Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

IRBIT, a Novel Inositol 1,4,5-Trisphosphate (IP3) Receptor-binding Protein, Is Released from the IP3 Receptor upon IP3 Binding to the Receptor

2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês

10.1074/jbc.m210119200

ISSN

1083-351X

Autores

Hideaki Ando, Akihiro Mizutani, Toru Matsuura, Katsuhiko Mikoshiba,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated Ca2+ channels on intracellular Ca2+ stores. Herein, we report a novel protein, termed IRBIT (IP3Rbinding protein released with inositol 1,4,5-trisphosphate), which interacts with type 1 IP3R (IP3R1) and was released upon IP3 binding to IP3R1. IRBIT was purified from a high salt extract of crude rat brain microsomes with IP3elution using an affinity column with the huge immobilized N-terminal cytoplasmic region of IP3R1 (residues 1–2217). IRBIT, consisting of 530 amino acids, has a domain homologous toS-adenosylhomocysteine hydrolase in the C-terminal and in the N-terminal, a 104 amino acid appendage containing multiple potential phosphorylation sites. In vitro binding experiments showed the N-terminal region of IRBIT to be essential for interaction, and the IRBIT binding region of IP3R1 was mapped to the IP3 binding core. IP3 dissociated IRBIT from IP3R1 with an EC50 of ∼0.5 μm, i.e. it was 50 times more potent than other inositol polyphosphates. Moreover, alkaline phosphatase treatment abolished the interaction, suggesting that the interaction was dualistically regulated by IP3 and phosphorylation. Immunohistochemical studies and co-immunoprecipitation assays showed the relevance of the interaction in a physiological context. These results suggest that IRBIT is released from activated IP3R, raising the possibility that IRBIT acts as a signaling molecule downstream from IP3R. The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated Ca2+ channels on intracellular Ca2+ stores. Herein, we report a novel protein, termed IRBIT (IP3Rbinding protein released with inositol 1,4,5-trisphosphate), which interacts with type 1 IP3R (IP3R1) and was released upon IP3 binding to IP3R1. IRBIT was purified from a high salt extract of crude rat brain microsomes with IP3elution using an affinity column with the huge immobilized N-terminal cytoplasmic region of IP3R1 (residues 1–2217). IRBIT, consisting of 530 amino acids, has a domain homologous toS-adenosylhomocysteine hydrolase in the C-terminal and in the N-terminal, a 104 amino acid appendage containing multiple potential phosphorylation sites. In vitro binding experiments showed the N-terminal region of IRBIT to be essential for interaction, and the IRBIT binding region of IP3R1 was mapped to the IP3 binding core. IP3 dissociated IRBIT from IP3R1 with an EC50 of ∼0.5 μm, i.e. it was 50 times more potent than other inositol polyphosphates. Moreover, alkaline phosphatase treatment abolished the interaction, suggesting that the interaction was dualistically regulated by IP3 and phosphorylation. Immunohistochemical studies and co-immunoprecipitation assays showed the relevance of the interaction in a physiological context. These results suggest that IRBIT is released from activated IP3R, raising the possibility that IRBIT acts as a signaling molecule downstream from IP3R. inositol 1,4,5-trisphosphate inositol 1,4,5-trisphosphate receptor type 1 inositol 1,4,5-trisphosphate receptor metabotropic glutamate receptors B2 bradykinin receptors IP3R-binding protein released with inositol 1,4,5-trisphosphate glutathione S-transferase green fluorescent protein inositol 4,5-bisphosphate inositol 1,3,4,5-tetrakisphosphate inositol 1,2,3,4,5,6-hexakisphosphate phosphate-buffered saline fluorescence resonance energy transfer 1,4-piperazinediethanesulfonic acid The hydrolysis of phosphatidylinositol 4,5-bisphosphate in response to cell surface receptor activation leads to the production of an intracellular second messenger, inositol 1,4,5-trisphosphate (IP3).1IP3 mediates the release of Ca2+ from intracellular Ca2+ storage organelles, mainly the endoplasmic reticulum, by binding to its receptor (IP3R). In these IP3/Ca2+ signaling cascades, IP3R works as a signal converter from IP3 to Ca2+ (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6158) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4397) Google Scholar, 3Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar). IP3R is a tetrameric intracellular IP3-gated Ca2+ release channel (3Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar, 4Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (370) Google Scholar). There are three distinct types of IP3R in mammals (5Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (824) Google Scholar, 6Südhof T.C. Newton C.L. Archer III, B.T. Ushkaryov U.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 7Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar). Type 1 IP3R (IP3R1) is highly expressed in the central nervous system, particularly in the cerebellum (8Worley P.F. Baraban J.M. Colvin J.S. Snyder S.H. Nature. 1987; 325: 159-161Crossref PubMed Scopus (189) Google Scholar, 9Furuichi T. Simon-Chazottes D. Fujino I. Yamada N. Hasegawa M. Miyawaki A. Yoshikawa S. Guénet J.-L. Mikoshiba K. Recept. Channels. 1993; 1: 11-24PubMed Google Scholar). Mouse IP3R1 is composed of 2749 amino acids (5Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (824) Google Scholar), and is divided into three functionally distinct regions: the IP3-binding domain near the N terminus, the channel-forming domain with six membrane-spanning regions close to the C terminus, and the regulatory domain separating the two regions (10Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (273) Google Scholar, 11Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Crossref PubMed Scopus (127) Google Scholar). Deletion mutagenesis analysis of the IP3-binding domain has shown that residues 226–578 of IP3R1 are close to the minimum for specific and high affinity ligand binding, thus assigned to the IP3 binding core (12Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The precise gating mechanism of IP3R triggered by IP3 remains unclear, but IP3 binding induces a substantial but as yet undefined conformational change, which may cause channel opening (10Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (273) Google Scholar). Besides this channel opening, such IP3-induced conformational change has been assumed to be responsible for degradation of IP3R (13Zhu C.-C. Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; 274: 3476-3484Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Zhu C.-C. Wojcikiewicz R.J.H. Biochem. J. 2000; 348: 551-556Crossref PubMed Scopus (32) Google Scholar). The increase in the cytoplasmic Ca2+ concentration resulting from IP3R activation regulates the activities of thousands of downstream targets that play key roles in many aspects of cellular processes, including fertilization, development, proliferation, secretion, and synaptic plasticity (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6158) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4397) Google Scholar, 15Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1760) Google Scholar). To control such a vast array of cell functions, Ca2+ signals need to be precisely regulated in terms of space, time and amplitude (2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4397) Google Scholar, 15Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1760) Google Scholar). Such a complex regulation of Ca2+ signals has been partly attributed to the diversity of IP3R isoform expression, assembly of heterotetrameric complexes of IP3R isoforms, subcellular distributions of IP3R, and regulation of IP3R by Ca2+ itself, ATP, and phosphorylation (3Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar, 4Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (370) Google Scholar, 16Thrower E.C. Hagar R.E. Ehrlich B.E. Trends Pharmacol. Sci. 2001; 22: 580-586Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). IP3R channels are also regulated by their interacting proteins (4Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (370) Google Scholar, 17Mackrill J.J. Biochem. J. 1999; 337: 345-361Crossref PubMed Scopus (192) Google Scholar), including calmodulin (18Patel S. Morris S.A. Adkins C.E. O'beirne G. Taylor C.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11627-11632Crossref PubMed Scopus (99) Google Scholar, 19Michikawa T. Hirota J. Kawano S. Hiraoka M. Yamada M. Furuichi T. Mikoshiba K. Neuron. 1999; 23: 799-808Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), FKBP12 (Refs. 20Cameron A.M. Steiner J.P. Sabatini D.M. Kaplin A.I. Walensky L.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1784-1788Crossref PubMed Scopus (269) Google Scholar, 21Cameron A.M. Nucifora Jr., F.C. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 22Dargan S.L. Lea E.J.A. Dawson A.P. Biochem. J. 2002; 361: 401-407Crossref PubMed Scopus (44) Google Scholar, but also see Refs. 23Bultynck G. De Smet P. Weidema A.F. Ver Heyen M. Maes K. Callewaert G. Missiaen L. Parys J.B. De Smedt H. J. Physiol. 2000; 525: 681-693Crossref PubMed Scopus (63) Google Scholar and 24Bultynck G. De Smet P. Rossi D. Callewaert G. Missiaen L. Sorrentino V. De Smedt H. Parys J.B. Biochem. J. 2001; 354: 413-422Crossref PubMed Scopus (81) Google Scholar), calcineurin (Refs. 21Cameron A.M. Nucifora Jr., F.C. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar and 25Cameron A.M. Steiner J.P. Roskams A.J. Ali S.M. Ronnett G.V. Snyder S.H. Cell. 1995; 83: 463-472Abstract Full Text PDF PubMed Scopus (448) Google Scholar, but also see Refs. 23Bultynck G. De Smet P. Weidema A.F. Ver Heyen M. Maes K. Callewaert G. Missiaen L. Parys J.B. De Smedt H. J. Physiol. 2000; 525: 681-693Crossref PubMed Scopus (63) Google Scholar and 24Bultynck G. De Smet P. Rossi D. Callewaert G. Missiaen L. Sorrentino V. De Smedt H. Parys J.B. Biochem. J. 2001; 354: 413-422Crossref PubMed Scopus (81) Google Scholar), ankyrin (26Joseph S.K. Samanta S. J. Biol. Chem. 1993; 268: 6477-6486Abstract Full Text PDF PubMed Google Scholar, 27Bourguignon L.Y.W. Jin H. Iida N. Brandt N.R. Zhang S.H. J. Biol. Chem. 1993; 268: 7290-7297Abstract Full Text PDF PubMed Google Scholar, 28Hayashi T. Su T.-P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 491-496Crossref PubMed Scopus (290) Google Scholar), sigma-1 receptor (28Hayashi T. Su T.-P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 491-496Crossref PubMed Scopus (290) Google Scholar), chromogranins A and B (29Yoo S.H. So S.H. Kweon H.S. Lee J.S. Kang M.K. Jeon C.J. J. Biol. Chem. 2000; 275: 12553-12559Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 30Yoo S.H. Jeon C.J. J. Biol. Chem. 2000; 275: 15067-15073Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 31Thrower E.C. Park H.Y. So S.H. Yoo S.H. Ehrlich B.E. J. Biol. Chem. 2002; 277: 15801-15806Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), IRAG (32Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.-X. Allescher H.-D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (381) Google Scholar), Fyn (33Jayaraman T. Ondrias K. Ondiasova E. Marks A.R. Science. 1996; 272: 1492-1494Crossref PubMed Scopus (202) Google Scholar), and BANK (34Yokoyama K. Su I. Tezuka T. Yasuda T. Mikoshiba K. Tarakhovsky A. Yamamoto T. EMBO J. 2002; 21: 83-92Crossref PubMed Scopus (149) Google Scholar). Moreover, a family termed CaBP has been shown to interact with IP3R in a Ca2+-dependent manner, and to directly activate IP3R in the absence of IP3 (35Yang J. McBride S. Mak D.-O.D. Vardi N. Palczewski K. Haeseleer F. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7711-7716Crossref PubMed Scopus (168) Google Scholar). IP3R has also been demonstrated to be physically coupled to its upstream or downstream signaling molecules by protein-protein interactions. For example, IP3R is coupled with group 1 metabotropic glutamate receptors (mGluRs) via the Homer family of proteins (36Tu J.C. Xiao B. Yuan J.P. Lanahan A.A. Leoffert K. Li M. Linden D.J. Worley P.F. Neuron. 1998; 21: 717-726Abstract Full Text Full Text PDF PubMed Scopus (737) Google Scholar) and with B2 bradykinin receptors (B2Rs) by an unknown mechanism (37Delmas P. Wanaverbecq N. Abogadie F.C. Mistry M. Brown D.A. Neuron. 2002; 14: 209-220Abstract Full Text Full Text PDF Scopus (230) Google Scholar). Activations of mGluRs and B2Rs lead to the production of IP3 in proximity to IP3R, the result being efficient and specific signal propagation. Another example is Trp3, a candidate for plasma membrane Ca2+ channels regulated by intracellular Ca2+ store depletion (capacitative calcium entry channels). IP3R has been shown to interact with Trp3 directly, and to activate it via a conformational coupling mechanism (38Kiselyov K. Mignery G.A. Zhu M.X. Muallem S. Mol. Cell. 1999; 4: 423-429Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 39Boulay G. Brown D.M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14955-14960Crossref PubMed Scopus (347) Google Scholar). These protein-protein interactions are supposed to regulate the IP3/Ca2+ signaling pathway and contribute to the specificity of intracellular Ca2+ dynamics. To gain further insights into regulation of the IP3/Ca2+ signaling pathway, we searched for IP3R-binding proteins. In particular, we focused on molecules that interact with IP3R in a manner regulated by IP3, because such molecules may recognize the conformational change in IP3R induced by IP3binding, and/or may function as novel upstream or downstream signaling molecules of IP3R. For this purpose, we used an affinity column conjugated with the N-terminal 2217 amino acid residues of IP3R1 containing most of the large cytoplasmic region of the receptor molecule. By eluting bound proteins with IP3from this affinity column, we identified a novel IP3R-binding protein, IRBIT (IP3Rbinding protein released with inositol 1,4,5-trisphosphate). IRBIT bound to IP3R1 in vitro and in vivo, and co-localized intensively with IP3R1. Moreover, IRBIT was released from IP3R1 at a physiological concentration of IP3. On the basis of these results, we consider herein the role of IRBIT in IP3/Ca2+signaling. The cDNA encoding the N-terminal region of mouse IP3R1 (residues 1–225) was inserted into glutathioneS-transferase (GST) fusion vector pGEX-KG (40Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1639) Google Scholar). The GST-IP3R1 (1–225) fragment was subcloned into the baculovirus transfer vector pBlueBac4.5 (Invitrogen). The 3′-region downstream from the SmaI site of GST-IP3R1-(1–225) was replaced with theSmaI-EcoRI fragment of mouse IP3R1 (corresponding to residues 79–2217) to generate GST-IP3R1-(1–2217) (termed GST-EL, for the E coRI Large fragment) construct. GST alone was subcloned into pBlueBac4.5 as a control. Sf9 cells were cultured in TNM-FH medium supplemented with 10% fetal bovine serum at 27 °C. Recombinant baculoviruses carrying GST-EL or GST were generated with the Bac-N-BlueTMtransfection kit (Invitrogen) according to the manufacturer's protocols. GST-EL and GST were expressed in 2 × 108Sf9 cells by infecting recombinant baculoviruses at a multiplicity of infection of 5, and incubating for 48 h. Cells were harvested and stored at −80 °C. Frozen cells were suspended in 10 ml of 10 mm Hepes (pH 7.4), 100 mm NaCl, 2 mm EDTA, 1 mm 2-mercaptoethanol, 0.1% Triton X-100, and protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, 2 μm pepstatin A, and 10 μm E-64), and were homogenized with a glass-Teflon homogenizer (1000 rpm, 10 strokes). The homogenate was centrifuged at 20,000 × g for 30 min. The supernatant was incubated with 3 ml of glutathione-Sepharose 4B (AmershamBiosciences) for 3 h at 4 °C. After washing eight times with 40 ml of 10 mm Hepes (pH 7.4), 250 mm NaCl, 2 mm EDTA, 1 mm 2-mercaptoethanol, and 0.1% Triton X-100, GST-EL or GST coupled with glutathione-Sepharose was packed into columns and equilibrated with 10 mm Hepes (pH 7.4), 100 mm NaCl, 2 mm EDTA, 1 mm2-mercaptoethanol, and 0.1% Triton X-100. About 5 mg of GST-EL was immobilized. Adult rat cerebella (∼5 g) were homogenized in 45 ml of homogenizer buffer (10 mm Hepes (pH 7.4), 320 mm sucrose, 2 mm EDTA, 1 mm2-mercaptoethanol, and protease inhibitors) with a glass-Teflon homogenizer (950 rpm, 10 strokes), and the homogenate was centrifuged at 1,000 × g for 10 min. The supernatant (S1 fraction) was centrifuged at 100,000 × g for 60 min to obtain the cytosolic fraction (the supernatant) and the crude microsome (the pellet). The crude microsome was homogenized in 25 ml of homogenizer buffer containing 500 mm NaCl with a glass-Teflon homogenizer (1,200 rpm, 10 strokes), incubated on ice for 15 min, and centrifuged at 100,000 × g for 60 min to obtain the high salt extract (the supernatant) and the stripped-crude microsome (the pellet). The high salt extract was diluted five times with 10 mm Hepes (pH 7.4), 2 mm EDTA, 1 mm2-mercaptoethanol, 0.01% Brij 35, and protease inhibitors. The diluted high salt extract was precleared with glutathione-Sepharose and loaded onto a GST-EL affinity column equilibrated with binding buffer (10 mm Hepes (pH 7.4), 100 mm NaCl, 2 mm EDTA, and 1 mm 2-mercaptoethanol). The GST column was used as a control. The columns were washed with 20 column volumes of binding buffer, and bound proteins were eluted with binding buffer containing 50 μm IP3 (Dojindo) and 0.05% Brij 35. The eluted material was concentrated, separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% gel, and stained with Coomassie Brilliant Blue. The 60-kDa protein band was excised from the gel and digested with lysyl endopeptidase (Wako) essentially according to the previously described method (41Rosenfeld J. Capdevielle J. Guillemot J.C. Ferrara P. Anal. Biochem. 1992; 203: 173-179Crossref PubMed Scopus (1127) Google Scholar). The polypeptides were separated by a C18 reversed-phase column (μRPC C2/C18 SC 2.1/10, Amersham Biosciences) connected on a SMART system (Amersham Biosciences). The amino acid sequence of each peptide was determined by 494 procise protein sequencer (Applied Biosystems). Two peptide sequences, N-YSFMATVTK-C and N-QIQFADDMQEFTK-C were obtained. BLAST searches of two peptide sequences derived from the 60-kDa protein against the non-redundant data base revealed that these sequences match the sequence of a human cDNA deposited in a patent (GenBankTM accession numberCAC09285). Based on the data bases of mouse expressed sequence tags (accession number AW229870 and BE282170) homologous to this cDNA, primers (5′-ATGTCGATGCCTGACGCGATGC-3′ and 5′-GCGTGGTTCATGTGGACTGGTC-3′) were synthesized. cDNA of IRBIT was amplified by polymerase chain reaction (PCR) using mouse cerebellum oligo(dT)-primed, first-strand cDNA as a template. PCR product was cloned into pBluescript II KS(+) (Stratagene) and sequenced. Sequences of three independent clones were confirmed. The cDNA encoding the N-terminal region (residues 1–104) of IRBIT was subcloned into the bacterial hexahistidine (His6) fusion vector pET-23a(+) (Novagen) to generate the IRBIT-(1–104)-His6 construct. The same cDNA was subcloned into the GST fusion vector pGEX-4T-1 (Amersham Biosciences) to generate the GST-IRBIT-(1–104) construct. The cDNA fragments corresponding to the amino acid residues 1–225, 1–343, 341–923, 600–1248, 916–1581, and 1553–1943 of mouse IP3R1 were inserted into pGEX-KG to generate the GST-Ia, GST-Iab, GST-IIab, GST-IIbIIIa, GST-IIIab, and GST-IV construct, respectively. Residues 1593–2217 of mouse IP3R1 were inserted into pGEX-4T-1 to generate the GST-IV-Va construct. These fusion proteins were expressed in Escherichia coli. GST-EL was expressed in Sf9 cells as described above. Expressed IRBIT-(1–104)-His6 was purified using ProBond resin (Invitrogen). GST fusion proteins were purified using glutathione-Sepharose. GST-IbIIa (residues 224–604 of mouse IP3R1) and its site-directed mutants K508A and R441Q were described previously (Ref. 42Uchiyama T. Yoshikawa F. Hishida A. Furuichi T. Mikoshiba K. J. Biol. Chem. 2002; 277: 8106-8113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, GST-IbIIa was termed G224 therein). A Japanese white rabbit was immunized with purified IRBIT-(1–104)-His6 by subcutaneous injection with the complete Freund's adjuvant at 14-day intervals. The anti-IRBIT antisera was affinity-purified by passing serum from the immunized rabbit over a GST-IRBIT-(1–104) column covalently coupled with cyanogen bromide-activated Sepharose 4B (Amersham Biosciences), and specific antibodies bound to the column were eluted with 100 mm glycine-HCl (pH 2.5). Cerebrum, cerebellum, heart, lung, liver, kidney, thymus, spleen, testis, and ovary were dissected from the adult mouse and S1 fraction were obtained essentially as described above. The cytosol, the crude microsome, the high salt extract, and the stripped-crude microsome of mouse cerebellum were obtained essentially as described above. Proteins with the amount indicated were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride membrane by electroblotting. After blocking, membranes were immunoblotted with anti-IRBIT antibody (1 μg/ml) for 1 h at room temperature, followed by horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences). Immunoreactive bands were visualized with the enhanced chemiluminescence detection system (Amersham Biosciences). The cDNA encoding full-length IRBIT was subcloned into the pcDNA3 (Invitrogen). The cDNA encoding full-length IRBIT or its deletion mutants (residues 1–277, 1–104, and 105–530) were subcloned into the pEGFP-C1 (Clontech) to generate green fluorescent protein (GFP) fusion protein constructs. Mouse IP3R1 expression vector pBact-STneoB-C1 was described previously (43Miyawaki A. Furuichi T. Maeda N. Mikoshiba K. Neuron. 1990; 5: 11-18Abstract Full Text PDF PubMed Scopus (109) Google Scholar). COS-7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, penicillin, and streptomycin at 37 °C. Transient transfections were performed using TransIT transfection reagents (Mirus) according to the manufacturer's instruction. Transfected cells were processed for immunoblotting, pull-down experiments, or immunostaining 2 days after transfection. Mouse cerebellar cytosolic fraction was diluted two times with 10 mm Hepes (pH 7.4), 200 mm NaCl, 2 mm EDTA, 1 mm2-mercaptoethanol, and 0.02% Triton X-100. The high salt extract was diluted five times with 10 mm Hepes (pH 7.4), 2 mm EDTA, 1 mm 2-mercaptoethanol, and 0.01% Triton X-100. Diluted fractions (the final NaCl concentration of both fractions was 100 mm) were incubated with 20 μg of GST-EL or GST for 2 h at 4 °C. After adding 10 μl of glutathione-Sepharose and another 2-h incubation, the resins were washed five times with wash buffer (10 mm Hepes, pH 7.4, 100 mm NaCl, 2 mm EDTA, 1 mm2-mercaptoethanol, and 0.01% Triton X-100), and bound proteins were eluted with 20 mm glutathione. Eluted proteins were analyzed by Western blotting with anti-IRBIT antibody. For dephosphorylation, the diluted high salt extract was incubated with or without bacterial alkaline phosphatase (Toyobo) in the presence of 2 mm MgCl2 for 30 min at 37 °C after which 5 mm EDTA was added, and the sample was processed for pull-down assay as described above. For the dissociation experiments, IRBIT in the diluted high salt extract was pulled down with GST-EL and washed as described above, and resins were added in 100 μl of wash buffer containing IP3, inositol 4,5-bisphosphate (IP2) (Dojindo), inositol 1,3,4,5-tetrakisphosphate (IP4) (Calbiochem), inositol 1,2,3,4,5,6-hexakisphosphate (IP6) (Calbiochem), or ATP (Amersham Biosciences) (0.1, 0.3, 1, 3, 10 μm, each). After incubation on ice for 10 min, samples were centrifuged at 10,000 rpm for 1 min, and the supernatant was subjected to immunoblot analysis with anti-IRBIT antibody or goat anti-GST antibody (AmershamBiosciences). For quantitation, Alexa 680-conjugated goat anti-rabbit IgG (Molecular Probes) was used as a secondary antibody. Intensity of fluorescence of immunoreactive bands of IRBIT was measured using Odyssey infrared imaging system (Aloka). Quantitative data (the mean ± S.D. from at least three independent experiments) are expressed as percentage of the amount of IRBIT in the 10 μm IP3 eluate. For the determination of the IRBIT binding region and the critical amino acids of IP3R1, the diluted high salt extract were processed for pull-down assay with 100 pmol of GST, GST-EL, GST-Ia, GST-Iab, GST-IbIIa, GST-IIab, GST-IIbIIIa, GST-IIIab, GST-IV, GST-IV-Va, K508A, or R441Q as described above and analyzed by Western blotting with anti-IRBIT antibody. For the determination of the IP3R1-interacting region of IRBIT, COS-7 cells expressing GFP-tagged full-length IRBIT or its truncated mutants were lysed in lysis buffer (10 mm Hepes pH 7.4, 100 mm NaCl, 2 mm EDTA, 1 mm 2-mercaptoethanol, 0.5% Nonidet P-40, and protease inhibitors) for 30 min at 4 °C, followed by centrifugation (100,000 × g, 30 min). The supernatants were processed for pull-down assay with GST-EL or GST as described above, and bound proteins were subjected to immunoblot analysis with anti-GFP antibody (Medical & Biological Laboratories). Transfected COS-7 cells grown on glass coverslips were washed once in phosphate-buffered saline (PBS), fixed in 4% formaldehyde in PBS for 15 min, permeabilized in 0.1% Triton X-100 in PBS for 5 min, and blocked in PBS containing 2% normal goat serum for 60 min at room temperature. For washing out cytosolic proteins, transfected cells were washed once in PBS, permeabilized in ice-cold permeabilization buffer (80 mm PIPES, pH 7.2, 1 mm MgCl2, 1 mm EGTA, and 4% polyethylene glycol) containing 0.1% saponin for 10 min on ice, and washed twice with ice-cold permeabilization buffer before fixation. Cells were then stained with rabbit anti-IRBIT antibody (1 μg/ml for 60 min at room temperature) and rat anti-IP3R1 antibody 18A10 (44Maeda N. Niinobe M. Nakahira K. Mikoshiba K. J. Neurochem. 1988; 51: 1724-1730Crossref PubMed Scopus (125) Google Scholar) overnight at 4 °C. Following four 5-min PBS washes, Alexa 488-conjugated goat anti-rabbit IgG and Alexa 594-conjugated goat anti-rat IgG (Molecular Probes) were applied for 45 min at 37 °C. Following four 5-min PBS washes, the coverslips were mounted with Vectashield (Vector Laboratories) and observed under IX-70 confocal fluorescence microscopy (Olympus) with a ×60 objective. Immunoprecipitation was performed as described (45Shen L. Liang F. Walensky L.D. Huganir R.L. J. Neurosci. 2000; 20: 7932-7940Crossref PubMed Google Scholar) with modifications. Adult mouse cerebellum was homogenized in 10 volumes of 4 mm Hepes (pH 7.4), 320 mm sucrose, and protease inhibitors with a glass-Teflon homogenizer. The homogenate was centrifuged at 800 × gfor 10 min, and the supernatant was subjected to another centrifugation at 9000 × g for 15 min. The supernatant from the second centrifugation was solubilized in 1% sodium deoxycholate at 36 °C for 30 min, followed by adding 0.1 volume of 1% Triton X-100 in 50 mm Tris-HCl (pH 9.0), and the preparation was centrifuged at 100,000 × g for 10 min. The supernatant was incubated with 5 μl of protein G-Sepharose 4 fast flow (AmershamBiosciences) for 2 h at 4 °C to clarify nonspecific binding to the protein G beads. At the same time, 3 μg of rabbit anti-IRBIT antibody, control rabbit IgG, rat anti-IP3R1 antibody 10A6 (46Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (232) Google Scholar), control rat IgG, mouse anti-IP3R2 antibody KM1083 (47Sugiyama T. Furuya A. Monkawa T. Yamamoto-Hino M. Satoh S. Ohmori K. Miyawaki A. Hanai N. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 354: 149-154Crossref PubMed Scopus (82) Google Scholar), or control mouse IgG was preincubated with 5 μl of protein G beads for 2 h, and the protein G-antibody complex was spun down at 3,000 rpm for 2 min. The clarified supernatant was then added to the antibody-bound protein G beads, and the mixture was incubated for 2 h at 4 °C. Beads were washed five times with 10 mm Hepes (pH 7.4), 100 mm NaCl, and 0.5% Triton X-100 and analyzed by Western blotting with anti-IRBIT antibody, mouse anti-IP3R1 antibody KM1112 (47Sugiyama T. Furuya A. Monkawa T. Yamamoto-Hino M. Satoh S. Ohmori K. Miyawaki A. Hanai N. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 354: 149-154Crossref PubMed Scopus (82) Google Scholar), KM1083, or mouse anti-IP3R3 antibody KM1082 (47Sugiyama T. Furuya A. Monkawa T. Yamamoto-Hino M. Satoh S. Ohmori K. Miyawaki A. Hanai N. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 354: 149-154Crossref PubMed Scopus (82) Google Scholar). To identify IP3R-interacting molecules, we used a GST fusion p

Referência(s)