IRBIT, Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3, Binds Na+/H+ Exchanger NHE3 and Activates NHE3 Activity in Response to Calcium
2008; Elsevier BV; Volume: 283; Issue: 48 Linguagem: Inglês
10.1074/jbc.m805534200
ISSN1083-351X
AutoresPeijian He, Huanchun Zhang, C. Chris Yun,
Tópico(s)Ion Channels and Receptors
ResumoCalcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition. Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition. The calcium ion (Ca2+) is a highly versatile second messenger that can regulate many different cellular functions (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 517-529Crossref PubMed Scopus (4065) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4319) Google Scholar). Ca2+ regulates various cellular processes by activating or inhibiting cellular signaling pathways and Ca2+-regulated proteins (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 517-529Crossref PubMed Scopus (4065) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4319) Google Scholar). A major intracellular store of Ca2+ is the endoplasmic reticulum. Extracellullar stimuli, including hormones, growth factors, and neurotransmitter, induce production of the second messenger, inositol 1,4,5-triphosphate (IP3), 2The abbreviations used are: IP3, inositol 1,4,5-triphosphate; IRBIT, IP3 receptor-binding protein released with IP3; CaM, calmodulin; CaMKII, calmodulin-dependent kinase II; NHE, Na+/H+ exchanger; NHERF2, Na+/H+ exchanger regulatory protein 2; NBC1, Na+-HCO3− cotransporter 1; VSVG, vesicular stomatitis virus glycoprotein; HA, hemagglutinin; AdoHcy, S-adenosylhomocysteine hydrolase; AdoHcyL1, S-adenosylhomocysteine hydrolase-like 1; PKC, protein kinase C; shRNA, small hairpin RNA; aa, amino acid(s); PBS, phosphate-buffered saline; TMA, tetramethylammonium; BCECF-AM, 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; SD, synthetic dropout. 2The abbreviations used are: IP3, inositol 1,4,5-triphosphate; IRBIT, IP3 receptor-binding protein released with IP3; CaM, calmodulin; CaMKII, calmodulin-dependent kinase II; NHE, Na+/H+ exchanger; NHERF2, Na+/H+ exchanger regulatory protein 2; NBC1, Na+-HCO3− cotransporter 1; VSVG, vesicular stomatitis virus glycoprotein; HA, hemagglutinin; AdoHcy, S-adenosylhomocysteine hydrolase; AdoHcyL1, S-adenosylhomocysteine hydrolase-like 1; PKC, protein kinase C; shRNA, small hairpin RNA; aa, amino acid(s); PBS, phosphate-buffered saline; TMA, tetramethylammonium; BCECF-AM, 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; SD, synthetic dropout. through phosphoinositide turnover, which engages with IP3 receptor (IP3R) to release Ca2+ from endoplasmic reticulum (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 517-529Crossref PubMed Scopus (4065) Google Scholar, 3Mikoshiba K. J. Neurochem. 2007; 102: 1426-1446Crossref PubMed Scopus (305) Google Scholar). Ca2+ is often viewed as a second messenger acting as a trigger for Ca2+-dependent events. However, for Ca2+ to regulate multiple cellular processes in different cells with different outcomes, Ca2+ homeostasis must be tightly regulated temporally and spatially (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 517-529Crossref PubMed Scopus (4065) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4319) Google Scholar). Recently, an IP3 receptor binding protein, IP3 receptor-binding protein released with IP3 (IRBIT), has been identified (4Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). IRBIT inhibits activation of IP3R and Ca2+ release by competing with IP3 (5Ando H. Mizutani A. Kiefer H. Tsuzurugi D. Michikawa T. Mikoshiba K. Mol. Cell. 2006; 22: 795-806Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Interestingly, IRBIT binds to the pancreas-type electrogenic Na+/HCO3− cotransporter 1 (pNBC1) and co-expression of IRBIT and pNBC1 in Xenopus oocytes activated Na+/HCO3− cotransporter activity (6Shirakabe K. Priori G. Yamada H. Ando H. Horita S. Fujita T. Fujimoto I. Mizutani A. Seki G. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9542-9547Crossref PubMed Scopus (130) Google Scholar). Na+/H+ exchange is a process present in all organisms from prokaryotes to human (7Brett C.L. Donowitz M. Rao R. Am. J. Physiol. 2005; 288: C223-C239Crossref PubMed Scopus (423) Google Scholar). Na+/H+ exchange in mammals is mediated by the Na+/H+ exchange gene family (NHE) consisting of 9 members (7Brett C.L. Donowitz M. Rao R. Am. J. Physiol. 2005; 288: C223-C239Crossref PubMed Scopus (423) Google Scholar, 8Zachos N.C. Tse M. Donowitz M. Annu. Rev. Physiol. 2005; 67: 411-443Crossref PubMed Scopus (297) Google Scholar). The NHE3 isoform is expressed in the apical membrane of epithelial cells located in the renal proximal tubule and intestine (9Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar, 10Hoogerwerf W.A. Tsao S.C. Devuyst O. Levine S. Yun C.H.C. Yip J.W. Cohen M. Wilson P.D. Lazenby A.J. Tse M. Donowitz M. Am. J. Physiol. 1996; 270: G29-G41PubMed Google Scholar). In the kidney, NHE3 is responsible for the majority of Na+ and HCO3− reabsorption in renal proximal tubule as evidenced by the studies using NHE3-null mice (11Schultheis P.J. Clarke L.L. Meneton P. Miller M.L. Soleimani M. Gawenis L.R. Riddle T.M. Duffy J.J. Doetschman T. Wang T. Giebisch G. Aronson P.S. Lorenz J.N. Shull G.E. Nat. Genet. 1998; 19: 282-285Crossref PubMed Scopus (683) Google Scholar, 12Wang T. Yang C.L. Abbiati T. Schultheis P.J. Shull G.E. Giebisch G. Aronson P.S. Am. J. Physiol. 1999; 277: F298-F302Crossref PubMed Google Scholar). In the intestine, NHE3 mediates electroneutral NaCl absorption by coupling to a Cl-/HCO3− exchanger (8Zachos N.C. Tse M. Donowitz M. Annu. Rev. Physiol. 2005; 67: 411-443Crossref PubMed Scopus (297) Google Scholar). Through osmotic coupling to passive water absorption, NHE3 is a major constituent in basal Na+ absorption in intestine and a frequent target of inhibition in many diarrheal diseases (8Zachos N.C. Tse M. Donowitz M. Annu. Rev. Physiol. 2005; 67: 411-443Crossref PubMed Scopus (297) Google Scholar). Among diverse stimuli and second messengers that regulate NHE3, Ca2+ remains an enigmatic second messenger because the effect on NHE3 by Ca2+ varies depending on cell types (13Choi J.W. Lee-Kwon W. Jeon E.S. Kang Y.J. Kawano K. Kim H.S. Suh P.-G. Donowitz M. Kim J.H. Biochim. Biophys. Acta. 2004; 1683: 59-68Crossref PubMed Scopus (18) Google Scholar, 14Chu T.S. Peng Y. Cano A. Yanagisawa M. Alpern R.J. J. Clin. Investig. 1996; 97: 1454-1462Crossref PubMed Scopus (70) Google Scholar, 15Kim J.H. Lee-Kwon W. Park J.B. Ryu S.H. Yun C.H. Donowitz M. J. Biol. Chem. 2002; 277: 23714-23724Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). NHE3 and other mammalian Na+/H+ exchangers consist of two structurally and functionally distinct domains, the NH2 terminus and COOH terminus (16Levine S.A. Nath S. Yun C.H.C. Yip J.W. Montrose M.H. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 17Yun C.H.C. Tse C.-M. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10723-10727Crossref PubMed Scopus (30) Google Scholar). The COOH-terminal domain has been shown to be necessary for all identified acute regulation. Part of this necessity is the ability of the COOH-terminal domain to interact with an increasing number of cellular proteins (8Zachos N.C. Tse M. Donowitz M. Annu. Rev. Physiol. 2005; 67: 411-443Crossref PubMed Scopus (297) Google Scholar, 16Levine S.A. Nath S. Yun C.H.C. Yip J.W. Montrose M.H. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 17Yun C.H.C. Tse C.-M. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10723-10727Crossref PubMed Scopus (30) Google Scholar). In this study, we report the identification of IRBIT as a NHE3 interacting protein. We provide evidence that IRBIT activates NHE3 activity in response to Ca2+. Plasmid Constructs—The bait plasmid, pGBKT7-C832, was constructed by cloning the coding region of the cytoplasmic COOH-terminal domain of rabbit NHE3 (aa 475-832) into the yeast DNA-binding vector pGBKT7 vector (Clontech). NHE3 COOH-terminal domains, aa 475-591 and 475-696, were cloned in pGBKT7 to generate pGBKT7-C591 and pGBKT7-C696, respectively. pACT-IRBIT is the GAL4 activation domain plasmid containing the entire coding sequence of IRBIT. The cDNAs corresponding to aa 1-105, 1-224, and 105-530 of IRBIT were cloned into the GAL4-activation domain plasmid, pACT2, to generate pACT-105R, pACT-SB, and pACT-105T, respectively. Yeast Two-hybrid—The bait plasmid, pGBKT7-C832, was transformed into the yeast strain AH109. The resulting AH109 (pGBKT7-C832) yeast strain was mated with >3 × 106 clones of the yeast strain Y187 pretransformed with a human kidney Matchmaker cDNA library (Clontech). The resulting yeast strain was plated on synthetic dropout plates lacking adenosine, histidine, leucine, and tryptophan (SD/-Ade/-His/-Leu/-Trp). Yeast colonies formed after 5 days were replated on SD/-Ade/-His/-Leu/-Trp plates containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Positive library plasmids were rescued from the yeast colonies and transformed together with pGBKT7-C832 into AH109 and double transformants were selected by growth on SD/-Leu/-Trp plates. Resulting yeast colonies were plated on SD/-Ade/-His/Leu/-Trp plates to reconfirm the interaction. Positive clones were sequenced to identify their cDNA inserts. Cell Cultures—PS120 cells stably expressing rabbit NHE3 fused at the COOH terminus with an antibody epitope derived from vesicular stomatitis virus glycoprotein (VSVG), PS120/NHE3V, was previously described (18Yun C.C. Lamprecht G. Forster D.V. Sidor A. J. Biol. Chem. 1998; 273: 25856-25863Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 19Yun C.C. Chen Y. Lang F. J. Biol. Chem. 2002; 277: 7676-7683Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). PS120/NHE3V truncation mutants with portions of the COOH-terminal cytoplasmic domain deleted, PS120/NHE3V-585 and PS120/NHE3V-689, were previously described and were kindly provided by Dr. Ming Tse, Johns Hopkins University (16Levine S.A. Nath S. Yun C.H.C. Yip J.W. Montrose M.H. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). All PS120 fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 1 mm sodium pyruvate, 50 units/ml penicillin, 50 μg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2 humidified incubator at 37 °C. Caco2BBE cells transfected with NHE3V, Caco2BBE/NHE3V, were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 25 mm NaHCO3, 50 μg/ml streptomycin, 50 units/ml penicillin, and 1% nonessential amino acids. Transfection and Gene Silencing—cDNA encoding IRBIT was tagged with a hemagglutinin (HA) epitope at the NH2 terminus by PCR and was subcloned into pcDNA3.1Hygro (Invitrogen). PS120 and Caco2BBE cells and their variants were transfected with pcDNA-HA-IRBIT by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendation and selected with 600 μg/ml hygromycin. pLKO.1 harboring small hairpin RNA (shRNA) constructs for IRBIT were obtained from Sigma. PS120/NHE3 cells were transfected with shRNA plasmids and selected with 4 μg/ml puromycin. Immunoprecipitation—PS120 cells were washed twice in cold phosphate-buffered saline (PBS), scraped, and lysed in lysis buffer (Cell Signaling, Danvers, MA), containing 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm β-glycerophosphate, 2.5 mm sodium pyrophosphate, 1 mm Na2EDTA, 1 mm EGTA, 1 mm Na3VO4, 1 μg/ml leupeptin, 1% Triton X-100, and protease inhibitor mixture tablets (Roche). The crude lysate was sonicated for 2 × 15 s and spun at 14,000 × g for 15 min. Protein concentration was determined by the bicinchoninic acid assay (Sigma). Lysate (500 μg) was pre-cleared by incubation with 30 μl of protein A-Sepharose beads for 1 h and the supernatant was then incubated overnight with either monoclonal anti-VSVG antibody or polyclonal anti-IRBIT antibody. Immunocomplex was purified by incubating with 50 μl of protein A-Sepharose beads for 1 h, followed by 3 washes in lysis buffer and 2 washes in PBS. All the above steps were performed at 4 °C or on ice. The bound immunocomplex was eluted by incubating the protein A beads in Laemmli sample buffer for 10 min at 95 °C and then separated by SDS-PAGE. The proteins were then transferred to nitrocellulose membrane for Western immunoblot. Surface Biotinylation—Surface biotinylation of NHE3 was performed as previously described (20Wang D. Sun H. Lang F. Yun C.C. Am. J. Physiol. 2005; 289: C802-C810Crossref PubMed Scopus (64) Google Scholar). Briefly, cells grown in 10-cm Petri dishes were treated with 100 nm thapsigargin, 500 nm ionomycin, or ethanol for 10 min, followed by rinsing twice in PBS and 10 min incubation in borate buffer composed of 154 mm NaCl, 7.2 mm KCl, 1.8 mm CaCl2, and 10 mm H3BO3 (pH 9.0). Cells were then incubated for 40 min with 0.5 mg/ml NHS-SS-biotin (Pierce) in borate buffer. Unbound NHS-SS-biotin was quenched with Tris buffer (20 mm Tris, 120 mm NaCl, pH 7.4). Cells were then rinsed with PBS, scraped, lysed in the lysis buffer described above, and sonicated for 2 × 15 s. The lysate was agitated for 30 min and spun at 14,000 × g for 15 min to remove the insoluble cell debris. An aliquot was retained as the total fraction representing the total cellular NHE3 and IRBIT. Protein concentration was determined and 1 mg of lysate was then incubated with streptavidin-agarose beads (Pierce) for 2 h. The strepavidin-agarose beads were washed 3 times in lysis buffer and twice in PBS. All the above procedures were performed at 4 °C or on ice. Biotinylated surface proteins were then eluted by boiling the beads at 95 °C for 10 min. Dilutions of the total and surface NHE3 were resolved by SDS-PAGE, and immunoblotted with anti-VSVG antibody or anti-HA monoclonal antibody. Densitometric analysis was performed using Scion Image software (National Institutes of Health). Na+-dependent Intracellular pH Recovery—The Na+-dependent changes in intracellular pH (pHi) by NHE3 was determined with the use of the ratio fluorometric, pH-sensitive dye 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester (BCECF-AM) as previously described (20Wang D. Sun H. Lang F. Yun C.C. Am. J. Physiol. 2005; 289: C802-C810Crossref PubMed Scopus (64) Google Scholar, 21Lamprecht G. Weinman E.J. Yun C.C. J. Biol. Chem. 1998; 273: 29972-29978Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Briefly, cells were seeded on coverslips, grown to 70-80% confluence for PS120 or 5 days post-confluence for Caco2 cells, and then serum starved overnight. Cells were washed in Na+ buffer (30 mm NaCl, 20 mm HEPES, 5 mm KCl, 1 mm tetramethylammonium-PO4 (TMA-PO4), 2 mm CaCl2, 1 mm MgSO4, and 18 mm glucose) and then were dye-loaded by incubating for 20 min with 6.5 μm BCECF-AM in the same solution. The coverslips were mounted on a perfusion chamber mounted on an inverted microscope, and were superfused with NH4+ buffer (40 mm NH4Cl, 90 mm NaCl, 20 mm HEPES, 5 mm KCl, 1 mm TMA-PO4, 2 mm CaCl2, 1 mm MgSO4, and 18 mm glucose) and subsequently with TMA+ buffer (130 mm TMA-Cl, 20 mm HEPES, 5 mm KCl, 1 mm TMA-PO4, 2 mm CaCl2, 1 mm MgSO4, and 18 mm glucose), with each containing 100 nm thapsigargin, 500 nm ionomycin, or ethanol as a control. Na+ buffer supplemented with (for Caco2BBE) or without (for PS120) 50 μm HOE-694 was then reintroduced to drive Na+-dependent pH recovery. Calibration of the fluorescence signal was performed using the K+/H+ ionophore nigericin as described previously (21Lamprecht G. Weinman E.J. Yun C.C. J. Biol. Chem. 1998; 273: 29972-29978Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The microfluorometry was performed on a Nikon TE200 inverted microscope with a Nikon CFI Super Fluor ×40 objective, coupled to a Lambda 10-2 filter wheel controller equipped with a multiwavelength filter set designed for BCECF. Photometric data were acquired using the Metafluor software (Molecular Devices). Na+/H+ exchange rate was described by the rate of pHi recovery, which was calculated by determining slopes along the pHi recovery by linear least-squares analysis over a minimum of 9 s (20Wang D. Sun H. Lang F. Yun C.C. Am. J. Physiol. 2005; 289: C802-C810Crossref PubMed Scopus (64) Google Scholar). Production of Anti-IRBIT Antiserum—The cDNA encoding the NH2-terminal region (aa 1-104) of IRBIT was subcloned into the bacterial hexahistidine (His6) fusion vector pET-16b (Novagen, Gibbstown, NJ) to generate the IRBIT-(1-104)-His6 recombinant protein. The expressed IRBIT-(1-104)-His6 protein was then purified using nickel-nitrilotriacetic acid-agarose resin (Qiagen, Valencia, CA) and used for immunization of New Zealand White rabbits by Convance Research Products (Denver, PA) to produce anti-IRBIT serum. Confocal Immunofluorescence—PS120/NHE3V/HA-IRBIT and PS120/NHE3V/pcDNA cells grown on coverslips were washed twice with cold PBS, fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized in 0.2% Triton X-100 in PBS for 5 min, and blocked in PBS containing 5% normal goat serum for 30 min at room temperature. Cells were then stained with polyclonal anti-HA (Sigma) or monoclonal anti-VSVG antibodies for 1 h at room temperature. Following three washes, 10 min each, with PBS, the cells were incubated with Alexa 488-conjugated donkey anti-mouse IgG or Alexa 555-conjugated goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature. After three 10-min washes with PBS, the coverslips were mounted with ProLong Gold Antifade Reagent (Invitrogen) and observed under a Zeiss LSM510 laser confocal microscope (Zeiss Microimaging, Thornwood, NY) coupled to a Zeiss Axioplan2e with ×63 Pan-Apochromat oil lenses. Statistical Analysis—Results were presented as mean ± S.E. Statistical significance was assessed by analysis of variance, with p < 0.05 considered significant. IRBIT Interacts with NHE3—To identify proteins interacting with NHE3, we screened a human kidney cDNA library under high stringency with the GAL4 DNA-binding domain fused to the cytoplasmic domain of NHE3 as bait. Screening of >3 × 106 independent clones yielded 4 positive clones containing cDNA encoding calcineurin homologous protein, which was previously shown to interact with NHE3 (22Di Sole F. Cerull R. Babich V. Quinones H. Gisler S.M. Biber J. Murer H. Burckhardt G. Helmle-Kolb C. Moe O.W. J. Biol. Chem. 2004; 279: 2962-2974Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Six other clones contained cDNA inserts encoding S-adenosylhomocysteine hydrolase-like 1 (AdoHcyL1). The relative strength of interaction of calcineurin homologous protein and AdoHcyL1 with NHE3 was robust and comparable based on the size of yeast colonies and the β-galactosidase activity (data not shown). AdoHcyL1 was recently shown to interact with IP3R and was termed IRBIT (4Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). AdoHcyL1 will be referred to as IRBIT hereafter. To confirm the interaction between NHE3 and IRBIT in a two-hybrid system, we tested co-immunoprecipitation of NHE3 and IRBIT in PS120/NHE3V fibroblasts transfected with HA-IRBIT. As shown in Fig. 1A, IRBIT co-immunoprecipitated with NHE3V in PS120/NHE3V/HA-IRBIT cells confirming the results from the two-hybrid screen. This interaction was specific as the presence of IRBIT was not detected in the absence of an anti-VSVG antibody or in sham-transfected cells. Fig. 1B shows an opposite experiment where IRBIT was pulled down with an anti-IRBIT antibody and co-immunoprecipitation of NHE3V was detected by Western blot. Having confirmed the interaction between NHE3 and IRBIT, we next determined domains of NHE3 and IRBIT mediating their interaction using yeast two-hybrid analysis. Results shown in Fig. 2A indicate that the truncation of a region distal to aa 696 of NHE3 (C696: aa 475-696) did not affect its interaction with IRBIT as compared with C832 (aa 475-832). A larger deletion of aa 592-832 of NHE3 (C591: aa 475-591) completely abrogated its ability to bind IRBIT, indicating that the region necessary for the interaction with IRBIT lies between aa 591 and 696. Fig. 2B shows that the binding of IRBIT to C832 was significantly attenuated by deletions of parts of IRBIT. The NH2-terminal 105 amino acids of IRBIT (105R) showed no interaction with C832. The 105T construct (aa 105-530) resulted in several isolated yeast colonies, suggesting a marginal capacity, if any, to bind NHE3. The SB construct of IRBIT (aa 1-224) weakly interacted with NHE3 compared with full-length IRBIT based on the number of yeast colonies. These results were reproducible in three independent matings. The domains involved in NHE3-IRBIT association were further explored by co-immunoprecipitation in PS120 cells. HA-IRBIT was expressed in PS120/NHE3V, PS120/NHE3V-689, and PS120/NHEV-585 cells and NHE3 and its variants were immunoprecipitated with anti-VSVG, followed by Western blot using an anti-HA antibody to detect co-immunoprecipitated HA-IRBIT. The upper and middle panels of Fig. 2C show the expression of NHE3 variants and IRBIT, respectively, in each cell line. The bottom panel shows Western blot of the immunocomplex pulled down by an anti-VSVG antibody. The results shown in Fig. 2C confirmed that IRBIT bound to NHE3V-689 as well as full-length NHE3V. Although the expression level of NHE3V-585 was significantly lower, no apparent interaction was observed between IRBIT and NHE3V-585. We confirmed the absence of interaction between NHE3V-585 and IRBIT by using as much as 4 mg of lysate with no sign of co-immunoprecipitated IRBIT (data not shown). Fig. 2D shows co-immunoprecipitation of IRBIT and its variants with NHE3V. The expression level of 105T protein in PS120 cells was extremely low relative to IRBIT and 105R, suggesting that the 105T protein may not be stable in these cells. Neither 105T nor 105R co-immunoprecipitated with NHE3V. On the other hand, a small amount of SB co-immunoprecipitated with NHE3V, but the relative intensity of the SB band compared with IRBIT indicated that its interaction was significantly compromised, which was consistent with the results from two-hybrid analysis. IRBIT-mediated Activation of NHE3 Dependent on Ca2+ Elevation—Recent studies have shown that IRBIT regulates intracellular Ca2+ via its ability to bind IP3R (4Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 5Ando H. Mizutani A. Kiefer H. Tsuzurugi D. Michikawa T. Mikoshiba K. Mol. Cell. 2006; 22: 795-806Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Ca2+ as a second messenger for regulation of NHE3 has been widely studied (14Chu T.S. Peng Y. Cano A. Yanagisawa M. Alpern R.J. J. Clin. Investig. 1996; 97: 1454-1462Crossref PubMed Scopus (70) Google Scholar, 15Kim J.H. Lee-Kwon W. Park J.B. Ryu S.H. Yun C.H. Donowitz M. J. Biol. Chem. 2002; 277: 23714-23724Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 23Donowitz M. Cohen M.E. Gould M. Sharp G.W.G. J. Clin. Investig. 1989; 83: 1953-1962Crossref PubMed Scopus (44) Google Scholar, 24Lee-Kwon W. Kim J.H. Choi J.W. Kawano K. Cha B. Dartt D.A. Zoukhri D. Donowitz M. Am. J. Physiol. 2003; 285: C1527-C1536Crossref PubMed Scopus (86) Google Scholar, 25Cinar A. Chen M.M. Riederer B. Bachmann O. Manns M. Kocher O. Seidler U. J. Physiol. 2007; 581: 1235-1246Crossref PubMed Scopus (54) Google Scholar). Hence, we explored the possibility that IRBIT may regulate NHE3 in a Ca2+-dependent manner by overexpressing HA-IRBIT in PS120/NHE3V cells (Fig. 3A). Ectopic expression of IRBIT did not affect the basal transport activity of NHE3 of PS120/NHE3V cells (Fig. 3B). We next examined whether the endoplasmic/sarcoplasmic reticulum Ca2+-ATPase pump inhibitor thapsigargin (100 nm) regulates NHE3 activity in PS120/NHE3V fibroblasts. Fig. 3, C and D, show that thapsigargin had no effect on NHE3 activity in PS120/NHE3V cells. Similarly, the Ca2+ ionophore, ionomycin (500 nm), did not affect NHE3 activity. On the other hand, both thapsigargin and ionomycin increased NHE3 transporter activity by ∼60% in PS120/NHE3V/IRBIT cells (Fig. 3, E and F). These results show that IRBIT mediates Ca2+-dependent stimulation of NHE3 activity. To affirm the importance of IRBIT in Ca2+-mediated activation of NHE3, we knocked down endogenous expression of IRBIT in PS120/NHE3V cells. Two shRNA constructs, sh-1 and sh-2, were able to knock down IRBIT expression by ∼75% in PS120/NHE3V cells (Fig. 4A). Importantly, in contrast to untransfected or control transfected cells in which thapsigargin exerted no effect on NHE3 activity, thapsigargin inhibited NHE3 activity in sh-1 or sh-2 transfected cells by ∼20% (Fig. 4B), confirming the role of IRBIT in up-regulation of NHE3 activity. Having shown the role of IRBIT in NHE3 regulation, we sought to determine whether binding of IRBIT to the COOH-terminal domain of NHE3 is necessary for the regulation of NHE3 activity. We examined the effect of thapsigargin on Na+/H+ exchange activity in PS120/NHE3V-585/IRBIT and PS120/NHE3V-689/IRBIT cells. Earlier we showed that IRBIT bound to NHE3V-689 but not to NHE3V-585 (Fig. 2). Consistent with the IRBIT binding, thapsigargin stimulated Na+/H+ exchange activity in PS120/NHE3V-689/IRBIT cells (Fig. 5B), but not in PS120/NHE3V-585/IRBIT (Fig. 5A), providing evidence that the interaction of IRBIT and NHE3 is necessary for IRBIT-dependent activation of NHE3 activity. To verify that IRBIT-dependent regulation of NHE3 is dependent on the rise in intracellular Ca2+, we prevented the change in Ca2+ by pretreating cells with the Ca2+ chelator, BAPTA-AM. Pretreatment with BAPTA-AM (40 μm) for 20 min during dye loading showed no inhibitory effect on basal NHE3 activity, but completely blocked thapsigargin-induced stimulation of NHE3 activity (Fig. 6). These findings suggest that the IRBIT-dependent stimulatory effect on NHE3 activity requires a change in intracellular Ca2+. Expression of IRBIT Reverses NHERF2-dependent Ca2+ Inhibition of NHE3 Activity—Previous reports suggested that NHERF2 is required for Ca2+-mediated inhibition of NHE3 activity (15Kim J.H. Lee-Kwon W. Park J.B. Ryu S.H. Yun C.H. Donowitz M. J. Biol. Chem. 2002; 277: 23714-23724Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 24Lee-Kwon W. Kim J.H. Choi J.W. Kawano K. Cha B. Dartt D.A. Zoukhri D. Donowitz M. Am. J. Physiol. 2003; 285: C1527-C1536Crossref PubMed Scopus (86) Google Scholar). We were curious what would happen to NHE3 activity wh
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