NaPi-IIa.
Kidney International 10/2003; 64(5):1733-1745. · 6.61 Impact Factor
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ABSTRACT: Inorganic phosphate (P(i)) reabsorption in the renal proximal tubule occurs mostly via the Na(+)/P(i) cotransporter type IIa (NaP(i)-IIa) located in the brush-border membrane (BBM) and is regulated, among other factors, by dietary P(i) intake and parathyroid hormone (PTH). The PTH-induced inhibition of P(i) reabsorption is mediated by endocytosis of Na/P(i)-IIa from the BBM and subsequent lysosomal degradation. Megalin is involved in receptor-mediated endocytosis of proteins from the urine in the renal proximal tubule. The recently identified receptor-associated protein (RAP) is a novel type of chaperone responsible for the intracellular transport of endocytotic receptors such as megalin. Gene disruption of RAP leads to a decrease of megalin in the BBM and to a disturbed proximal tubular endocytotic machinery. Here we investigated whether the distribution of NaP(i)-IIa and/or its regulation by dietary P(i) intake and PTH is affected in the proximal tubules of RAP-deficient mice as a model for megalin loss. In RAP-deficient mice megalin expression was strongly reduced and restricted to a subapical localization. NaP(i)-IIa protein distribution and abundance in the kidney was not altered. The localization and abundance of the NaP(i)-IIa interacting proteins MAP17, PDZK-1, D-AKAP2, and NHE-RF1 were also normal. Other transport proteins expressed in the BBM such as the Na(+)/H(+) exchanger NHE-3 and the Na(+)/sulphate cotransporter NaSi were normally expressed. In whole animals and in isolated fresh kidney slices the PTH-induced internalization of NaP(i)-IIa was strongly delayed in RAP-deficient mice. PTH receptor expression in the proximal tubule was not affected by the RAP knock-out. cAMP, cGMP or PKC activators induced internalization which was delayed in RAP-deficient mice. In contrast, both wildtype and RAP-deficient mice were able to adapt to high-, normal, and low-P(i) diets appropriately as indicated by urinary P(i) excretion and NaP(i)-IIa protein abundance.
Pflügers Archiv - European Journal of Physiology 08/2003; 446(4):475-84. · 4.46 Impact Factor
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ABSTRACT: Type IIa Na/P(i) cotransporters are expressed in renal proximal brush border and are the major determinants of inorganic phosphate (P(i)) reabsorption. Their carboxyl-terminal tail contains information for apical expression, and interacts by means of its three terminal amino acids with several PSD95/DglA/ZO-1-like domain (PDZ)-containing proteins. Two of these proteins, NaPi-Cap1 and Na/H exchanger-regulatory factor 1 (NHERF1), colocalize with the cotransporter in the proximal brush border. We used opossum kidney cells to test the hypothesis of a potential role of PDZ-interactions on the apical expression of the cotransporter. We found that opossum kidney cells contain NaPi-Cap1 and NHERF1 mRNAs. For NHERF1, an apical location of the protein could be documented; this location probably reflects interaction with the cytoskeleton by means of the MERM-binding domain. Overexpression of PDZ domains involved in interaction with the cotransporter (PDZ-1/NHERF1 and PDZ-3/NaPi-Cap1) had a dominant-negative effect, disturbing the apical expression of the cotransporter without affecting the actin cytoskeleton or the basolateral expression of Na/K-ATPase. These data suggest an involvement of PDZ-interactions on the apical expression of type IIa cotransporters.
Proceedings of the National Academy of Sciences 10/2002; 99(18):11957-62. · 9.68 Impact Factor
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Serge M Gisler,
Saranya Kittanakom,
Daniel Fuster,
Victoria Wong,
Mia Bertic,
Tamara Radanovic,
Randy A. Hall,
Heini Murer,
Jurg Biber,
Daniel Markovich,
Orson W Moe,
Igor Stagljar
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ABSTRACT: PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.
