Identification of BSPRY as a Novel Auxiliary Protein Inhibiting TRPV5 Activity
Stan F.J. van de Graaf,
Annemiete W.C.M. van der Kemp,
Dennis van den Berg,
Mijke van Oorschot,
Joost G.J. Hoenderop and
René J.M. Bindels
Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Address correspondence to: Dr. René J.M. Bindels, 286 Cell Physiology, Radboud University Nijmegen Medical Centre, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. Phone: +31-24-361-4211; Fax: +31-24-361-6413; E-mail: r.bindels{at}ncmls.ru.nl
Transient receptor potential vallinoid 5 (TRPV5) and TRPV6 arethe most Ca2+-selective members of the TRP superfamily and areessential for active Ca2+ (re)absorption in epithelia. However,little is known about intracellular proteins that regulate theactivity of these channels. This study identified BSPRY (B-boxand SPRY-domain containing protein) as a novel factor involvedin the control of TRPV5. The interaction between BSPRY and TRPV5by GST pull-down and co-immunoprecipitation assays was demonstrated.BSPRY showed co-localization with TRPV5 in mouse kidney. Expressionof BSPRY resulted in a significant reduction of the Ca2+ influxin Madin-Darby Canine Kidney cells that stably express TRPV5without affecting channel cell-surface abundance. Finally, BSPRYexpression in kidney was increased in 25-hydroxyvitamin D3-1-hydroxylaseknockout mice, suggesting an inverse regulation by vitamin D3.Together, these results demonstrate the physiologic role ofthe novel protein BSPRY in the regulation of epithelial Ca2+transport via negative modulation of TRPV5 activity.
The epithelial Ca2+ channels transient receptor potential vallinoid5 (TRPV5) and TRPV6 form two highly Ca2+-permeable members ofthe TRP superfamily of cation channels. Recent studies usingTRPV5 knockout mice have demonstrated the pivotal gatekeeperrole of TRPV5 in active Ca2+ reabsorption (1). These mice showa robust renal Ca2+ loss and display considerable bone abnormalities.In addition, TRPV5 knockout mice exhibit a significant Ca2+hyperabsorption, associated with an increased duodenal TRPV6expression level, resulting from elevated 1,25-dihydroxyvitaminD3 levels (1,2). The preliminary characterization of TRPV6 knockoutmice showed decreased intestinal Ca2+ absorption and bone mineraldensity, further substantiating the role of this channel inintestinal Ca2+ absorption (3). Together, TRPV5 and TRPV6 formthe gatekeepers of transcellular Ca2+ transport, and understandingtheir regulation is pivotal for our insight into the (patho)physiologyof Ca2+ homeostasis (4). However, the molecular mechanisms underlyingchannel trafficking and regulation of its activity at the plasmamembrane are incompletely understood.
The aim of this study, therefore, was to identify proteins thatmodulate the activity of TRPV5 or TRPV6 to regulate transcellularCa2+ transport. Here, we describe the identification of BSPRY(B-box and SPRY-domain containing protein), a novel proteinwith previously unknown function, as a channel-associated regulatoryprotein of the epithelial Ca2+ channel TRPV5.
DNA Constructs and cRNA Synthesis
Full-length BSPRY was obtained from mouse expressed sequencetag (EST) I.M.A.G.E. 5116439, cloned into the pTLN oocyte expressionvector (5) in frame with an amino-terminal VSV tag and subclonedinto pGEX6p2 (Amersham Biosciences, Uppsala, Sweden) and pCB7(6). Full-length and carboxyl-termini of TRPV5 and TRPV6 wereobtained as described previously (7).
Yeast Two-Hybrid System
Y153 yeast strain transformation and subsequently screeningof a mouse kidney cDNA library (Clontech, Palo Alto, CA) wasperformed as described previously (7).
Protein–Protein Interaction Assays
Glutathione S-transferase (GST) fusion proteins were expressedin Escherichia coli BL21 and purified according to the manufacturer’sprotocol (Amersham Biosciences). BSPRY cRNA was synthesizedin vitro using SP6 RNA polymerase as described previously (7)and injected into Xenopus laevis oocytes. After 2 d, oocyteswere lysed in PBS that contained 0.4% (vol/vol) Triton X-100.[35S]Methionine-labeled full-length TRPV5/TRPV6 protein wasprepared using a reticulocyte lysate system. Xenopus oocytelysates or in vitro–translated proteins were incubatedwith GST or GST-fusion proteins that were immobilized on glutathione-Sepharose4B beads (Amersham Biosciences) in PBS that contained 0.4% (vol/vol)Triton X-100 for 2 h at room temperature, and bound proteinswere visualized by autoradiography or immunoblotting. MDCK cellsthat were stably transfected with green fluorescent protein(GFP)-fused TRPV5 and VSV-BSPRY were lysed in sucrose bufferthat fused contained 20 mM Tris (pH 7.4), 5 mM EDTA, 135 mMNaCl, 0.5% (vol/vol) NP-40, 0.2% (vol/vol) Triton X-100, and10% (wt/vol) sucrose, incubated on ice for 60 min, and centrifugedfor 30 min at 16,000 x g. Supernatants were incubated with guineapig anti-TRPV5 or monoclonal anti-VSV antibodies (Sigma, St.Louis, MO) immobilized on protein A-agarose beads (Kem-En-TecA/S, Copenhagen, Denmark) for 16 h at 4°C. Immunoprecipitatedproteins were analyzed by immunoblot analysis.
Reverse Transcription–PCR Analysis
Total RNA was isolated using TRIzol (Life Technologies/BRL,Life Technologies, Breda, The Netherlands). Total RNA (2 µg)was subjected to reverse transcription (RT) using Moloney murineleukemia virus reverse transcriptase, and a PCR for BSPRY wasperformed using the primers 5'-GATTCGGAGAAATTAAGC-3' and 5'-GGCTGCAGTTACAGAGTGC-3'.BSPRY mRNA expression was quantified by real-time PCR usingSYBRGreen dye and shown as relative expression to mouse hypoxanthine-guaninephosphoribosyl transferase (detected with the forward primer5'-TTATCAGACTGAAGAGCTACTGTAATGATC-3', reverse primer 5'-TTACCAGTGTCAATTATATCTTCAACAATC-3'),which was used as an endogenous control to normalize variationsin RNA extractions, the degree of RNA degradation, and variabilitiesin RT efficiencies. The primers that were used to amplify -actinwere 5'-ACCATTTCCCTCTCAGCTGTG-3' and 5'-GTATGCCTCTGGTCGTACCAC-3'.
Preparation of Antibodies and Immunohistochemistry
Rabbit anti-BSPRY antibodies were raised against the peptideH2N-LFPVFAVADQLISIV-COOH of mouse BSPRY and used for immunohistochemistryas described. For semiquantitative determination of proteinlevels, images were analyzed with Image J (http://rsb.info.nih.gov/ij/),resulting in quantification of the protein levels as the meanof integrated optical density as described (2). All negativecontrols, including preimmune serum and serum depleted of anti-BSPRYantibodies by incubation for 16 h with GST-BSPRY immobilizedon glutathione-Sepharose 4B beads, were devoid of staining.
45Ca2+ Uptake Assay
Ca2+ uptake was determined using MDCK cells by incubation inuptake medium that contained 1.0 mM CaCl2 for 5 min at roomtemperature as described previously.
Cell-Surface Biotinylation
Proteins that were present at the cell surface of confluentstably transfected MDCK cells were biotinylated at 4°C usingNHS-LC-LC-biotin (0.5 mg/ml; Pierce, Etten-Leur, The Netherlands),precipitated using neutravidin-coupled beads (Pierce), and analyzedby immunoblot analyses.
Statistical Analyses
In all experiments, the data are expressed as mean ±SEM. Overall statistical significance was determined by ANOVA.P < 0.05 was considered significant.
Our study provides the first evidence of the physiologic roleof BSPRY as a negative modulator of the epithelial Ca2+ channelTRPV5. This conclusion is based on four independent observations.First, the specific interaction between this channel and BSPRYwas demonstrated by yeast two-hybrid, GST pull-down and co-immunoprecipitationassays. Second, BSPRY showed complete co-localization with TRPV5in Ca2+-transporting tubular segments of the kidney. Third,stable expression of BSPRY significantly inhibited Ca2+ influxin confluent layers of MDCK cells expressing TRPV5. Fourth,BSPRY expression was inversely regulated by the calciotropichormone vitamin D3.
Identification of BSPRY as a Novel TRPV5- and TRPV6-Associated Protein
To discover novel TRPV5- and TRPV6-associated proteins, we performeda yeast two-hybrid screen on a mouse kidney cDNA library. Usingthe mouse TRPV6 carboxyl-terminus as bait, five clones thatwere identified as BSPRY were isolated. BSPRY contains a B-boxand SPRY domain, whose tentative functions are protein–proteininteraction modules (8,9). The association of full-length BSPRYwith GST-TRPV5 or TRPV6 carboxyl-termini, immobilized on Sepharose4B beads, confirmed the yeast two-hybrid results and showedthat TRPV5 (Figure 1A) also interacts with BSPRY (Figure 1A).Furthermore, the reverse approach, whereby full-length, GST-fusedBSPRY was immobilized on Sepharose beads and incubated within vitro–translated full-length TRPV5 or TRPV6, resultedin significant binding of both Ca2+ channels to BSPRY (Figure 1B).The binding of BSPRY was equal in the presence of 1 mMCa2+ or in Ca2+-free (2 mM EDTA) conditions, demonstrating aCa2+-independent association (data not shown). No interactionwas observed with GST alone, indicating the specificity of thebinding.
Figure 1. Interaction of transient receptor potential vallinoid 5 (TRPV5) or TRPV6 with BSPRY (B-box and SPRY-domain containing protein) as shown by glutathione S-transferase (GST) pull-down and co-immunoprecipitation analyses. (A) Lysates of Xenopus laevis oocytes that were injected with 10 ng of VSV-tagged BSPRY cRNA were incubated with GST or GST fused to the carboxyl-terminus of TRPV5 or TRPV6 immobilized on glutathione-Sepharose 4B beads. BSPRY interacted specifically with TRPV5 and TRPV6 but not with GST alone. (B) [35S]Methionine-labeled full-length TRPV5 or TRPV6 was incubated with GST or GST-BSPRY immobilized on glutathione-Sepharose 4B beads. Both TRPV5 and TRPV6 interacted with BSPRY, whereas no binding to GST alone was observed. (C) MDCK cells that stably expressed green fluorescent protein (GFP)-fused TRPV5 were transfected with pCB7-VSV-BSPRY of the empty pCB7 vector, and five clones with equal growth rate and TRPV5 expression of both conditions were pooled to eliminate clonal variation. MDCK lysates were incubated with anti-VSV antibodies. Co-immunoprecipitated TRPV5 was demonstrated using anti-GFP antibodies. TRPV5 could not be precipitated in the absence of BSPRY (right), although TRPV5 expression was similar in both conditions (left), demonstrating the specificity of the interaction. (D) Expression of VSV-BSPRY was determined by immunoblot analysis using monoclonal anti-VSV. Expression of -actin was used to show equal loading. Lysates were subjected to immunoprecipitation using anti-TRPV5 antibodies. Co-immunoprecipitated BSPRY was demonstrated by immunoblot analysis using anti-VSV antibodies. -Actin was not co-immunoprecipitated, showing the specificity of the interaction. None-transfected MDCK cells (NT) were used to demonstrate the specificity of the antibodies.
To substantiate further the BSPRY interaction, we generatedMDCK cell lines that were stably transfected with GFP-fusedTRPV5 and subsequently with VSV-BSPRY or the empty pCB7 vector.These cell lines allowed the investigation of TRPV5 in a polarizedepithelium, whereas endogenous expression levels of these channelsin native tissues, including kidney, were below the detectionlimit of immunoblot analysis. Furthermore, these polarized cellsexhibit apical uptake of Ca2+ and provide a valuable model tostudy TRPV5-mediated Ca2+ influx (10). Using these stably transfectedcells, TRPV5 could be co-immunoprecipitated with anti-VSV antibodiesupon expression of VSV-BSPRY, as indicated by the specific bandsat approximately 95 kD for the core-glycosylated GFP-TRPV5 andapproximately 115 kD for complex-glycosylated GFP-TRPV5 (Figure 1C).TRPV5 was not detected in the immunoprecipitated samplein the absence of VSV-BSPRY, confirming the specificity of theprocedure. Similarly, BSPRY was co-immunoprecipitated with TRPV5in the reverse reaction using anti-TRPV5 antibodies that wereimmobilized on Sepharose beads, as represented by an immunopositiveband of approximately 65 kD (Figure 1D). Here, -actin was usedas a control to demonstrate equal input of the samples. -Actinwas absent in the immunoprecipitated sample, indicating thespecificity of the interaction.
Co-Localization of BSPRY and TRPV5
The expression of BSPRY in mouse tissues was analyzed by RT-PCR.BSPRY was detected in several tissues, including kidney, smallintestine, prostate, lung, and uterus. BSPRY was less abundantlyexpressed in heart, whereas skeletal muscle and liver were negative(Figure 2A, top). All samples expressed -actin, which was usedas a positive control to confirm cDNA integrity (Figure 2A,bottom). Using anti-BSPRY antibodies, raised in rabbits usinga conserved 15–amino acid peptide of the carboxyl-terminusof mouse BSPRY, we demonstrated that the localization of BSPRYin kidney is strikingly similar to the localization of TRPV5(Figure 2B, bottom). BSPRY was present in the apical domainof all TRPV5-immunopositive tubules, previously identified asthe second part of the distal convoluted tubule and connectingtubule (11,12). Proximal tubules and glomeruli were negative,and preimmune serum did not show any staining. Furthermore,immunopositive BSPRY staining was absent upon incubation ofthe serum with immobilized GST-BSPRY, whereas incubation withGST alone had no effect (Figure 2B, top). Similar co-localizationwith TRPV5 in distal convoluted tubule and connecting tubulewas observed previously for calbindin-D28K and the Na+/Ca2+exchanger (12,13). These proteins play an essential role inrenal transcellular Ca2+ transport as they respectively facilitatethe diffusion of Ca2+ from the apical to the basolateral sideand the extrusion into the blood. Similarly, the robust co-localizationbetween BSPRY and TRPV5 strongly supports a physiologic andspecific function of BSPRY in the regulation of epithelial Ca2+transport by direct association with TRPV5 in the kidney.
Figure 2. Localization and tissue distribution of BSPRY. (A) RNA was extracted from several mouse tissues, and BSPRY expression was determined by reverse transcription–PCR. The BSPRY-specific band was amplified in brain, lung, duodenum, ileum, kidney, prostate, and uterus (top). A faint signal was detected in heart, whereas skeletal muscle and liver were negative (top). -Actin was used as a control to ensure cDNA integrity and was positive in all tested tissues (bottom). (B) Immunohistochemical analysis of BSPRY and TRPV5 in mouse kidney sections. To verify the specificity of the BSPRY antibody, we incubated polyclonal rabbit BSPRY antiserum for 16 h with GST-BSPRY, immobilized on glutathione-Sepharose 4B beads. Depletion of the BSPRY antibodies abolished all immunopositive signal, whereas incubation with immobilized GST alone (not depleted) did not affect the reactivity of the antiserum. Furthermore, kidney sections were co-stained with antibodies against BSPRY and TRPV5. BSPRY and TRPV5 have a remarkable similarity in localization and show significant overlap in the apical region of the cell. No signal was detected with preimmune serum or with fluorophore-conjugated antibodies only.
BSPRY-Mediated TRPV5 Inhibition of Ca2+ Influx
The effect of BSPRY co-expression on TRPV5-mediated Ca2+ influxwas measured using confluent monolayers of MDCK cells. TRPV5expression in these cells resulted in an approximately 2.5-foldincreased Ca2+ influx compared with nontransfected cells. Uponco-expression with BSPRY, Ca2+ influx was inhibited by 41 ±13% (Figure 3C), approaching levels of nontransfected cells.Total cellular TRPV5 expression levels were identical in thepresence or absence of BSPRY, as was demonstrated by immunoblotanalysis using anti-GFP antibodies (Figure 3, A and B). Furthermore,the correct size of the bands at approximately 95 to 115 kDin Figure 3A confirmed the integrity of the GFP-TRPV5 fusionprotein. Finally, flow cytometry analysis of the GFP signalshowed that >98% of the MDCK cells forming the confluentmonolayer contributed to the TRPV5-dependent Ca2+ influx, bothin the presence and in the absence of BSPRY (data not shown).These data provide the first evidence of a functional role ofBSPRY. So far, only two studies have provided information aboutBSPRY. First, BSPRY was identified recently in a yeast two-hybridscreen using zyxin as bait. In epithelial cells, zyxin is involvedin the formation of cell–cell contacts, which requireactin cytoskeleton rearrangements (14). Schenker et al. (15)provided no data about the localization and function of BSPRY.However, the association with zyxin might hint at a role ofthe cytoskeleton in the BSPRY-mediated regulation of TRPV5.Second, it was shown that BSPRY interacts with 14-3-3 proteins.Although the exact role of 14-3-3 proteins remains to be elucidatedfully, their role in the recognition of phosphorylated proteinsis well established (16). It has been demonstrated that 14-3-3proteins bind to specific motifs that contain a phosphorylatedserine residue and have been implicated in the binding to andactivation of signaling proteins (16,17). Furthermore, a roleof 14-3-3 proteins in K+ channel trafficking was postulated(18). However, cell-surface biotinylation did not provide evidencefor TRPV5 trafficking as an explanation for the observed inhibitoryfunction of BSPRY (Figure 3D). Furthermore, we demonstratedthat a significant fraction of BSPRY is membrane associatedand present at the plasma membrane (Figure 3, E and F, SupplementalFigure). Therefore, it is tempting to speculate that BSPRY isinvolved in inhibitory signaling cascades that control the activityof the epithelial Ca2+ channels at the cell surface.
Figure 3. BSPRY operates as a negative modulator of TRPV5 activity. (A) The expression level of GFP-tagged TRPV5 in stably transfected MDCK cells was determined by immunoblot using anti-GFP antibodies and shows equal expression levels in the presence or absence of BSPRY. (B) BSPRY expression was verified using monoclonal anti-VSV antibodies. (C) TRPV5 activity was determined by radioactive Ca2+ uptake in MDCK cells that were grown to confluence, demonstrating a significant inhibition of TRPV5-mediated Ca2+ influx upon BSPRY co-expression. (D) Intact GFP-TRPV5–expressing MDCK cells, stably transfected with pCB7-VSV-BSPRY or the empty pCB7 vector, were incubated with NHS-LC-LC-biotin to label cell-surface proteins. Biotinylated proteins were isolated using neutravidin-agarose beads, separated by SDS-PAGE, and TRPV5 was subsequently quantified by immunoblot analysis. TRPV5 plasma membrane abundance was equal in the presence or absence of BSPRY. TRPV5 and BSPRY were not detected in precipitates from cells that were not exposed to biotin, showing the specificity of the precipitation for biotinylated proteins. Similarly, BSPRY was detected in the plasma membrane by biotinylation (E) and immunocytochemistry (F, arrowheads) using anti-VSV antibodies on stably BSPRY-transfected MDCK cells. -Actin was found in the lysate from the biotinylated MDCK monolayers but not in the fraction recovered from the neutravidin beads, demonstrating that intracellular proteins are not isolated using this procedure (data not shown). *P < 0.05 versus all. (G) The role of vitamin D3 on BSPRY expression was determined by quantitative real-time PCR in kidneys of wild-type (WT; n = 11) or 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) knockout (KO; n = 6) mice. (H) Effect of vitamin D3 on BSPRY protein abundance in mouse kidney. Representative images showing kidney sections stained for BSPRY in WT or 1-OHase KO mice. Protein abundance was determined by computerized analysis of immunohistochemical images and is presented as mean optical density (arbitrary units) for n = 24 to 30 pictures from five mice for each condition. *P < 0.05 versus WT.
Vitamin D–Dependent Regulation of BSPRY Expression in Kidney
It was demonstrated previously that TRPV5 expression is stronglyregulated by 1,25-dihydroxyvitamin D3, the biologic active formof vitamin D (4,19). Therefore, we assessed the expression ofBSPRY in wild-type and 25-hydroxyvitamin D3-1-hydroxylase (1-OHase)knockout mice. These knockout mice are unable to synthesize1,25-dihydroxyvitamin D3 and are a valuable animal model tostudy vitamin D deficiency rickets type I (20). Quantitativereal-time PCR showed significantly enhanced BSPRY mRNA expressionin the 1-OHase knockout mice compared with wild-type mice, suggestingthat vitamin D negatively regulates the BSPRY expression (Figure 3G).Because the antibodies that we generated did not recognizeBSPRY on immunoblot, the protein expression of BSPRY in theseanimals was semiquantified by immunohistochemistry. Depictedin Figure 3H are representative images of BSPRY expression inwild-type and 1-OHase knockout mice. Computerized analysis ofthe immunohistochemical staining showed significantly enhancedBSPRY protein expression in the 1-OHase knockout mice comparedwith wild-type mice, confirming the inverse regulation of BSPRYexpression by circulating vitamin D on the protein level (Figure 3H).Together with the Ca2+ uptake results, this suggests thatBSPRY operates as a negative modulator for TRPV5 and that thismechanism will be downregulated when vitamin D levels increaseto stimulate active Ca2+ transport. These findings demonstratea novel target for vitamin D3 in the regulation of active Ca2+transport and provide new insight into the factors involvedin the Ca2+-related (patho)physiology.
We have identified BSPRY as a novel auxiliary protein of theepithelial Ca2+ channels. The association, co-localization,and functional analyses described in this study demonstratethe first physiologic role of the novel protein BSPRY in thedirect regulation of TRPV5.
Acknowledgments
This work was supported by the Dutch Organization of ScientificResearch (Zon-MW 016.006.001) and the Human Frontier ScienceProgram RGP 32/2004.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Hoenderop JG, van Leeuwen JP, van der Eerden BC, Kersten FF, van der Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E, Bindels RJ: Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5.
J Clin Invest 112
: 1906
–1914, 2003[CrossRef][Medline]
Renkema KY, Nijenhuis T, van der Eerden BC, van der Kemp AW, Weinans H, van Leeuwen JP, Bindels RJ, Hoenderop JG: Hypervitaminosis D mediates compensatory Ca2+ hyperabsorption in TRPV5 knockout mice.
J Am Soc Nephrol 16
, 3188
–3195, 2005[Abstract/Free Full Text]
Bianco S, Peng JB, Takanaga H, Kos CH, Crescenzi A, Brown EM, Hediger MA: Mice lacking the epithelial calcium channel CaT1 (TRPV6) show a deficiency in intestinal calcium absorption despite high plasma levels of 1,25-dihydroxyvitamin D.
FASEB J 18
: A706
, 2004
Hoenderop JG, Nilius B, Bindels RJ: Calcium absorption across epithelia.
Physiol Rev 85
: 373
–422, 2005[Abstract/Free Full Text]
Steinmeyer K, Schwappach B, Bens M, Vandewalle A, Jentsch TJ: Cloning and functional expression of rat CLC-5, a chloride channel related to kidney disease.
J Biol Chem 270
: 31172
–31177, 1995[Abstract/Free Full Text]
Brewer CB, Roth MG: A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin.
J Cell Biol 114
: 413
–421, 1991[Abstract/Free Full Text]
Van de Graaf SF, Hoenderop JG, Gkika D, Lamers D, Prenen J, Rescher U, Gerke V, Staub O, Nilius B, Bindels RJ: Functional expression of the epithelial Ca2+ channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex.
EMBO J 22
: 1478
–1487, 2003[CrossRef][Medline]
Borden KL: RING fingers and B-boxes: Zinc-binding protein-protein interaction domains.
Biochem Cell Biol 76
: 351
–358, 1998[CrossRef][Medline]
Den Dekker E, Schoeber J, Topala C, van de Graaf SF, Hoenderop JG, Bindels RJ: Characterization of a Madin-Darby Canine Kidney cell line stably expressing TRPV5.
Pflugers Arch 450
: 236
–244, 2005[CrossRef][Medline]
Loffing J, Loffing-Cueni D, Valderrabano V, Klausli L, Hebert SC, Rossier BC, Hoenderop JG, Bindels RJ, Kaissling B: Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron.
Am J Physiol Renal Physiol 281
: F1021
–F1027, 2001[Abstract/Free Full Text]
Hoenderop JG, Hartog A, Stuiver M, Doucet A, Willems PH, Bindels RJ: Localization of the epithelial Ca2+ channel in rabbit kidney and intestine.
J Am Soc Nephrol 11
: 1171
–1178, 2000[Abstract/Free Full Text]
Hoenderop JG, Dardenne O, Van Abel M, Van Der Kemp AW, Van Os CH, St-Arnaud R, Bindels RJ: Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3–1alpha-hydroxylase knockout mice.
FASEB J 16
: 1398
–1406, 2002[Abstract/Free Full Text]
Vasioukhin V, Bauer C, Yin M, Fuchs E: Directed actin polymerization is the driving force for epithelial cell-cell adhesion.
Cell 100
: 209
–219, 2000[CrossRef][Medline]
Birkenfeld J, Kartmann B, Anliker B, Ono K, Schlotcke B, Betz H, Roth D: Characterization of zetin 1/rBSPRY, a novel binding partner of 14-3-3 proteins.
Biochem Biophys Res Commun 302
: 526
–533, 2003[CrossRef][Medline]
Muslin AJ, Tanner JW, Allen PM, Shaw AS: Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.
Cell 84
: 889
–897, 1996[CrossRef][Medline]
O’Kelly I, Butler MH, Zilberberg N, Goldstein SA: Forward transport. 14-3-3 binding overcomes retention in endoplasmic reticulum by dibasic signals.
Cell 111
: 577
–588, 2002[CrossRef][Medline]
Hoenderop JG, Muller D, Van Der Kemp AW, Hartog A, Suzuki M, Ishibashi K, Imai M, Sweep F, Willems PH, Van Os CH, Bindels RJ: Calcitriol controls the epithelial calcium channel in kidney.
J Am Soc Nephrol 12
: 1342
–1349, 2001[Abstract/Free Full Text]
Dardenne O, Prud’homme J, Arabian A, Glorieux FH, St-Arnaud R: Targeted inactivation of the 25-hydroxyvitamin D3–1alpha-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.
Endocrinology 142
: 3135
–3141, 2001[Abstract/Free Full Text]
This article has been cited by other articles:
J. G. J. Hoenderop and R. J. M. Bindels Calciotropic and Magnesiotropic TRP Channels
Physiology,
February 1, 2008;
23(1):
32 - 40.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
-hydroxylase knockout mice, suggesting an inverse regulation by vitamin D3. Together, these results demonstrate the physiologic role of the novel protein BSPRY in the regulation of epithelial Ca2+ transport via negative modulation of TRPV5 activity. 
-actin were 5'-ACCATTTCCCTCTCAGCTGTG-3' and 5'-GTATGCCTCTGGTCGTACCAC-3'. 
