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• W2074273208 abstract "The membrane-associated guanylate kinase proteins have been known to interact various membrane receptors with their N-terminal segments designated the PDZ domains and to cluster these receptors at the target site of the cell membrane. NE-dlg/SAP102, a neuronal and endocrine tissue-specific MAGUK family protein, was found to be expressed in both dendrites and cell bodies in neuronal cells. Although NE-dlg/SAP102 localized at dendrites was shown to interact with N-methyl-d-aspartate receptor 2B via the PDZ domains to compose postsynaptic density, the binding proteins existing in the cell body of the neuron are still unknown. Here we report the isolation of a novel NE-dlg/SAP102-associated protein, p51-nedasin. Nedasin has a significant homology with amidohydrolase superfamily proteins and shows identical sequences to a recently identified protein that has guanine aminohydrolase activity. Nedasin has four alternative splice variants (S, V1, V2, and V3) that exhibited different C-terminal structures. NE-dlg/SAP102 is shown to interact with only the S form of nedasin which is predominantly expressed in brain. The expression of nedasin in neuronal cells increases in parallel with the progress of synaptogenesis and is mainly detected in cell bodies where it co-localizes with NE-dlg/SAP102. Furthermore, nedasin interferes with the association between NE-dlg/SAP102 and NMDA receptor 2B in vitro. These findings suggest that alternative splicing of nedasin may play a role in the formation and/or structural change in synapses during neuronal development by modifying clustering of neurotransmitter receptors at the synaptic sites. The membrane-associated guanylate kinase proteins have been known to interact various membrane receptors with their N-terminal segments designated the PDZ domains and to cluster these receptors at the target site of the cell membrane. NE-dlg/SAP102, a neuronal and endocrine tissue-specific MAGUK family protein, was found to be expressed in both dendrites and cell bodies in neuronal cells. Although NE-dlg/SAP102 localized at dendrites was shown to interact with N-methyl-d-aspartate receptor 2B via the PDZ domains to compose postsynaptic density, the binding proteins existing in the cell body of the neuron are still unknown. Here we report the isolation of a novel NE-dlg/SAP102-associated protein, p51-nedasin. Nedasin has a significant homology with amidohydrolase superfamily proteins and shows identical sequences to a recently identified protein that has guanine aminohydrolase activity. Nedasin has four alternative splice variants (S, V1, V2, and V3) that exhibited different C-terminal structures. NE-dlg/SAP102 is shown to interact with only the S form of nedasin which is predominantly expressed in brain. The expression of nedasin in neuronal cells increases in parallel with the progress of synaptogenesis and is mainly detected in cell bodies where it co-localizes with NE-dlg/SAP102. Furthermore, nedasin interferes with the association between NE-dlg/SAP102 and NMDA receptor 2B in vitro. These findings suggest that alternative splicing of nedasin may play a role in the formation and/or structural change in synapses during neuronal development by modifying clustering of neurotransmitter receptors at the synaptic sites. At the sites of cell-cell contacts of epithelial cells or the synaptic junctions of neuronal cells, several membrane receptors and channels are clustered into multiprotein complexes linked to the cytoskeleton via interactions of their C-terminal cytoplasmic tails with a novel protein family called membrane-associated guanylate kinase homologues (MAGUK) 1The abbreviations used are:MAGUKmembrane-associated guanylate kinase homologuesdlglethal (1Woods D.F. Bryant P.J. Mech. Dev. 1993; 44: 85-89Crossref PubMed Scopus (188) Google Scholar)-discs largeGSTglutathione S-transferaseGAHguanine aminohydrolaseHAhemagglutininNE-dlgneuronal and endocrine dlgNMDAN-methyl-d-aspartateNR2BNMDA-type glutamate receptor subunit 2BPDZPSD95/Dlg/ZO-1bpbase pairRT-PCRreverse transcriptase-polymerase chain reactionPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salinehIBMhereditary inclusion body myopathyGKguanylate kinasesCRcentirary1The abbreviations used are:MAGUKmembrane-associated guanylate kinase homologuesdlglethal (1Woods D.F. Bryant P.J. Mech. Dev. 1993; 44: 85-89Crossref PubMed Scopus (188) Google Scholar)-discs largeGSTglutathione S-transferaseGAHguanine aminohydrolaseHAhemagglutininNE-dlgneuronal and endocrine dlgNMDAN-methyl-d-aspartateNR2BNMDA-type glutamate receptor subunit 2BPDZPSD95/Dlg/ZO-1bpbase pairRT-PCRreverse transcriptase-polymerase chain reactionPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salinehIBMhereditary inclusion body myopathyGKguanylate kinasesCRcentirary (1Woods D.F. Bryant P.J. Mech. Dev. 1993; 44: 85-89Crossref PubMed Scopus (188) Google Scholar). The MAGUK family proteins contain three distinct domains as follows: an N-terminal segment comprised of one or three copies of an 80–90-amino acid motif called the PDZ (PSD-95/Dlg/ZO-1) domain, ansrc homology 3 (SH3) domain, and a region with high similarity to guanylate kinases (GK) (2Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Abstract Full Text PDF PubMed Scopus (763) Google Scholar, 3Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (993) Google Scholar). The PDZ domain is utilized as a module for interacting with the C-terminal Xaa-(Ser/Thr)-Xaa-Val (X(S/T)XV) motif of various proteins and generating multiprotein complexes (4Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 5Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1206) Google Scholar). membrane-associated guanylate kinase homologues lethal (1Woods D.F. Bryant P.J. Mech. Dev. 1993; 44: 85-89Crossref PubMed Scopus (188) Google Scholar)-discs large glutathione S-transferase guanine aminohydrolase hemagglutinin neuronal and endocrine dlg N-methyl-d-aspartate NMDA-type glutamate receptor subunit 2B PSD95/Dlg/ZO-1 base pair reverse transcriptase-polymerase chain reaction polyacrylamide gel electrophoresis phosphate-buffered saline hereditary inclusion body myopathy guanylate kinases centirary membrane-associated guanylate kinase homologues lethal (1Woods D.F. Bryant P.J. Mech. Dev. 1993; 44: 85-89Crossref PubMed Scopus (188) Google Scholar)-discs large glutathione S-transferase guanine aminohydrolase hemagglutinin neuronal and endocrine dlg N-methyl-d-aspartate NMDA-type glutamate receptor subunit 2B PSD95/Dlg/ZO-1 base pair reverse transcriptase-polymerase chain reaction polyacrylamide gel electrophoresis phosphate-buffered saline hereditary inclusion body myopathy guanylate kinases centirary Each MAGUK protein is thought to perform a distinct function depending upon its tissue distribution, cellular localization, and associated molecules. For instance, PSD-95/SAP90, which is one of the MAGUK proteins, is predominantly expressed in the brain and localizes at the postsynaptic membrane and presynaptic axon terminals of inhibitory neurons (6Kistner U. Wenzel B.M. Veh R.W. Cases-Langhoff C. Garner A.M. Appeltauer U. Voss B. Gundelfinger E.D. Garner C.C. J. Biol. Chem. 1993; 268: 4580-4583Abstract Full Text PDF PubMed Google Scholar, 7Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (892) Google Scholar, 8Hunt C.A. Schenker L.J. Kennedy M.B. J. Neurosci. 1996; 16: 1380-1388Crossref PubMed Google Scholar). PSD-95/SAP90 binds to the cytoplasmic tail of both Shaker-type voltage-gated K+ channels and the 2B subunit ofN-methyl-d-aspartate (NMDA)-type glutamate receptors (7Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (892) Google Scholar, 9Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1608) Google Scholar, 10Muller B.M. Kistner U. Kindler S. Chung W.J. Kuhlendahl S. Fenster S.D. Lau L.F. Veh R.W. Huganir R.L. Gundelfinger E.D. Garner C.C. Neuron. 1996; 17: 255-265Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 11Niethammer M. Kim E. Sheng M. J. Neurosci. 1996; 16: 2157-2163Crossref PubMed Google Scholar). PSD-95/SAP90 expressed in neuronal cells is therefore thought to contribute to clustering ion channels and neurotransmitter receptors at the synaptic membranes. Mutations of the lethal (1Woods D.F. Bryant P.J. Mech. Dev. 1993; 44: 85-89Crossref PubMed Scopus (188) Google Scholar)-discs large ( dlg ) gene in Drosophila, which also encodes a MAGUK family protein, was shown to cause postsynaptic structural defects (12Perrimon N. Dev. Biol. 1988; 127: 392-407Crossref PubMed Scopus (68) Google Scholar, 13Lahey T. Gorczyca M. Jia X.X. Budnik V. Neuron. 1994; 13: 823-835Abstract Full Text PDF PubMed Scopus (256) Google Scholar), suggesting that the dlg protein and its associated proteins are involved in the maturation of neuronal cells. These lines of evidence indicate that some MAGUKs play a role in synaptic organization in neuronal cells by linking interacting receptors to downstream signal molecules and regulating the structure of the synaptic junction. We recently identified a novel member of human MAGUK protein, NE-dlg (neuronal and endocrine dlg) (14Makino K. Kuwahara H. Masuko N. Nishiyama Y. Morisaki T. Sasaki J. Nakao M. Kuwano A. Nakata M. Ushio Y. Saya H. Oncogene. 1997; 14: 2425-2433Crossref PubMed Scopus (56) Google Scholar). NE-dlg is considered to be a human homologue of the rat postsynaptic protein SAP102 (10Muller B.M. Kistner U. Kindler S. Chung W.J. Kuhlendahl S. Fenster S.D. Lau L.F. Veh R.W. Huganir R.L. Gundelfinger E.D. Garner C.C. Neuron. 1996; 17: 255-265Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 15Lau L.F. Mammen A. Ehlers M.D. Kindler S. Chung W.J. Garner C.C. Huganir R.L. J. Biol. Chem. 1996; 271: 21622-21628Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), since the two proteins share 86% amino acid identity. NE-dlg/SAP102 contains three PDZ domains, an SH3 domain and a GK domain as do PSD-95/SAP90, and is highly expressed in neuronal and endocrine tissues. In the neurons, NE-dlg/SAP102 has been shown to be expressed in axons and dendrites (10Muller B.M. Kistner U. Kindler S. Chung W.J. Kuhlendahl S. Fenster S.D. Lau L.F. Veh R.W. Huganir R.L. Gundelfinger E.D. Garner C.C. Neuron. 1996; 17: 255-265Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 14Makino K. Kuwahara H. Masuko N. Nishiyama Y. Morisaki T. Sasaki J. Nakao M. Kuwano A. Nakata M. Ushio Y. Saya H. Oncogene. 1997; 14: 2425-2433Crossref PubMed Scopus (56) Google Scholar) and to bind to NMDA receptor subunit 2B (NR2B) at the synaptic membrane sites (15Lau L.F. Mammen A. Ehlers M.D. Kindler S. Chung W.J. Garner C.C. Huganir R.L. J. Biol. Chem. 1996; 271: 21622-21628Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Furthermore, NE-dlg/SAP102 has been found to interact with PSD-95/SAP90 in the presence of calmodulin and Ca2+ and is speculated to regulate the clustering of NMDA receptors to form the synapses at the specific site of membrane (16Masuko N. Makino K. Kuwahara H. Fukunaga K. Sudo T. Araki N. Yamamoto H. Yamada Y. Miyamoto E. Saya H. J. Biol. Chem. 1999; 274: 5782-5790Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). However, the NE-dlg/SAP102 is abundantly expressed also in cytoplasm of the matured neuron, which is not co-localized with NR2B (16Masuko N. Makino K. Kuwahara H. Fukunaga K. Sudo T. Araki N. Yamamoto H. Yamada Y. Miyamoto E. Saya H. J. Biol. Chem. 1999; 274: 5782-5790Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Therefore, it is possible that NE-dlg/SAP102 has some interactive molecules in the cytoplasm of the neuron, and it may modulate the NE-dlg/SAP102-related signaling in neuronal cells. In this study, we tried to identify a cytoplasmic NE-dlg/SAP102-interacting protein using GST-NE-dlg/SAP102 affinity column chromatography. From a bovine brain cytosol, we purified and determined a novel amidohydrolase superfamily protein, termed nedasin, that interacts with the PDZ domains of NE-dlg/SAP102 both in vitro and in vivo. Immunolocalization study shows that nedasin and NE-dlg/SAP102 co-localize at cell bodies of neuronal cells. Nedasin was shown to have four alternative splicing isoforms that have diversity at their C-terminal tails, and one isoform, called nedasin S, specifically binds to NE-dlg/SAP102. We also found that the nedasin S isoform competitively inhibits the binding between the NR2B subunit of NMDA receptors and the PDZ domains of NE-dlg/SAP102. These results suggest that nedasin modifies the dlg-related molecular clustering at the synaptic sites during development of neuronal cells and that alternative splicing of the nedasin transcript may affect this interaction. The cDNA fragments coding full-length NE-dlg/SAP102 and six deletion variants ΔGK, PDZ1 + 2 + 3, PDZ1 + 2, PDZ3, PDZ2 and PDZ1, as illustrated in Fig. 4, were amplified by PCR, subcloned into a pCR2 TA cloning vector (Invitrogen, San Diego, CA), digested with only EcoRI or with both EcoRI and HindIII, excised an inserted cDNA, and subcloned into a pGEX-2TH bacterial expression vector. The cDNA fragments coding full-length nedasin S and nedasin V1 were also amplified by PCR and subcloned into a pGEX-2TH vector. The expression and purification of GST fusion proteins were described previously (17Takeshima H. Izawa I. Lee P.S. Safdar N. Levin V.A. Saya H. Oncogene. 1994; 9: 2135-2144PubMed Google Scholar). Cytosol of bovine brain was prepared as described (18Yamamoto T. Matsui T. Nakafuku M. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1995; 270: 30557-30561Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In brief, bovine brain gray matter was cut into small pieces and suspended in homogenizing buffer A (25 mm Tris-HCl, pH 7.5, 1 mmdithiothreitol, 5 mm EGTA, 10 mmMgCl2, 10% sucrose). The suspension was homogenized with a Potter-Elvehjem Teflon glass homogenizer and filtered through gauze. The homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. Solid ammonium sulfate was added to the supernatant to a final concentration of 40% saturation. After being stirred for 1 h, the precipitate was collected by centrifugation and dissolved in 4 ml of buffer A, dialyzed against buffer A three times, and stored at −80 °C as the 0–40% cytosolic fraction. Subsequently, the supernatant was saturated by adding solid ammonium sulfate to a final concentration of 80%. The precipitate was collected, dissolved in buffer A, dialyzed as described above, and stored as the 40–80% cytosol fraction. All procedures were performed at 4 °C. Cytosol of rat brain was prepared as described (19Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1423) Google Scholar, 20Garcia E.P. Mehta S. Blair L.A. Wells D.G. Shang J. Fukushima T. Fallon J.R. Garner C.C. Marshall J. Neuron. 1998; 21: 727-739Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) with minor modifications. In brief, adult rat brain was homogenized in 5 volumes of buffer B (150 mm NaCl, 50 mm Tris-HCl, pH 7.5) containing 1 mm sodium orthovanadate, 1 mmaminoethylbenzenesulfonyl fluoride, 10 mm pepstatin, 3% aprotinin, and 10 mg/ml leupeptin with a Potter-Elvehjem Teflon glass homogenizer and centrifuged at 1,000 rpm for 10 min. The supernatant was added to Nonidet P-40 to a final concentration of 1%, lysed for 60 min, and then centrifuged at 14,000 rpm for 60 min. The resulting supernatant was used as the crude cytosol of rat brain. All procedures were performed at 4 °C. The GST-NE-dlgΔGK fusion protein (400 μg) was immobilized on GSH-agarose, which was packed into a column. The column was equilibrated with buffer B (30 mm Tris-HCl, pH 7.5, 1 mm EDTA, 5 mm MgCl2, 1 mm dithiothreitol). Bovine brain cytosolic fraction (800 μl) was first precleared by passing it through a GSH column and then was loaded onto the GST-NE-dlgΔGK affinity column. The column was washed with 2 ml of buffer B, and the protein bound to the column was eluted by the addition of 5 ml of buffer C (buffer A containing 0.5m NaCl), and fractions of 1 ml each were collected. The second and third fractions were mixed and loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To purify p51, 4 ml of the cytosol fraction of bovine brain was applied to 2 ml of GST-NE-dlgΔGK immobilized to GSH beads at a half-slurry with phosphate-buffered saline (PBS). Eluates were collected and dialyzed against distilled water. After being concentrated to a 100-μl solution by freeze-drying, the sample was loaded on SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was stained with Ponceau S, and immobilized p51 was cut out from membrane. After being reduced andS-carboxymethylated, p51 was digested byAchromobacter protease I. After sonication, the supernatant was loaded into a C20 chromatography column. Fractionated samples were subjected to amino acid sequencing using the 492 Procise protein sequencing system (Perkin-Elmer). The PCR-based full-length nedasin cDNA cloning was performed as previously reported (14Makino K. Kuwahara H. Masuko N. Nishiyama Y. Morisaki T. Sasaki J. Nakao M. Kuwano A. Nakata M. Ushio Y. Saya H. Oncogene. 1997; 14: 2425-2433Crossref PubMed Scopus (56) Google Scholar). Primers for cloning (A1, 5′-TGATTTGCACATTCAGAGCCATAT-3′; A2, 5′-CTTATACCCCAGTTATAAAAACTA-3′; A3, 5′-CATCACTGTCTTATTTGTCAAAAG-3′; A4, 5′-CACAGATGTGTAGTTTTT ATAACT-3′) were designed based on the sequence information of the EST clone R34820whose open reading frames contained two peptide sequences of the purified p51 protein. For amplification of the 5′ region, the first PCR was performed using only primer A1 to amplify single-strand cDNA from a HeLa cell cDNA library (Marathon-Ready™ cDNA,CLONTECH, Palo Alto, CA). The first PCR product was used as a template in the second run, where the A2 primer and adaptor primer 1 (5′-CCATCCTAATACGACT CACTATAGGGC-3′), based on the sequence of the Marathon cDNA adaptor, were utilized as primers to amplify the 5′ region cDNA. Amplification of the 3′ region was also performed by the same two-step PCR procedure. A3 primer was used for the first-round PCR to amplify the 3′ region, and A4 and adaptor primer 1 were used for the second-round PCR. The PCR fragments were ligated into a pGEM-T Easy cloning vector (Promega, Madison, WI) and sequenced. All PCR procedures were performed by using rTth DNA polymerase (Perkin-Elmer), which has proof-reading activity. The nucleotide sequence was confirmed by sequencing several clones that were generated by independent PCR to avoid errors introduced during the PCR reaction. PCR was performed to detect nedasin sequences in the GeneBridge 4 Radiation Hybrid Screening Panel (Research Genetics, Huntsville, AL) using a set of primers (B1, 5′-ATTGAAGAGGTTTATGTGGGC-3′, and B2, 5′-CAAGGGAGATGCACAACCACGCTA-3′) that were designed based on a partial genomic sequence of thenedasin gene. PCR was carried out as described previously (14Makino K. Kuwahara H. Masuko N. Nishiyama Y. Morisaki T. Sasaki J. Nakao M. Kuwano A. Nakata M. Ushio Y. Saya H. Oncogene. 1997; 14: 2425-2433Crossref PubMed Scopus (56) Google Scholar), and the PCR results were sent to the Whitehead Institute/MIT Center for Genome Research for the mapping of the gene (21Hudson T.J. Stein L.D. Gerety S.S. Ma J. Castle A.B. Silva J. Slonim D.K. Baptista R. Kruglyak L. Xu S.H. Hu X. Colbert A.M.E. Rosenberg C. Reeve-Daly M.P. Rozen S. Hui L. Wu X. Vestergaard C. Wilson K.M. Bae J.S. Maitra S. Ganiatsas S. Evans C.A. DeAngelis M.M. Ingalls K.A. Nahf R.W. Horton Jr., L.T. Anderson M.O. Collymore A.J. Ye W. Kouyoumjian V. Zemsteva I.S. Tam J. Devine R. Courtney D.F. Renaud M.T. Nguyen H. O'Connor T.J. Fizames C. Faure S. Gyapay G. Dib C. Morissette J. Orlin J.B. Birren B.W. Goodman N. Weissenback J. Hawkins T.L. Foote S. Page D.C. Lander E.S. Science. 1995; 270: 1945-1954Crossref PubMed Scopus (728) Google Scholar). A Northern blot derived from various human tissues (CLONTECH) was probed with an 848-bp cDNA fragment of nedasin that had been labeled with [α-32P]dCTP as described previously (14Makino K. Kuwahara H. Masuko N. Nishiyama Y. Morisaki T. Sasaki J. Nakao M. Kuwano A. Nakata M. Ushio Y. Saya H. Oncogene. 1997; 14: 2425-2433Crossref PubMed Scopus (56) Google Scholar). Tissue samples were frozen immediately after surgical resection and stored at −80 °C. Poly(A)+ mRNA was extracted using a Micro Fast Track kit (Invitrogen). First-strand cDNA was synthesized from mRNA with Superscript II reverse transcriptase and random primers. The cDNA was used for the subsequent RT-PCR reaction. Human fetal brain and HeLa cell cDNA libraries (Marathon-Ready™ cDNA) were purchased from CLONTECH. The sequences of the primers for RT-PCR were P1 (5′-ATTGAAGAGGTTTATGTGGGC-3′) and P2 (5′-CAAGGGAGATGCACAACCACGCTA-3′). The PCR reaction was performed in a volume of 15 μl containing 0.5 μl of cDNA, 10 pm primers, 10× PCR mixture, 1.5 μl of 25 mm MgCl2, 1.5 μl of GeneAmp dNTP mixture, and 0.5 units of AmpliTaq Gold™ (Perkin-Elmer) polymerase. The amplification sequence consisted of an initial denaturation at 96 °C/9 min and 40 cycles of 96 °C/30 s, 60 °C/30 s, 72 °C/30 s and a final extension at 72 °C/7 min using the Perkin-Elmer PCR thermocycler 2400. Amplified products (5 μl) were resolved on 2.5% ethidium bromide-stained TBE-agarose gels. An antibody against the C-terminal region of nedasin was raised by the subcutaneous immunization of a rabbit with a synthetic peptide (RNIEEVYVGGKQVVPFSSSV) coupled to the keyhole limpet hemocyanin. Both the S form and V1 form of nedasin were amplified by PCR from the HeLa cDNA library using rTth DNA polymerase and a set of primers, P3 (5′-TGCGCGAATTCGGATCCATGTGTGCCGCTCAGATGCCG-3′) and P4 (5′-AAATAGGATCCAAGCTTAAGGAAATGGTGGAGGATGGGG-3′), and subcloned into pGEM-T Easy vector. The nedasin S and V1 cDNA were then digested with BamHI and ligated into pBj-Myc to construct pBj-Myc/nedasin S or pBj-Myc/nedasin V1. The S and V1 cDNA were also ligated into pCALNL5 vector, which has a Cre-recombinase mediated activation unit (22Kanegae Y. Lee G. Sato Y. Tanaka M. Nakai M. Sakaki T. Sugano S. Saito I. Nucleic Acids Res. 1995; 23: 3816-3821Crossref PubMed Scopus (598) Google Scholar, 23Kanegae Y. Takamori K. Sato Y. Lee G. Nakai M. Saito I. Gene ( Amst. ). 1996; 181: 207-212Crossref PubMed Scopus (102) Google Scholar), to construct pCALNL5/nedasin S or pCALNL5/nedasin V1. The HA-tagged full-length NE-dlg/SAP102-expressing plasmid, pCGN/full-length NE-dlg, was constructed as described previously (14Makino K. Kuwahara H. Masuko N. Nishiyama Y. Morisaki T. Sasaki J. Nakao M. Kuwano A. Nakata M. Ushio Y. Saya H. Oncogene. 1997; 14: 2425-2433Crossref PubMed Scopus (56) Google Scholar). The pCGN/NE-dlgΔGK construct lacking a guanylate kinase domain was obtained by digesting the pCGN/full-length NE-dlg plasmid with BamHI and then self-ligated. The plasmids were transfected into COS-7 cells by the liposome-mediated gene transfer method. Various deletion derivatives of NE-dlg/SAP102 fused to GST or GST immobilized to GSH-agarose beads were incubated with the lysates of COS-7 cells, which were transfected with pBj-Myc/nedasin S or pBj-Myc/nedasin V1, for 2 h at 4 °C. After washing with TNN buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.5% Nonidet P-40) 3 times, the precipitates were probed with the anti-Myc antibody. COS-7 cells transfected with pBj-Myc/nedasin S (or pBj-Myc/nedasin V1) and pCGN/NE-dlg ΔGK were lysed in TNN buffer containing 1 mm sodium orthovanadate, 1 mmaminoethylbenzenesulfonyl fluoride, 10 mm pepstatin, 3% aprotinin, and 10 mg/ml leupeptin for 45 min on ice. Cell lysates were centrifuged at 14,000 rpm for 30 min at 4 °C, and the supernatant was immunoprecipitated with the anti-Myc or HA antibody. The precipitates were then probed with the anti-HA or Myc antibody. Crude cytosolic fraction of adult rat brain was immunoprecipitated with rabbit anti-nedasin antibody, rabbit anti-NE-dlg/SAP102 antibody (Affinity BioReagents, Inc.), or control rabbit IgG. The immunoprecipitates were probed with goat anti-NE-dlg/SAP102 (Santa Cruz Biotechnology, Inc.) or rabbit anti-nedasin antibody. COS-7 cells that were transfected by pBj-Myc/nedasin S or pBj-Myc/nedasin V1 were harvested at 48 h after the transfection and boiled in SDS loading dye. The pCALNL5/nedasin (S or V1), which has the Cre-mediated gene activation unit, was transfected into COS-7 cells. Twenty-four hours after the transfection, the cells were infected with the Cre recombinase producing recombinant adenovirus (AxCANCre) (23Kanegae Y. Takamori K. Sato Y. Lee G. Nakai M. Saito I. Gene ( Amst. ). 1996; 181: 207-212Crossref PubMed Scopus (102) Google Scholar). The cells were harvested and boiled in SDS loading dye at 48 h after the infection. Cell lysates were separated on SDS-PAGE and transferred to a nitrocellulose filter. The filters were probed with an anti-Myc monoclonal antibody (9E10), anti-HA monoclonal antibody (12CA5), anti-GST monoclonal antibody (MBL, Nagoya, Japan), goat anti-NE-dlg/SAP102 antibody, and rabbit anti-nedasin antibody by using the method previously described (14Makino K. Kuwahara H. Masuko N. Nishiyama Y. Morisaki T. Sasaki J. Nakao M. Kuwano A. Nakata M. Ushio Y. Saya H. Oncogene. 1997; 14: 2425-2433Crossref PubMed Scopus (56) Google Scholar). Neonatal rat neuronal cell cultures were prepared according to the method described previously (24Fukunaga K. Soderling T.R. Miyamoto E. J. Biol. Chem. 1992; 267: 22527-22533Abstract Full Text PDF PubMed Google Scholar). Cells were harvested at the indicated days in culture and were subjected to Western blot analysis using the rabbit anti-nedasin antibody and125I-labeled protein A (NEN Life Science Products) for detecting the nedasin protein. Normal human neural progenitor cells were purchased from Clonetics. Normal human neural progenitor cells were cultured and induced differentiation according to the manufacturer's protocol. For the immunofluorescence analysis, cells at 21 days in culture were fixed with 4% paraformaldehyde in PBS for 10 min, followed by permeabilization with 0.2% Triton X-100 in PBS for 5 min. Fixed cells were stained with rabbit anti-nedasin polyclonal antibody (1:250) and goat anti-NE-dlg/SAP102 antibody (1:100). After being washed with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated swine anti-rabbit IgG antibody and Cy3-conjugated donkey anti-goat IgG antibody. After being washed with PBS, samples were mounted in 80% glycerol and visualized with a confocal laser microscope (Fluoview, Olympus) as described previously (16Masuko N. Makino K. Kuwahara H. Fukunaga K. Sudo T. Araki N. Yamamoto H. Yamada Y. Miyamoto E. Saya H. J. Biol. Chem. 1999; 274: 5782-5790Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The interference of the interaction between NE-dlg/SAP102 and NMDA receptor subunit NR2B by nedasin was evaluated by means of surface plasmon resonance responses in BIAcore instrument (BIAcore AB, Uppsala, Sweden) based on the basic principles and detection method as described (25Jonsson U. Fagerstam L. Ivarsson B. Johnsson B. Karlsson R. Lundh K. Lofas S. Persson B. Roos H. Ronnberg I. Sjoelander S. Sternberg E. Stahlberg R. Urbaniczky C. Oestlin H. Malmqvist M. BioTechniques. 1991; 11: 620-627PubMed Google Scholar, 26Marfatia S.M. Cabral J.H. Lin L. Hough C. Bryant P.J. Stolz L. Chishti A.H. J. Cell Biol. 1996; 135: 753-766Crossref PubMed Scopus (80) Google Scholar). The biotinylated peptides PEP7154 (biotin-NGHVYEKLSSIESDV-COOH) corresponding to the C terminus of rat NMDA receptor subunit NR2B and PEP7153 (biotin-NGHVYEKLSSIESD-COOH) for a negative control were supplied by Iwaki Glass Co. (Chiba, Japan). These peptides were immobilized on the flow cell of the sensor tip SA (Amersham Pharmacia Biotech). The indicated concentration of GST-NE-dlg PDZ1+2 and GST-nedasin was mixed and incubated at 4 °C for 2 h and then added to a final volume of 200 μl with HBS buffer (0.01 m HEPES, pH 7.4, 0.15m NaCl, 3 mm EDTA, 0.005% (v/v) surfactant P-20, 1 mm dithiothreitol) and injected at a flow rate of 6 μl/min. The sensorgram of the PEP7153 flow cell was subtracted from that of the PEP7154 flow cell. To identify NE-dlg/SAP102 interacting cytosolic molecules, we loaded bovine brain cytosolic fraction onto a GST-NE-dlgΔGK affinity column. The proteins bound to the affinity columns were eluted by 500 mm NaCl. A protein with a mass of about 51 kDa (p51) was detected in the 500 mm NaCl eluate from the GST-NE-dlgΔGK affinity column but not from the GST affinity column (Fig.1). To clarify the molecular identity of p51, the purified protein was subjected to amino acid sequencing, and 10 peptide sequences derived from p51 were determined. These peptide sequences did not match any previously identified molecules, but two peptide sequences (NLYPSYK and NYTSVYD) were found in one of the open reading frames of a human EST clone, R34820. Based on the sequence of this EST clone, we performed a two-step polymerase chain reaction (PCR) (14Makino K. Kuwahara H. Masuko N. Nishiyama Y. Morisaki T. Sasaki J. Nakao M. Kuwano A. Nakata M. Ushio Y. Saya H. Oncogene. 1997; 14: 2425-2433Crossref PubMed Scopus (56) Google Scholar) to clone the full-length cDNA. We identified a 2,040-bp cDNA that contains one large open reading frame encoding a polypeptide of 454" @default.
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