Serge M Gisler

University of Zurich, Zürich, ZH, Switzerland

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Publications (23)97.95 Total impact

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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. · 1.59 Impact Factor
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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: Epidural adhesion formation is believed to be a central governing factor in the prevalence of pain after spinal surgery and is regarded as being the primary instigator of neural tethering, leading to complications during revision surgery. In this study, we assess the effectiveness and safety of fibrin sealant supplemented with tributyrin, termed Medicated Adhesion Barrier (MAB), as an alternative means of reducing the incidence of posterior spinal epidural adhesion formation. Laminectomy defects in sheep were treated with MAB, fibrin sealant alone, ADCONGel, or remained untreated. At 12 weeks postoperatively, the extent of fibrosis and epidural adhesion formation was evaluated using magnetic resonance imaging (MRI), peel-off testing, and histological examination. Initial invitro analysis revealed that tributyrin was retained in fibrin gel in a time-dependent manner and was an effective inhibitor of fibroblast proliferation. Treatment of sheep with MAB significantly reduced both the prevalence (p < 0.05) and tenacity (p < 0.05) of epidural adhesions. The effectiveness of MAB in preventing epidural adhesions was found to be comparable with that of ADCONGel. No adverse events were reported after the use of MAB. The MAB preparation seems to be an effective resorbable barrier for the prevention of epidural adhesions.
    Journal of Biomedical Materials Research Part B Applied Biomaterials 11/2009; 92(2):439-46. · 2.31 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. · 3.61 Impact Factor
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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.
    Molecular &amp Cellular Proteomics 01/2008; · 7.25 Impact Factor
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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. · 3.71 Impact Factor
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    ABSTRACT: The epithelial Ca(2+) channels TRPV5 and TRPV6 constitute the apical Ca(2+) influx pathway in epithelial Ca(2+) transport. PDZ proteins have been demonstrated to play a crucial role in the targeting or anchoring of ion channels and transporters in the apical domain of the cell. In this study, we describe the identification of NHERF4 (Na-P(i) Cap2/IKEPP/PDZK2) as a novel TRPV5- and TRPV6-associated PDZ protein. NHERF4 was identified using two separate yeast two-hybrid screens with the carboxyl termini of TRPV5 and TRPV6 as bait. Binding of the carboxyl termini of TRPV5 and TRPV6 with NHERF4 was confirmed by GST pull-down assays using in-vitro-translated NHERF4 or lysates of Xenopus laevis oocytes expressing NHERF4. Furthermore, the interaction was confirmed by GST pull-down and co-immunoprecipitation assays using in-vitro-translated full-length TRPV5 and Xenopus oocytes or HEK293 cells co-expressing NHERF4 and TRPV5/TRPV6, respectively. The fourth PDZ domain of NHERF4 was sufficient for the interaction, although PDZ domain 1 also contributed to the binding. The binding site for NHERF4 localized in a conserved region in the carboxyl terminus of TRPV5 and was distinct from the binding site of the PDZ protein NHERF2. NHERF4 predominantly localized at the plasma membrane of X. laevis oocytes and HeLa cells. This localization was independent of the presence of TRPV5. Therefore, we hypothesize a role for this novel PDZ protein as a putative plasma membrane scaffold for the epithelial Ca(2+) channels.
    Pflügers Archiv - European Journal of Physiology 08/2006; 452(4):407-17. · 4.87 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. · 2.48 Impact Factor
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    ABSTRACT: Regulation of renal proximal tubular reabsorption of phosphate (Pi) is one of the critical steps in Pi homeostasis. Experimental evidence suggests that this regulation is achieved mainly by controlling the apical expression of the Na+-dependent Pi cotransporter type IIa (NaPi-IIa) in proximal tubules. Only recently have we started to obtain information regarding the molecular mechanisms that control the apical expression of NaPi-IIa. The first critical observation was the finding that truncation of only its last three amino acid residues has a strong effect on apical expression. A second major finding was the observation that the last intracellular loop of NaPi-IIa contains sequence information that confers parathyroid hormone (PTH) sensitivity. The use of the above domains of the cotransporter in yeast two-hybrid (Y2H) screening allowed the identification of proteins interacting with NaPi-IIa. Biochemical and morphological, as well as functional, analyses have allowed us to obtain insights into the physiological roles of such interactions, although our present knowledge is still far from complete.
    The Journal of Physiology 09/2005; 567(Pt 1):21-6. · 4.38 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. · 3.71 Impact Factor
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    J Biber, S M Gisler, N Hernando, H Murer
    Journal of Membrane Biology 03/2005; 203(3):111-8. · 2.48 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. · 3.61 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. · 3.96 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. · 4.65 Impact Factor
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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. · 7.92 Impact Factor
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    ABSTRACT: An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells. Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1. Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the NH2-terminal part of NaPi-IIa. We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys. Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders. As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments. No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets). In agreement, no change in the amount of MAP17 mRNA was observed. Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1. In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the NH2 terminus of NaPi-IIa within the PDZK1/NaPi-IIa/MAP17 complex, and 3) acts as an apical anchoring site for PDZK1.
    American journal of physiology. Renal physiology 11/2003; 285(4):F784-91. · 3.61 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. · 7.92 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. · 4.87 Impact Factor
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    ABSTRACT: In renal proximal brush borders the Na/Pi cotransporter NaPi-IIa is part of a heteromultimeric complex including the PDZ proteins PDZK1 and NHERF1, which interact with the C terminus of NaPi-IIa. In this study, a yeast two-hybrid screen against the N terminus of the Na/Pi cotransporter NaPi-IIa was performed. Thereby we identified visinin-like protein-3 (VILIP-3), a member of neuronal calcium sensors. In this study, expression and protein localization of VILIP-3 in the mouse kidney was performed by immunofluorescence and RT-PCR using laser-assisted microdissected nephron segments. VILIP-3 was found to be abundant in distal and collecting ducts where it partly colocalized with calbindin D28K. In addition VILIP-3 was observed in the brush borders of proximal tubular S1 and S3 segments of both superficial and deep nephrons.
    Nephron Physiology 02/2003; 95(4):p76-82. · 1.57 Impact Factor