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• W2005114787 abstract "Slp4-a/granuphilin-a was originally described as a protein specifically associated with insulin-containing granules in pancreatic β-cells, but it was subsequently found to be present on amylase-containing granules in parotid acinar cells. Although Slp4-a has been suggested to control insulin secretion through interaction with syntaxin-1a and/or Munc18-1, nothing is known about the binding partner(s) of Slp4-a during amylase release from parotid acinar cells, which do not endogenously express either syntaxin-1a or Munc18-1. In this study we systematically investigated the interaction between syntaxin-1-5 and Munc18-1-3 by co-immunoprecipitation assay using COS-7 cells and discovered that Slp4-a interacts with a closed conformation of syntaxin-2/3 in a Munc18-2-dependent manner, whereas Munc18-2 itself hardly interacts with Slp4-a at all. By contrast, Slp4-a was found to strongly interact with Munc18-1 regardless of the presence of syntaxin-2/3, and syntaxin-2/3 co-immunoprecipitated with Slp4-a only in the presence of Munc18-1/2. Deletion analysis showed that the syntaxin-2/3 (or Munc18-1/2)-binding site is a linker domain of Slp4-a (amino acid residues 144-354), a previously uncharacterized region located between the N-terminal Rab27A binding domain and the C2A domain. We also found that the Slp4-a·syntaxin-2 complex is actually present in rat parotid glands and that introduction of the antibody against Slp4-a linker domain into streptolysin O-permeabilized parotid acinar cells severely attenuates isoproterenol-stimulated amylase release, possibly by disrupting the interaction between Slp4-a and syntaxin-2/3 (or Munc18-2). These results suggest that Slp4-a modulates amylase release from parotid acinar cells through interaction with syntaxin-2/3 on the apical plasma membrane. Slp4-a/granuphilin-a was originally described as a protein specifically associated with insulin-containing granules in pancreatic β-cells, but it was subsequently found to be present on amylase-containing granules in parotid acinar cells. Although Slp4-a has been suggested to control insulin secretion through interaction with syntaxin-1a and/or Munc18-1, nothing is known about the binding partner(s) of Slp4-a during amylase release from parotid acinar cells, which do not endogenously express either syntaxin-1a or Munc18-1. In this study we systematically investigated the interaction between syntaxin-1-5 and Munc18-1-3 by co-immunoprecipitation assay using COS-7 cells and discovered that Slp4-a interacts with a closed conformation of syntaxin-2/3 in a Munc18-2-dependent manner, whereas Munc18-2 itself hardly interacts with Slp4-a at all. By contrast, Slp4-a was found to strongly interact with Munc18-1 regardless of the presence of syntaxin-2/3, and syntaxin-2/3 co-immunoprecipitated with Slp4-a only in the presence of Munc18-1/2. Deletion analysis showed that the syntaxin-2/3 (or Munc18-1/2)-binding site is a linker domain of Slp4-a (amino acid residues 144-354), a previously uncharacterized region located between the N-terminal Rab27A binding domain and the C2A domain. We also found that the Slp4-a·syntaxin-2 complex is actually present in rat parotid glands and that introduction of the antibody against Slp4-a linker domain into streptolysin O-permeabilized parotid acinar cells severely attenuates isoproterenol-stimulated amylase release, possibly by disrupting the interaction between Slp4-a and syntaxin-2/3 (or Munc18-2). These results suggest that Slp4-a modulates amylase release from parotid acinar cells through interaction with syntaxin-2/3 on the apical plasma membrane. Small GTPase Rab27A is expressed in a wide variety of secretory cells (1Tolmachova T. Anders R. Stinchcombe J. Bossi G. Griffiths G.M. Huxley C. Seabra M.C. Mol. Biol. Cell. 2004; 15: 332-344Crossref PubMed Scopus (137) Google Scholar) and has been suggested to control secretion by these cells through interaction with cell type- or tissue-specific Rab27A effector(s) (for review, see Refs. 2Fukuda M. Recent Res. Dev. Neurochem. 2002; 5: 297-309Google Scholar, 3Izumi T. Gomi H. Kasai K. Mizutani S. Torii S. Cell Struct. Funct. 2003; 28: 465-474Crossref PubMed Scopus (102) Google Scholar, 4Fukuda M. J. Biochem. (Tokyo). 2005; 137: 9-16Crossref PubMed Scopus (188) Google Scholar). To date three distinct groups of Rab27A-binding proteins have been reported in humans and mice (4Fukuda M. J. Biochem. (Tokyo). 2005; 137: 9-16Crossref PubMed Scopus (188) Google Scholar). The first group includes the members of the synaptotagmin-like protein (Slp) 2The abbreviations used are:Slpsynaptotagmin-like proteinGSTglutathione S-transferaseGTPγSguanosine 5′-O-(3-thiotriphosphate)HRPhorseradish peroxidaseIPRisoproterenolSHDSlp homology domainSlac2Slp homologue lacking C2 domainsSLOstreptolysin OHAhemagglutininSNAREsoluble N-ethylmaleimide factor attachment proteinSNAP-25synaptosome-associated protein of 25 kDaVAMP-2vesicle-associated membrane protein 2 2The abbreviations used are:Slpsynaptotagmin-like proteinGSTglutathione S-transferaseGTPγSguanosine 5′-O-(3-thiotriphosphate)HRPhorseradish peroxidaseIPRisoproterenolSHDSlp homology domainSlac2Slp homologue lacking C2 domainsSLOstreptolysin OHAhemagglutininSNAREsoluble N-ethylmaleimide factor attachment proteinSNAP-25synaptosome-associated protein of 25 kDaVAMP-2vesicle-associated membrane protein 2 family (Slp1-5) and rabphilin (5Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (79) Google Scholar, 6Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar, 7Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 8Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. Biochem. Biophys. Res. Commun. 2002; 293: 899-906Crossref PubMed Scopus (63) Google Scholar, 9Yi Z. Yokota H. Torii S. Aoki T. Hosaka M. Zhao S. Takata K. Takeuchi T. Izumi T. Mol. Cell. Biol. 2002; 22: 1858-1867Crossref PubMed Scopus (182) Google Scholar, 10Strom M. Hume A.N. Tarafder A.K. Barkagianni E. Seabra M.C. J. Biol. Chem. 2002; 277: 25423-25430Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 11Fukuda M. Kanno E. Yamamoto A. J. Biol. Chem. 2004; 279: 13065-13075Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 12Fukuda M. J. Biol. Chem. 2003; 278: 15373-15380Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), all of which contain an N-terminal Rab27A binding domain (also called Slp homology domain (SHD)) and C-terminal tandem C2 domains that potentially bind phospholipids (5Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (79) Google Scholar, 13Fukuda M. Biochem. J. 2002; 366: 681-687Crossref PubMed Google Scholar, 14Kuroda T.S. Fukuda M. Nat. Cell Biol. 2004; 6: 1195-1203Crossref PubMed Scopus (131) Google Scholar). The second group of Rab27A-binding proteins includes the members of the Slac2 family (Slac2-a-c) and Noc2, all of which contain an N-terminal Rab27A-binding domain but lack tandem C2 domains (6Fukuda M. Saegusa C. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 283: 513-519Crossref PubMed Scopus (68) Google Scholar, 7Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 11Fukuda M. Kanno E. Yamamoto A. J. Biol. Chem. 2004; 279: 13065-13075Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 15Matesic L.E. Yip R. Reuss A.E. Swing D.A. O'Sullivan T.N. Fletcher C.F. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10238-10243Crossref PubMed Scopus (201) Google Scholar, 16El-Amraoui A. Schonn J.S. Kussel-Andermann P. Blanchard S. Desnos C. Henry J.P. Wolfrum U. Darchen F. Petit C. EMBO Rep. 2002; 3: 463-470Crossref PubMed Scopus (150) Google Scholar, 17Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 18Cheviet S. Coppola T. Haynes L.P. Burgoyne R.D. Regazzi R. Mol. Endocrinol. 2004; 18: 117-126Crossref PubMed Scopus (71) Google Scholar). Both Slac2-a/melanophilin and Slac2-c/MyRIP contain a myosin binding domain at the middle of the molecule (10Strom M. Hume A.N. Tarafder A.K. Barkagianni E. Seabra M.C. J. Biol. Chem. 2002; 277: 25423-25430Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 19Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 20Wu X.S. Rao K. Zhang H. Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer J.A. II I Nat. Cell Biol. 2002; 4: 271-278Crossref PubMed Scopus (380) Google Scholar, 21Nagashima K. Torii S. Yi Z. Igarashi M. Okamoto K. Takeuchi T. Izumi T. FEBS Lett. 2002; 517: 233-238Crossref PubMed Scopus (117) Google Scholar, 22Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (104) Google Scholar, 23Kuroda T.S. Fukuda M. J. Biol. Chem. 2005; 280: 28015-28022Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and an actin binding domain at the C terminus (17Fukuda M. Kuroda T.S. J. Biol. Chem. 2002; 277: 43096-43103Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 22Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (104) Google Scholar). The last Rab27A-binding protein identified is Munc13-4, a putative priming factor for exocytosis (24Shirakawa R. Higashi T. Tabuchi A. Yoshioka A. Nishioka H. Fukuda M. Kita T. Horiuchi H. J. Biol. Chem. 2004; 279: 10730-10737Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 25Neeft M. Wieffer M. de Jong A.S. Negroiu G. Metz C.H. van Loon A. Griffith J. Krijgsveld J. Wulffraat N. Koch H. Heck A.J. Brose N. Kleijmeer M. van der Sluijs P. Mol. Biol. Cell. 2005; 16: 731-741Crossref PubMed Scopus (172) Google Scholar). synaptotagmin-like protein glutathione S-transferase guanosine 5′-O-(3-thiotriphosphate) horseradish peroxidase isoproterenol Slp homology domain Slp homologue lacking C2 domains streptolysin O hemagglutinin soluble N-ethylmaleimide factor attachment protein synaptosome-associated protein of 25 kDa vesicle-associated membrane protein 2 synaptotagmin-like protein glutathione S-transferase guanosine 5′-O-(3-thiotriphosphate) horseradish peroxidase isoproterenol Slp homology domain Slp homologue lacking C2 domains streptolysin O hemagglutinin soluble N-ethylmaleimide factor attachment protein synaptosome-associated protein of 25 kDa vesicle-associated membrane protein 2 Although Rab27A has been shown to be involved in the control of hormone secretion by endocrine cells through interaction with Slp4-a (9Yi Z. Yokota H. Torii S. Aoki T. Hosaka M. Zhao S. Takata K. Takeuchi T. Izumi T. Mol. Cell. Biol. 2002; 22: 1858-1867Crossref PubMed Scopus (182) Google Scholar, 26Torii S. Zhao S. Yi Z. Takeuchi T. Izumi T. Mol. Cell. Biol. 2002; 22: 5518-5526Crossref PubMed Scopus (87) Google Scholar, 27Torii S. Takeuchi T. Nagamatsu S. Izumi T. J. Biol. Chem. 2004; 279: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 28Fukuda M. Kanno E. Saegusa C. Ogata Y. Kuroda T.S. J. Biol. Chem. 2002; 277: 39673-39678Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 29Fukuda M. J. Biol. Chem. 2003; 278: 15390-15396Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), rabphilin (11Fukuda M. Kanno E. Yamamoto A. J. Biol. Chem. 2004; 279: 13065-13075Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), Noc2 (11Fukuda M. Kanno E. Yamamoto A. J. Biol. Chem. 2004; 279: 13065-13075Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 18Cheviet S. Coppola T. Haynes L.P. Burgoyne R.D. Regazzi R. Mol. Endocrinol. 2004; 18: 117-126Crossref PubMed Scopus (71) Google Scholar), and/or Slac2-c (30Waselle L. Coppola T. Fukuda M. Iezzi M. El-Amraoui A. Petit C. Regazzi R. Mol. Biol. Cell. 2003; 14: 4103-4113Crossref PubMed Scopus (133) Google Scholar, 31Desnos C. Schonn J.S. Huet S. Tran V.S. El-Amraoui A. Raposo G. Fanget I. Chapuis C. Ménasché G. de Saint Basile G. Petit C. Cribier S. Henry J.P. Darchen F. J. Cell Biol. 2003; 163: 559-570Crossref PubMed Scopus (133) Google Scholar) and of secretion by certain immune cells through interaction with Munc13-4 (24Shirakawa R. Higashi T. Tabuchi A. Yoshioka A. Nishioka H. Fukuda M. Kita T. Horiuchi H. J. Biol. Chem. 2004; 279: 10730-10737Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 25Neeft M. Wieffer M. de Jong A.S. Negroiu G. Metz C.H. van Loon A. Griffith J. Krijgsveld J. Wulffraat N. Koch H. Heck A.J. Brose N. Kleijmeer M. van der Sluijs P. Mol. Biol. Cell. 2005; 16: 731-741Crossref PubMed Scopus (172) Google Scholar), very little is known about the expression and function of Rab27A effectors in exocrine tissues. We have recently found that Slp4-a and Slac2-c are localized on amylase-containing granules in rat parotid acinar cells and that the latter protein is involved in amylase release from acinar cells through interaction with Rab27B, a closely related isoform of Rab27A, and actin filaments (32Imai A. Yoshie S. Nashida T. Shimomura H. Fukuda M. J. Cell Sci. 2004; 117: 1945-1953Crossref PubMed Scopus (93) Google Scholar). Although Slp4-a has been suggested to promote docking of dense-core vesicles with the plasma membrane of pancreatic β-cell lines through interaction with syntaxin-1a (26Torii S. Zhao S. Yi Z. Takeuchi T. Izumi T. Mol. Cell. Biol. 2002; 22: 5518-5526Crossref PubMed Scopus (87) Google Scholar, 27Torii S. Takeuchi T. Nagamatsu S. Izumi T. J. Biol. Chem. 2004; 279: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and/or Munc18-1 (29Fukuda M. J. Biol. Chem. 2003; 278: 15390-15396Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 33Coppola T. Frantz C. Perret-Menoud V. Gattesco S. Hirling H. Regazzi R. Mol. Biol. Cell. 2002; 13: 1906-1915Crossref PubMed Scopus (114) Google Scholar), Slp4-a must be involved in the control of amylase release in a manner different from its involvement in hormone secretion because neither syntaxin-1a nor Munc18-1 is expressed in rat parotid glands (34Imai A. Nashida T. Shimomura H. Arch. Oral Biol. 2001; 46: 955-962Crossref PubMed Scopus (19) Google Scholar, 35Imai A. Nashida T. Shimomura H. Arch. Biochem. Biophys. 2004; 422: 175-182Crossref PubMed Scopus (35) Google Scholar). Because other syntaxin isoforms (e.g. syntaxin-2-4) and other Munc18 isoforms (e.g. Munc18-2-3) are expressed in the rat parotid gland and have been suggested to control amylase release (34Imai A. Nashida T. Shimomura H. Arch. Oral Biol. 2001; 46: 955-962Crossref PubMed Scopus (19) Google Scholar, 35Imai A. Nashida T. Shimomura H. Arch. Biochem. Biophys. 2004; 422: 175-182Crossref PubMed Scopus (35) Google Scholar, 36Imai A. Nashida T. Yoshie S. Shimomura H. Arch. Oral Biol. 2003; 48: 597-604Crossref PubMed Scopus (39) Google Scholar, 37Takuma T. Arakawa T. Tajima Y. Arch. Oral Biol. 2000; 45: 369-375Crossref PubMed Scopus (28) Google Scholar), Slp4-a may regulate amylase release through interaction with these syntaxins and/or Munc18s. However, these possibilities have never even been investigated in vitro. In this study we systematically investigated the interaction between syntaxin-1-5 and Munc18-1-3 in vitro and found that Slp4-a interacts with syntaxin-2/3 (specifically the closed conformation of syntaxin-2/3) only in the presence of Munc18-2 and not with syntaxin-2/3 alone or Munc18-2 alone. By contrast, Slp4-a interacts with Munc18-1 regardless of the presence of syntaxin-1a (29Fukuda M. J. Biol. Chem. 2003; 278: 15390-15396Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) or syntaxin-2/3. Systematic deletion analysis further showed that syntaxin-2/3 (or Munc18-1/2) binds the linker domain of Slp4-a (amino acid residues 144-354) located between the SHD and the C2A domain. We also found that Slp4-a·syntaxin-2 complex is actually present in rat parotid glands and that introduction of the recombinant linker domain of Slp4-a or the antibody against the Slp4-a linker domain (i.e. anti-Slp4-a-linker antibody) into streptolysin O (SLO)-permeabilized parotid acinar cells severely attenuates isoproterenol (IPR)-stimulated amylase release. Based on these results, we propose that Slp4-a is a novel regulator of the closed conformation of syntaxin-2/3 in amylase release from parotid acinar cells. Materials—Anti-syntaxin-2 rabbit polyclonal antibody was obtained from Synaptic Systems (Göttingen, Germany). Anti-Munc18 and anti-syntaxin-3 rabbit polyclonal antibodies were obtained from Merck Biosciences Calbiochem. Anti-T7 tag antibody-conjugated agarose and horseradish peroxidase (HRP)-conjugated anti-T7 tag mouse monoclonal antibody were from Merck Biosciences Novagen. HRP-conjugated anti-glutathione S-transferase (GST) antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-FLAG M2 affinity gel, HRP-conjugated anti-FLAG M2 mouse monoclonal antibody, and HRP-conjugated anti-HA tag mouse monoclonal antibody were from Sigma. Anti-Rab27B, anti-Slp4-a-C2B, anti-Slp4-a-SHD rabbit polyclonal antibodies were prepared as described previously (28Fukuda M. Kanno E. Saegusa C. Ogata Y. Kuroda T.S. J. Biol. Chem. 2002; 277: 39673-39678Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 32Imai A. Yoshie S. Nashida T. Shimomura H. Fukuda M. J. Cell Sci. 2004; 117: 1945-1953Crossref PubMed Scopus (93) Google Scholar). Plasmid Constructions—cDNAs encoding mouse SNAP-23, syntaxin-2-5, vesicle-associated membrane protein (VAMP)-3/8, and Slp4-b were amplified from Marathon-Ready adult mouse brain cDNA (BD Clontech; Palo Alto, CA) or mouse cerebellum cDNA (38Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar) by PCR using the following pairs of oligonucleotides with a BamHI linker (underlined) or a stop codon (boldface) as described previously (39Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar): 5′-CGGATCCATGGATAATCTGTCATCAGA-3′ (SNAP-23-Met primer, sense) and 5′-TTAGCTGTCAATGAGTTTCT-3′ (SNAP-23-stop primer, antisense); 5′-GCGGATCCATGCGGGACCGGCTGCCCGA-3′ (syntaxin-2-Met primer, sense) and 5′-TCATTTGCCAACCGACAAGC-3′ (syntaxin-2-stop primer, antisense); 5′-GCGGATCCATGAAGGACCGGCTGGAGCA-3′ (syntaxin-3-Met primer, sense) and 5′-TTATTTCAGCCCAACGGACA-3′ (syntaxin-3-stop primer, antisense); 5′-GCGGATCCATGCGCGACAGGACCCACGA-3′ (syntaxin-4-Met primer, sense) and 5′-TTATCCAACGGTTATGGTGA-3′ (syntaxin-4-stop primer, antisense); 5′-GCGGATCCATGTCCTGCCGGGATCGGAC-3′ (syntaxin-5-Met primer, sense) and 5′-TCAGGCAAGGAAGACCACAA-3′ (syntaxin-5-stop primer, antisense); 5′-GCGGATCCATGTCTACAGGTGTGCCTTC-3′ (VAMP-3-Met primer, sense) and 5′-TTAAGAGACACACCACACGA-3′ (VAMP-3-stop primer, antisense); 5′-GCGGATCCATGGAGGAGGCCAGTGGGAG-3′ (VAMP-8-Met primer, sense) and 5′-TTAAGTGGGGATGGTACCAG-3′ (VAMP-8-stop primer, antisense); 5′-CGGATCCATGTCGGAGATACTAGACCT-3′ (Slp4-b-Met primer, sense) and 5′-TTACTTCTTCACCAGTCGAA-3′ (Slp4-b-stop primer, antisense). Purified PCR products were directly inserted into the pGEM-T Easy vector (Promega; Madison, WI) as described previously (39Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). After verification by DNA sequencing, cDNA inserts were transferred to the pEF-FLAG or pEF-T7-tag mammalian expression vectors (modified from pEF-BOS) (38Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 39Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 40Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar, 41Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and the resultant plasmids are referred to as pEF-FLAG-SNAP-23, pEF-FLAG-syntaxin-2-5, pEF-FLAG-VAMP-3/8, and pEF-T7-Slp4-b, respectively. cDNA encoding rat Munc18-2 or Munc18-3 was prepared as described previously (35Imai A. Nashida T. Shimomura H. Arch. Biochem. Biophys. 2004; 422: 175-182Crossref PubMed Scopus (35) Google Scholar) and similarly subcloned into the pEF-FLAG or pEF-T7 tag expression vectors. pEF-FLAG-syntaxin-1a, pEF-FLAG/T7-Munc18-1, pEF-FLAG/T7-Slp4-a, pEF-T7-GST-Slp4-a, pEF-T7-Slp5, and pEF-HA-Rab27A were prepared as described previously (7Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. J. Biol. Chem. 2002; 277: 9212-9218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 8Kuroda T.S. Fukuda M. Ariga H. Mikoshiba K. Biochem. Biophys. Res. Commun. 2002; 293: 899-906Crossref PubMed Scopus (63) Google Scholar, 22Kuroda T.S. Ariga H. Fukuda M. Mol. Cell. Biol. 2003; 23: 5245-5255Crossref PubMed Scopus (104) Google Scholar, 28Fukuda M. Kanno E. Saegusa C. Ogata Y. Kuroda T.S. J. Biol. Chem. 2002; 277: 39673-39678Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 29Fukuda M. J. Biol. Chem. 2003; 278: 15390-15396Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 42Fukuda M. J. Biol. Chem. 2002; 277: 30351-30358Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Plasmid DNA for transfection into mammalian cell cultures was prepared by using Qiagen (Chatsworth, CA) maxiprep kits according to the manufacturer's notes. Construction of Deletion Mutants of Slp4-a and Site-directed Mutagenesis—pEF-T7-Slp4-a-ΔSHD, pEF-T7-Slp4-a-linker, pEF-T7-GST-Slp4-a-linker, pGEX-4T-3-Slp4-a-linker, pEF-T7-Slp4-a-C2AB, pEF-T7-Slp4-a-C2A, and pEF-T7-Slp4-a-C2B were constructed by conventional PCR techniques as described previously (19Fukuda M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 29Fukuda M. J. Biol. Chem. 2003; 278: 15390-15396Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The sequences of the oligonucleotides used are available from the authors upon request. Slp4-a-ΔSHD contains amino acid residues 144-673 of mouse Slp4-a; Slp4-a-linker contains amino acid residues 144-354 of mouse Slp4-a; Slp4-a-C2AB contains amino acid residues 355-673 of mouse Slp4-a; Slp4-a-C2A contains amino acid residues 355-489 of mouse Slp4-a, and Slp4-a-C2B contains amino acid residues 489-673 of mouse Slp4-a. Site-directed mutagenesis and construction of a syntaxin-2/3 point mutant (pEF-FLAG-syntaxin-2/3(L165A/E166A)) were performed by two-step PCR techniques as described previously (43Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) using the following oligonucleotides with a XhoI linker (underlined) and substituted nucleotides (in italics): 5′-CTCGAGCATCTCTGCCGCCTCGTCGTCAGT-3′ (syntaxin-2-L165A/E166A-5′ primer, antisense) and 5′-CTCGAGAGCGGGAAGCCGTC-3′ (syntaxin-2-L165A/E166A-3′ primer, sense) and 5′-CTCGAGCATCTCTGCCGCCTCCTCATC-3′ (syntaxin-3-L165A/E166A-5′ primer, antisense) and 5′-CTCGAGAGTGGCAACCCAGCC-3′ (syntaxin-3-L165A/E166A-3′ primer, sense). Co-immunoprecipitation Assay in COS-7 Cells—COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin at 37 °C under 5% CO2. pEF-T7, pEF-FLAG, and/or pEF-HA vectors (a total of 4 μg of plasmids) were transfected into COS-7 cells (7.5 × 105 cells, the day before transfection/10-cm dish) by using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's notes. Three days after transfection cells were harvested and homogenized, and total cell lysates were prepared as described previously (39Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 41Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 44Fukuda M. Kanno E. Methods Enzymol. 2005; 403: 445-457Crossref PubMed Scopus (39) Google Scholar). The total cell lysates (400 μl) were incubated with either anti-T7-tag antibody-conjugated agarose beads or anti-FLAG M2 affinity gel (wet volume 20 μl) with gentle agitation at 4 °C for 1 h, and the proteins bound to the beads were analyzed by 10% (or 12.5%) SDS-PAGE followed by immunoblotting with HRP-conjugated anti-T7-tag antibody (1/10,000 dilution), HRP-conjugated anti-FLAG M2 antibody (1/10,000 dilution), and/or HRP-conjugated anti-HA tag antibody (1/10,000 dilution) as described previously (39Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The immunoreactive bands were visualized by means of enhanced chemiluminescence (Amersham Biosciences). Direct and Munc18-2-dependent Interaction between Slp4-a and Syntaxin-2—Agarose beads coupled with T7-Slp4-a were prepared as described above. FLAG-Munc18-2 and FLAG-syntaxin-2 were affinity-purified with anti-FLAG M2 affinity gel, and bound proteins were eluted with 0.1 mg/ml FLAG peptide (Sigma). The T7-Slp4-a beads were incubated for 1 h at 4°C with the purified FLAG-Munc18-2 and FLAG-syntaxin-2 in 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, 1 mm MgCl2, 0.2% Triton X-100, and protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, and 10 μm pepstatin A; “protease inhibitors” refers to these three inhibitors throughout the text). After washing 3 times with the binding buffer, the proteins bound to the beads were analyzed by 10% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-FLAG M2 antibody (1/10,000 dilution). Immunoprecipitation and Immunoblotting—Rat parotid glands were homogenized in a buffer containing 5 mm HEPES-NaOH, pH 7.2, 50 mm mannitol, 0.25 mm MgCl2, 25 mm β-mercaptoethanol, 0.1 mm EGTA, 2 μm leupeptin, 2.5 μg/ml trypsin inhibitor, 0.1 mm phenylmethylsulfonyl fluoride, 5 mm benzamidine, and 2 μg/ml aprotinin, and the protein concentrations were adjusted to 10 mg/ml. After solubilization with 1% Triton X-100 in the presence of 0.5 mm GTPγS at 4 °C for 1 h, the insoluble material was removed by centrifugation at 15,000 rpm for 10 min. The supernatant was first incubated at 4 °C for 1 h with 30 μl (wet volume) of protein A-Sepharose beads (Amersham Biosciences) for preabsorption. The resultant supernatant was incubated with anti-Slp4-a-C2B IgG or control rabbit IgG (20 μg/ml) for 1 h at4 °Cand then incubated with 15 μl (wet volume) of protein A-Sepharose beads for 1 h at 4 °C. After washing the beads 5 times with 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, 1 mm MgCl2, 0.2% Triton X-100, and protease inhibitors, the proteins bound to the beads were analyzed by 10% (or 7.5%) SDS-PAGE followed by immunoblotting with anti-syntaxin-2 (1/500 dilution), anti-syntaxin-3 (1/250 dilution), anti-Munc18 (1/250 dilution), anti-Rab27B (6 μg/ml), and anti-Slp4-a-SHD rabbit polyclonal antibodies (2.6 μg/ml). SDS-PAGE and the immunoblot analysis were performed as described previously (32Imai A. Yoshie S. Nashida T. Shimomura H. Fukuda M. J. Cell Sci. 2004; 117: 1945-1953Crossref PubMed Scopus (93) Google Scholar, 39Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The immunoreactive bands were visualized by means of enhanced chemiluminescence. GST Pull-down Assay—T7-GST-Slp4-a-linker was expressed in COS-7 cells as described above, and the expressed GST fusion proteins were affinity-purified on glutathione-Sepharose beads (wet volume 20 μl) as described previously (44Fukuda M. Kanno E. Methods Enzymol. 2005; 403: 445-457Crossref PubMed Scopus (39) Google Scholar). The GST-Slp4-a-linker beads or beads coupled with GST alone as a control were incubated with ∼100 μl of total lysates of rat parotid glands (10 mg/ml; see above). After washing the beads 3 times with 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, 1 mm MgCl2, 0.2% Triton X-100, and protease inhibitors, proteins bound to the beads were analyzed by 10% SDS-PAGE followed by immunoblotting with anti-syntaxin-2, anti-syntaxin-3, anti-Munc18, anti-Rab27B rabbit polyclonal antibody, and HRP-conjugated anti-GST antibody (1/10,000 dilution) as described above. Inhibition of the Slp4-a·Syntaxin-2 Interaction by Anti-Slp4-a Linker IgG in Vitro—GST-Slp4-a-linker was expressed in bacteria and affinity-purified on glutathione-Sepharose beads as described previously (44Fukuda M. Kanno E. Methods Enzymol. 2005; 403: 445-457Crossref PubMed Scopus (39) Google Scholar). New Zealand White rabbits were immunized with the purified GST-Slp4-a-linker proteins, and anti-Slp4-a-linker IgG was affinity-purified as described previously (44Fukuda M. Kanno E. Methods Enzymol. 2005; 403: 445-457Crossref PubMed Scopus (39) Google Scholar). Antibody inhibition experiments in COS-7 cells were performed essentially as described previously (32Imai A. Yoshie S. Nashida T. Shimomura H. Fukuda M. J. Cell Sci. 2004; 117: 1945-1953Crossref PubMed Scopus (93) Google Scholar). In brief, agarose beads coupled with T7-Slp4-a were preincubated for 30 min at 4 °C with 10 μg of anti-Slp4-a-linker IgG or control IgG and then incubated for 1 h at 4 °C with COS-7 cell lysates containing FLAG-Munc18-2 and FLAG-syntaxin-2 (or FLAG-Rab27A). Proteins bound to the beads were analyzed as described above. Preparation of Parotid Acinar Cells and Measurement of A" @default.
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• W2005114787 title "Slp4-a/Granuphilin-a Interacts with Syntaxin-2/3 in a Munc18-2-dependent Manner" @default.
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