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• W2014295084 abstract "Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-α. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-α displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-α is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain. Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-α. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-α displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-α is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain. Receptor-stimulated phosphoinositide (PI) 1The abbreviations used are: PIphosphoinositideInsPninositol polyphosphateIns(1,4,5)P3inositol 1,4,5-trisphosphateDAGdiacylglycerolInsP4inositol 1,3,4,5-tetrakisphosphateInsP5inositol pentakisphosphateInsP6inositol hexakisphosphatePtdIns(3)Pphosphatidylinositol 3-phosphatePtdIns(4)Pphosphatidylinositol 4-phosphatePtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphatePtdInsP3phosphatidylinositol 3,4,5-trisphosphateTEABtriethylammonium bicarbonate[125I]ASA-InsP41-O-[125I](3-[2-iodo-4-azidosalicylamidyl]propyl)-inositol tetrakisphosphate[3H]BZDC-InsP41-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-inositol tetrakisphosphateBZDC-NHS4-benzoyldihydrocinnamoyl N-hydroxysuccinimide esterHPLChigh performance liquid chromatographygPInsP3glycerophosphoinositol 3,4,5-trisphosphatedaPtdInsP3diacetylphosphatidylinositol 3,4,5-trisphosphategPInsP2glycerophosphoinositol 4,5-bisphosphateGAPGTPase-activating proteinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reactionbpbase pair(s)kbkilobase(s)ORFopen reading frame. metabolism generates numerous inositol polyphosphates (InsPns) and inositol phospholipids, many of which may function as potential second messengers (1Michell R.H. Trends Biochem. Sci. 1992; 17: 274-276Abstract Full Text PDF PubMed Scopus (72) Google Scholar). Of the possible PI metabolites, Ins(1,4,5)P3 (InsP3) and diacylglycerol (DAG) are the best characterized second messengers. Generated by receptor-stimulated phospholipase C, hydrolysis of PtdIns(4,5)P2 (2Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6153) Google Scholar), Ins(1,4,5)P3 binds to and gates an InsP3 receptor calcium channel on the endoplasmic reticulum (2Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6153) Google Scholar, 3Ferris C.D. Snyder S.H. Annu. Rev. Physiol. 1992; 54: 469-488Crossref PubMed Scopus (196) Google Scholar). The lipid DAG remains in the membrane where it activates several protein kinase C isoforms and may regulate other targets (4Nakamura S. Nishizuka Y. J. Biochem. 1994; 115: 1029-1034Crossref PubMed Scopus (137) Google Scholar, 5Liscovitch M. Cantley L.C. Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (311) Google Scholar). In the membrane, DAG is metabolized rapidly to monoacylglycerol and to several phospholipids. In the cytoplasm, Ins(1,4,5)P3 can be phosphorylated to Ins(1,3,4,5)P4 by an InsP3 3-kinase. Other isomers of InsP4, InsP5, and InsP6, some of which are synthesized independently of Ins(1,4,5)P3, have been identified (for review, see Ref. 6Menniti F.S. Oliver K.G. Putney J.W. Shears S.B. Trends Biochem. Sci. 1993; 18: 53-56Abstract Full Text PDF PubMed Scopus (122) Google Scholar), and their production may also be regulated by receptors or during cell growth. Information from receptor binding studies, using radioactive InsP4 and InsP6, have demonstrated that a number of important regulatory proteins contain high affinity InsPn binding sites. InsPns have been implicated in the regulation of clathrin assembly proteins AP-2 (7Voglmaier S.M. Keen J.H. Murphy J.E. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. 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Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar). phosphoinositide inositol polyphosphate inositol 1,4,5-trisphosphate diacylglycerol inositol 1,3,4,5-tetrakisphosphate inositol pentakisphosphate inositol hexakisphosphate phosphatidylinositol 3-phosphate phosphatidylinositol 4-phosphate phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 3,4,5-trisphosphate triethylammonium bicarbonate 1-O-[125I](3-[2-iodo-4-azidosalicylamidyl]propyl)-inositol tetrakisphosphate 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-inositol tetrakisphosphate 4-benzoyldihydrocinnamoyl N-hydroxysuccinimide ester high performance liquid chromatography glycerophosphoinositol 3,4,5-trisphosphate diacetylphosphatidylinositol 3,4,5-trisphosphate glycerophosphoinositol 4,5-bisphosphate GTPase-activating protein 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid polyacrylamide gel electrophoresis polymerase chain reaction base pair(s) kilobase(s) open reading frame. Inositol phospholipids have also been postulated as messenger molecules. From in vivo, genetic, and permeabilized cell studies, evidence for critical roles for the inositol phospholipids PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P2 as regulators of membrane vesicle trafficking and cytoskeletal rearrangements is accumulating rapidly (5Liscovitch M. Cantley L.C. Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 13Janmey P.A. Annu. Rev. Physiol. 1994; 56: 169-191Crossref PubMed Scopus (471) Google Scholar, 14Liscovitch M. Cantley L.C. Cell. 1995; 81: 659-662Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 15De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (659) Google Scholar). One inositol phospholipid, PtdIns(3,4,5)P3, has emerged as a potential messenger molecule in receptor-stimulated cells (16Traynor-Kaplan A.E. Harris A.L. Thompson B.L. Taylor P. Sklar L.A. Nature. 1988; 334: 353-356Crossref PubMed Scopus (208) Google Scholar, 17Stephens L.R. Hughes K.T. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (386) Google Scholar, 18Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 358: 157-159Crossref PubMed Scopus (198) Google Scholar). Synthesized by receptor-stimulated PI 3-kinase phosphorylation of PtdIns(4,5)P2 (18Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 358: 157-159Crossref PubMed Scopus (198) Google Scholar), PtdInsP3 is not a substrate for PI-specific phospholipase Cs (19Serunian L.A. Haber M.T. Fukui T. Kim J.W. Rhee S.G. Lowenstein J.M. Cantley L.C. J. Biol. Chem. 1989; 264: 17809-17815Abstract Full Text PDF PubMed Google Scholar). PtdInsP3 production is stimulated by numerous inflammatory, growth, and trophic factors, including insulin, epidermal growth factor, platelet-derived growth factor, nerve growth factor, fMLP, platelet-activating factor, and the interleukins IL-2, IL-3, and IL-4, via activation of receptor-regulated PI 3-kinase (for review, see Ref. 20Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). Numerous studies have implicated receptor-stimulated PI 3-kinase in regulating a variety of cellular activities, including mitogenesis (21Fantl W.J. Escobedo J.A. Martin G.A. Turck C.W. Rosario M. McCormick F. Willians L.T. Cell. 1992; 69: 413-423Abstract Full Text PDF PubMed Scopus (472) Google Scholar), membrane ruffling, and actin cytoskeletal reorganization (22Wennstrom S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 23Wymann M. Acaro A. Biochem. J. 1994; 298: 517-520Crossref PubMed Scopus (124) Google Scholar, 24Kotani K. Yanezawa K. Hara K. Ueda H. Kitamura Y. EMBO J. 1994; 13: 2313-2321Crossref PubMed Scopus (326) Google Scholar) particularly via the small GTPase rac (25Hawkins P.T. Equinoa A. Qiu R.-G. Stokoe D. Cooke F.T. Walters R. Wennstrom S. Claesson-Welsh L. Evans T. Symons M. Stephens L. Curr. Biol. 1995; 5: 393-403Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar), glucose transporter translocation (26Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 27Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar, 28Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (999) Google Scholar), membrane trafficking (29Joly M. Kazlauskas A. Corvera S. J. Biol. Chem. 1995; 270: 13225-13230Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), stimulation of p70 ribosomal S6-kinase (28Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (999) Google Scholar, 30Chung J. Grammer T.C. Lemon K.P. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (656) Google Scholar), regulation of Akt kinase (protein kinase B) (31Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1873) Google Scholar, 32Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 33Kohn A.D. Kovacina K.S. Roth R. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar), and neurite outgrowth (34Kimura K. Hattori S. Kabuyama Y. Shizawa Y. Takayanagi J. Nakamura S. Toki S. Matsuda Y. Onodera K. Fukui Y. J. Biol. Chem. 1994; 269: 18961-18967Abstract Full Text PDF PubMed Google Scholar, 35Jackson T.R. Blader I.J. Hammonds-Odie L.P. Burga C.R. Cooke F. Hawkins P.T. Wolf A.G. Heldman K.A. Theibert A.B. J. Cell Sci. 1996; 109: 289-300Crossref PubMed Google Scholar). Although the molecular basis by which PI 3-kinase regulates these cellular activities is still unclear, the existence of specific receptor targets for PtdInsP3, analogous to the receptors for InsP3 and DAG, has been proposed (35Jackson T.R. Blader I.J. Hammonds-Odie L.P. Burga C.R. Cooke F. Hawkins P.T. Wolf A.G. Heldman K.A. Theibert A.B. J. Cell Sci. 1996; 109: 289-300Crossref PubMed Google Scholar, 36Ogawa W. Roth R.A. Biochim. Biophys. Acta. 1994; 1224: 533-540Crossref PubMed Scopus (2) Google Scholar). In vitro, PtdInsP3 competes with phosphotyrosine for binding to the SH2 domain of the p85 subunit of PI 3-kinase (37Rameh L.E. Chen C.-S. Cantley L.C. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar). PtdInsP3 has been shown to stimulate pleckstrin phosphorylation in permeabilized platelets (38Zhang J. Falck J.R. Reddy K.K. Abrams C.S. Zhao W. Rittenhouse S.E. J. Biol. Chem. 1995; 270: 22807-22810Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and there is evidence that 3-phosphorylated inositol phospholipids may interact with Akt kinase (protein kinase B) (32Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D. Tsichlis P.N. 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To isolate and identify specific, high affinity PtdInsP3 receptors from mammalian brain, we have used affinity chromatography with a tethered aminopropyl-InsP4 linked to Affi-Gel 10 and a hydrophobic InsP4 photoaffinity probe. Here, we report the identification, characterization, and cloning of a novel candidate PtdInsP3 receptor that is highly enriched in rat brain. Fine chemicals were purchased, if not specified, from Sigma. The [α-32P]dCTP and 5′-[α-35S]dA(thio)TP were obtained from DuPont NEN and Amersham, respectively. Restriction enzymes and DNA/RNA-modifying enzymes were obtained from Promega, Stratragene, and Clontech. The InsP4 affinity resin (aminopropyl-InsP4 linked to Affi-Gel 10 (Bio-Rad)) was synthesized as described previously (43Estevez V.A. Prestwich G.D. J. Am. Chem. Soc. 1991; 113: 9885-9887Crossref Scopus (96) Google Scholar, 44Theibert A.B. Estevez V.A. Ferris C.D. Danoff S.K. Barrow R.K. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3165-3169Crossref PubMed Scopus (105) Google Scholar). The precursor 1-O-(3-aminopropyl)-D-myo-Ins(1,3,4,5)P4 was synthesized in 12 steps from α-D-glucose (43Estevez V.A. Prestwich G.D. J. Am. Chem. Soc. 1991; 113: 9885-9887Crossref Scopus (96) Google Scholar) and was coupled with tritium-labeled 4-benzoyldihydrocinnamoyl N-hydroxysuccinimide ester (BZDC-NHS) (45Olszewski J.D. Dorman G. Elliot J.T. Hong Y. Ahern D.G. Prestwich G.D. Bioconjugate Chem. 1995; 6: 395-400Crossref PubMed Scopus (48) Google Scholar, 46Chaudhary A. Dorman G. Prestwich G.D. Tetrahedron Lett. 1994; 35: 7521-7524Crossref Scopus (17) Google Scholar). An ethyl acetate solution of 2.76 mCi (80 nmol) of this reagent was evaporated to dryness under N2 and dissolved in 200 µl of N,N-dimethylformamide. To this solution, 100 µl of 0.25 M triethylammonium bicarbonate (TEAB) buffer was added, followed by the addition of 100 µl of a 0.56 mg/ml solution of aminopropyl-InsP4 (0.81 µmol) in 0.25 M TEAB buffer. The reaction was kept in the dark and stirred overnight at room temperature. The mixture was then concentrated in vacuo; the residue was dissolved in 500 µl of deionized water and applied to a Pasteur pipette (4 × 0.5 cm) column of DEAE-cellulose (HCO3− form). The column was washed with 1 ml of deionized or distilled water and eluted sequentially with the following: 1 ml of 0.1 M TEAB, 1 ml of 0.2 M TEAB, 1 ml of 0.3 M TEAB, 1 ml of 0.35 M TEAB, 1 ml of 0.4 M TEAB, 1 ml of 0.4 M TEAB, 1 ml of 0.5 M TEAB, and 1 ml of 0.6 M TEAB. Fractions were analyzed by reverse phase HPLC (15% acetonitrile in 0.05 M KH2PO4 buffer) and monitored with a radiochemical detector. The highly radioactive fractions (generally, the 0.35, 0.4, and 0.5 M TEAB buffer fractions) had a retention time of 8.30 min, which corresponded to the retention time of nonradiolabeled BZDC-InsP4 detected by UV absorption. The radiochemical yield was 40%. Centaurin 2The chimaeric nature of centaurin-α and its homology to a family of GTPase-activating proteins is reminiscent of another distinct family of molecules named the chimaerins (5Liscovitch M. Cantley L.C. Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (311) Google Scholar), and we have therefore named our protein after another chimaera, the centaur (half-man, half-horse) of Greek mythology. -α was purified from whole rat brain of 150-300-g male Sprague-Dawley rats (Charles River) using methods described previously with all procedures at 0-4°C (44Theibert A.B. Estevez V.A. Ferris C.D. Danoff S.K. Barrow R.K. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3165-3169Crossref PubMed Scopus (105) Google Scholar, 47Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar). Briefly, whole brains were homogenized in 50 mM Tris/HCl, pH 7.7, containing 1 mM EDTA; 1 mM EGTA; 1 mMβ-mercaptoethanol; 100 mg/liter phenylmethylsulfonyl fluoride; 5 mg/liter each of chymostatin, antipain, and pepstatin; 10 mg/liter aprotinin and leupeptin; and 250 mg/liter CBZ-phenylalanine (PB). After centrifugation at 45,000 × g for 15 min, the soluble fraction was diluted 3-5-fold with PB containing 250 mM NaCl. Resuspended membranes were solubilized for 45 min with PB plus 1% CHAPS and 250 mM NaCl and centrifuged for 30 min at 45,000 × g. All buffers for membrane-associated fractions contained 1% CHAPS. CHAPS-solubilized membranes and soluble proteins each were incubated with heparin agarose for 1 h. The resin was washed with PB plus 250 mM NaCl and eluted with PB containing 1 M NaCl. The eluates were concentrated in Amicon Centripreps to a final volume of 1-2 ml for each preparation of 10-15 rat brains for photolabeling experiments. For further purification by InsP4 affinity chromatography, the concentrated heparin-agarose eluates were diluted with 50 mM Tris/HCl, pH 7.4, containing 1 mM EDTA (ACB) and loaded onto an InsP4 affinity column (44Theibert A.B. Estevez V.A. Ferris C.D. Danoff S.K. Barrow R.K. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3165-3169Crossref PubMed Scopus (105) Google Scholar) adapted to an FPLC (Pharmacia, column dimensions, 10 × 3 cm) at a rate of 0.2 ml/min. The column was washed with 10 ml of 200 mM NaCl in ACB and eluted with a gradient of 0.2 M to 1.5 M NaCl at 0.2 ml/min. Fractions (2 ml) were collected and assessed for protein or [3H]BZDC-InsP4 photoaffinity labeling. InsP4 column fractions from the soluble fraction of 100 whole rat brains were concentrated for tryptic digestion and peptide sequencing. Concentrated heparin-agarose column eluate (5-30 µl) was incubated with 40-60 µl of 25 mM Tris, pH 7.4, containing 1 mM EDTA, 0.5 mM potassium phosphate, 0.5 mM potassium pyrophosphate, and 20-70 nM [3H]BZDC-InsP4. To determine the affinity and specificity of photolabeled centaurin-α, 10 nM to 3 µM concentrations of the indicated inositol phosphate or phospholipid were included with the [3H]BZDC-InsP4. Samples were equilibrated for 10 min at 0°C before exposure to UV light (360 nm) for 1 h at 0°C. SDS sample buffer was then added to the samples and the proteins separated by SDS-PAGE on 10% Laemmli gels. Gels were fixed and fluorographed with the Entensify System (Amersham Corp.), dried, autoradiographed, and digitized. All of the photolabeling studies were performed in at least three independent experiments. Approximately 50 µg of concentrated centaurin-α purified by InsP4 affinity column chromatography was subjected to SDS-PAGE and transferred to Immobilon-P membrane. The membrane was stained with Coomassie to localize the protein band with an apparent molecular size of 46 kDa, which was then excised and digested with trypsin. The resultant tryptic peptides were fractionated via reverse phase HPLC with a linear gradient of acetonitrile, 0.1% trifluoroacetic acid (90 min). Individual tryptic peptides were then subjected to automated Edman degradation on a Procise 492 protein sequencer (Perkin-Elmer, ABD division). The nine unique peptide sequences that were obtained were compared with sequences in the GenBank data base using the TFASTA and BLAST DNA analysis software package of the University of Wisconsin Genetics Computer Group. Rat brain template cDNA was prepared by reverse transcription of rat brain poly(A)+ RNA (Clontech). Degenerate oligonucleotide primers were designed based on two tryptic peptides having 100% homology to putative peptide sequences at the ends of a human-expressed sequence tag (GenBank accession number T09325). The sequences of the primers synthesized (DNA International) were as follows: 5′-TT(T/C)CA(T/C)TA(T/C)(T/C)TICA(A/G)GTIGCITT(T/C)CC-3′ (26-mer, 64-fold degeneracy) and 5′-C(C/T)TGIGGIAGCATIGG(C/T)CT(G/A)TCIAC-3′ (25-mer, 8-fold degeneracy). Using these primers in PCR on the rat brain template cDNA, we were able to obtain a product corresponding to a predicted size of approximately 400 bp. The PCR product was subsequently cloned using the TA cloning system (Invitrogen). A total of 1.6 × 106 independent recombinants of a λZap II postnatal day 7 rat brain cDNA library (kindly provided by Dr. Craig Garner, University of Alabama at Birmingham) were screened with a [α-32P]dCTP-labeled 400-bp PCR product as a probe (Promega Prime-a-Gene System). Twenty-five strongly positive plaques were picked and excised with the Stratagene Solr System. The clone containing the longest insert of 2,450 bp was sequenced and called centaurin-α. Using a nested deletion based strategy, both strands of the centaurin-α clone were sequenced as double-stranded plasmids with M13 forward and reverse primers by the dideoxynucleotide chain termination method using deoxyadenosine 5′-[α-35S]thiotriphosphate and Sequenase 7.0 (U. S. Biochemical Corp.). The multiple tissue Northern blot containing poly(A)+ RNA from the indicated rat tissues was obtained from Clontech. According to the company's catalog, “Multiple tissue Northern blots are premade Northern blots of quality poly(A)+ RNA from different (rat) tissues. The poly(A)+ RNA is purified by several passages through oligo(dT)-cellulose columns, then run on a denaturing formaldehyde, 1.2% agarose gel, and blotted onto a positively charged nylon membrane. The lanes of the blots each contain approximately 2 µg of pure poly(A)+ RNA from specific tissues. The RNA loaded in each lane is adjusted so that a visible β-actin signal is present in every lane.” Prehybridization was performed at 42°C in 6 × SSPE (1.08 M NaCl, 60 mM sodium phosphate, 6 mM EDTA, pH 8.0, 5 × Denhardt's solution, 50% formamide, 100 µg/ml sheared salmon sperm DNA, and 0.1% SDS for 4 h. An [α-32P]dCTP-labeled AvaI/BamHI fragment (4.5 × 109 cpm/µg, 1,240 bp) from the 3′ end of centaurin-α was hybridized to the membrane for 24 h at 42°C (protocol recommended by Clontech). The membrane was washed four times in 1 liter of 2 × SSC (0.9 M NaCl, 99 mM sodium citrate), 0.1% SDS for 15 min at 50°C, followed by two 15-min washes in 1 liter of 1 × SSC, of 0.5 × SSC, and of distilled deionized water at 50°C. Autoradiography was performed at −80°C with Kodak X-Omat-AR film with intensifying screens for 24 h. A COOH-terminal fusion protein construct (containing ankyrin repeats 7-10) was made by digesting the 2.5-kb pair centaurin-α cDNA with BglII and KpnI to generate a 1.6-kb pair fragment that was cloned into the Pinpoint Xa2 expression vector (Promega). The expression of recombinant centaurin-α in the bacterial strain DH5α was induced by inositol-1-thio-β-D-galactopyranoside. The COOH-terminal fusion protein was extracted with 8 M urea from insoluble bacterial pellets. The extract was clarified by centrifugation. Then, the supernatant was dialyzed to remove the urea. The COOH-terminal fusion protein was then purified using the Soft-link avidin resin (Promega). Approximately 1 mg of purified fusion protein was obtained from 1 liter of bacterial culture. A peptide containing the NH2-terminal amino acids 2-19 of centaurin-α was synthesized and conjugated to PPDT (purified protein derivative of tuberculin). The bacterially expressed COOH-terminal Pinpoint centaurin-α fusion protein and the PPDT-conjugated NH2-terminal peptide were injected into rabbits following a standard booster immunization protocol. The NH2-terminal antipeptide antiserum was designated J49 and the COOH-terminal fusion protein antiserum, J4. COS-7 cells were routinely cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The EagI/SphI fragment of the centaurin-α cDNA was subcloned into the p63d2 vector downstream of the cytomegalovirus promoter (22Wennstrom S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) to give the centaurin-α vector named p63d.2.13. Transient transfections of COS-7 cells (plated at 50-80% confluence) were performed using 10-20 µg of recombinant centaurin-α DNA and 50 µl of lipofectamine (Life Technologies, Inc.)/10-cm dish, according to the manufacturer's protocol. Following transfection, the cells were cultured for an additional 48-60 h. The expressed recombinant centaurin-α protein was isolated and partially purified by heparin-agarose and InsP4 affinity column chromatography under the same conditions as for the native rat brain centaurin-α protein. Approximately 20 µg of purified recombinant centaurin-α protein was obtained from one 10-cm dish of cells. Fractions from rat brain homogenate and cell lysates were separated by SDS-PAGE and transferred to Protean nitrocellulose membranes (Schleicher & Schuell). Membranes were probed with either the rabbit polyclonal NH2-terminal J49 antipeptide or the J4 antiserum at 1:500 dilution. Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were detected using 3,3′-diaminobenzidine. Previously, we have used an InsP4 affinity column to isolate specific, high affinity InsPn-binding proteins from the particulate fraction of rat cerebella (44Theibert A.B. Estevez V.A. Ferris C.D. Danoff S.K. Barrow R.K. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3165-3169Crossref PubMed Scopus (105) Google Scholar, 47Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar). This strategy led to the identification of one high affinity InsPn-binding protein, the clathrin-associated protein AP-2 (7Voglmaier S.M. Keen J.H. Murphy J.E. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar). In addition to the proteins characterized for [3H]InsP4 binding and photolabeling, we noted the presence of a 46-kDa protein (47Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar). We predicted that this 46-kDa protein would have the highest affinity for InsP4, since the other InsPn-binding proteins eluted from the resin in rank order of their affinity for InsP4, and the 46-kDa protein eluted from the column last. However, under the conditions used for reversible [3H]InsP4 binding and photoaffinity labeling with an radioiodinated arylazido probe, [125I]ASA-InsP4 (43Estevez V.A. Prestwich G.D. J. Am. Chem. Soc. 1991; 113: 9885-9887Crossref Scopus (96) Google Scholar), we could not detect binding to this protein (47Theibert A.B. Estevez V.A. Mourey R.J. Marecek J.F. Barrow R.K. Prestwich G.D. Snyder S.H. J. Biol. Chem. 1992; 267: 9071-9079Abstract Full Text PDF PubMed Google Scholar). Thus, we were unable to characterize its specificity and affinity or de" @default.
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• W2014295084 title "Identification and Cloning of Centaurin-α" @default.
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