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• W2014228231 abstract "The widely expressed Sec/Munc18 (SM) protein Munc18c is required for SNARE-mediated insulin granule exocytosis from islet beta cells and GLUT4 vesicle exocytosis in skeletal muscle and adipocytes. Although Munc18c function is known to involve binding to the t-SNARE Syntaxin 4, a paucity of Munc18c-binding proteins has restricted elucidation of the mechanism by which it facilitates these exocytosis events. Toward this end, we have identified the double C2 domain protein Doc2β as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2β was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2β-Munc18c binding to be direct and complexes to be devoid of Syntaxin 4. Supporting the notion of Munc18c binding with Syntaxin 4 and Doc2β in mutually exclusive complexes, in vitro competition with Syntaxin 4 effectively displaced Munc18c from binding to Doc2β. The second C2 domain (C2B) of Doc2β and an N-terminal region of Munc18c were sufficient to confer complex formation. Disruption of endogenous Munc18c-Doc2β complexes by addition of the Doc2β binding domain of Munc18c (residues 173–255) was found to selectively inhibit glucose-stimulated insulin release. Moreover, increased expression of Doc2β enhanced glucose-stimulated insulin secretion by ∼40%, whereas siRNA-mediated depletion of Doc2β attenuated insulin release. All changes in secretion correlated with parallel alterations in VAMP2 granule docking with Syntaxin 4. Taken together, these data support a model wherein Munc18c transiently switches from association with Syntaxin 4 to association with Doc2β at the plasma membrane to facilitate exocytosis. The widely expressed Sec/Munc18 (SM) protein Munc18c is required for SNARE-mediated insulin granule exocytosis from islet beta cells and GLUT4 vesicle exocytosis in skeletal muscle and adipocytes. Although Munc18c function is known to involve binding to the t-SNARE Syntaxin 4, a paucity of Munc18c-binding proteins has restricted elucidation of the mechanism by which it facilitates these exocytosis events. Toward this end, we have identified the double C2 domain protein Doc2β as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2β was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2β-Munc18c binding to be direct and complexes to be devoid of Syntaxin 4. Supporting the notion of Munc18c binding with Syntaxin 4 and Doc2β in mutually exclusive complexes, in vitro competition with Syntaxin 4 effectively displaced Munc18c from binding to Doc2β. The second C2 domain (C2B) of Doc2β and an N-terminal region of Munc18c were sufficient to confer complex formation. Disruption of endogenous Munc18c-Doc2β complexes by addition of the Doc2β binding domain of Munc18c (residues 173–255) was found to selectively inhibit glucose-stimulated insulin release. Moreover, increased expression of Doc2β enhanced glucose-stimulated insulin secretion by ∼40%, whereas siRNA-mediated depletion of Doc2β attenuated insulin release. All changes in secretion correlated with parallel alterations in VAMP2 granule docking with Syntaxin 4. Taken together, these data support a model wherein Munc18c transiently switches from association with Syntaxin 4 to association with Doc2β at the plasma membrane to facilitate exocytosis. Glucose homeostasis is maintained by a balance of insulin secretion and insulin action. Insulin is secreted from islet beta cells filled with mature insulin-containing granules which traffic to and fuse with the cell surface upon stimulation by elevated blood glucose. Upon detection of insulin and elevated circulating glucose levels by the skeletal muscle and adipose tissues the intracellular vesicles containing the insulin-responsive glucose transporter GLUT4 translocate to the plasma membrane and facilitate glucose uptake into the cell (1Thurmond D.C. Pessin J.E. Mol. Membr. Biol. 2001; 18: 237-245Crossref PubMed Scopus (61) Google Scholar, 2Thurmond D.C. Saltiel J.E. Mechanisms of Insulin Action. Lands Bioscience, Georgetown, TX2006: 52-73Google Scholar). These “vesicle exocytosis” events are known to be regulated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 2The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Gran, granule; GFP, green fluorescence protein; MKRBB, modified Krebs-Ringer bicarbonate buffer; RIA, radioimmunoassay; MOI, multiplicity of infection; PM, plasma membrane; GST, glutathione S-transferase; PVDF, polyvinylidene fluoride; siRNA, small interfering RNA; BSA, bovine serum albumin; VAMP, vesicle-associated membrane protein; SM, Sec/Munc18. proteins (2Thurmond D.C. Saltiel J.E. Mechanisms of Insulin Action. Lands Bioscience, Georgetown, TX2006: 52-73Google Scholar, 3Jahn R. Scheller R.H. Nat. Rev. Mol. Cell. Biol. 2006; 7: 631-643Crossref PubMed Scopus (1942) Google Scholar). Vesicle exocytosis entails the specific pairing of a vesicle-associated membrane protein v-SNARE (VAMP) with a binary cognate receptor complex t-SNARE composed of SNAP-25/23 and Syntaxin proteins at the target membrane to form the SNARE core complex (3Jahn R. Scheller R.H. Nat. Rev. Mol. Cell. Biol. 2006; 7: 631-643Crossref PubMed Scopus (1942) Google Scholar, 4Rizo J. Sudhof T.C. Nat. Rev. Neurosci. 2002; 3: 641-653Crossref PubMed Scopus (436) Google Scholar). SNARE protein functions are further regulated by interaction with the Sec1/Munc18 (SM) secretory proteins, of which three plasma membrane-localized homologues exist in mammalian cells: Munc18a, Munc18b, and Munc18c (5Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2003-2007Crossref PubMed Scopus (224) Google Scholar, 6Tellam J.T. McIntosh S. James D.E. J. Biol. Chem. 1995; 270: 5857-5863Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). However among these, only the Munc18c isoform can bind with and regulate the t-SNARE protein Syntaxin 4 (7Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 8Tamori Y. Kawanishi M. Niki T. Shinoda H. Araki S. Okazawa H. Kasuga M. J. Biol. Chem. 1998; 273: 19740-19746Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and Syntaxin 4 is the singular functional Syntaxin isoform in insulin-stimulated GLUT4 vesicle exocytosis (9Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 10Yang C. Coker K.J. Kim J.K. Mora S. Thurmond D.C. Davis A.C. Yang B. Williamson R.A. Shulman G.I. Pessin J.E. J. Clin. Investig. 2001; 107: 1311-1318Crossref PubMed Scopus (97) Google Scholar, 11Oh E. Spurlin B.A. Pessin J.E. Thurmond D.C. Diabetes. 2005; 54: 638-647Crossref PubMed Scopus (69) Google Scholar) and one of two functional Syntaxin isoforms identified in glucose-stimulated insulin exocytosis (12Volchuk A. Wang Q. Ewart H.S. Liu Z. He L. Bennett M.K. Klip A. Mol. Biol. Cell. 1996; 7: 1075-1082Crossref PubMed Scopus (126) Google Scholar, 13Saito T. Okada S. Yamada E. Ohshima K. Shimizu H. Shimomura K. Sato M. Pessin J.E. Mori M. J. Biol. Chem. 2003; 278: 36718-36725Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 14Spurlin B.A. Park S.Y. Nevins A.K. Kim J.K. Thurmond D.C. Diabetes. 2004; 53: 2223-2231Crossref PubMed Scopus (51) Google Scholar, 15Spurlin B.A. Thurmond D.C. Mol. Endocrinol. 2006; 20: 183-193Crossref PubMed Scopus (74) Google Scholar). Whereas we and others (11Oh E. Spurlin B.A. Pessin J.E. Thurmond D.C. Diabetes. 2005; 54: 638-647Crossref PubMed Scopus (69) Google Scholar, 16Spurlin B.A. Thomas R.M. Nevins A.K. Kim H.J. Noh H.J. Kim J.A. Shulman G.I. Thurmond D.C. Diabetes. 2003; 52: 1910-1917Crossref PubMed Scopus (38) Google Scholar) have shown that either depletion or overexpression of Munc18c in vivo dramatically alters glucose homeostasis via disruption of skeletal muscle GLUT4 translocation and pancreatic islet function, the mechanism by which Munc18c regulates these particular exocytotic events remains unknown. Crystallographic and NMR studies support the concept that the Munc18 protein may keep its cognate Syntaxin in a “closed” conformation (17Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (553) Google Scholar, 18Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (617) Google Scholar, 19Yang B. Steegmaier M. Gonzalez Jr., L.C. Scheller R.H. J. Cell Biol. 2000; 148: 247-252Crossref PubMed Scopus (228) Google Scholar). Very recent studies further suggest that the Munc18 protein assists the transition of Syntaxin to the open state, possibly by stabilizing a labile transition half-open state of Syntaxin (20Zilly F.E. Sorensen J.B. Jahn R. Lang T. PLoS Biol. 2006; 4: e330Crossref PubMed Scopus (98) Google Scholar, 21Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Previous studies of Munc18c-Syntaxin 4 kinetics in 3T3L1 adipocytes are consistent with this model (9Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 22Araki S. Tamori Y. Kawanishi M. Shinoda H. Masugi J. Mori H. Niki T. Okazawa H. Kubota T. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 234: 257-262Crossref PubMed Scopus (85) Google Scholar). In addition, we have recently demonstrated that Munc18c becomes tyrosine-phosphorylated and transiently dissociates from Syntaxin 4 upon stimulation in both islet beta cells and in 3T3L1 adipocytes (23Oh E. Thurmond D.C. J. Biol. Chem. 2006; 281: 17624-17634Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and this dissociation was correlated with increased SNARE docking. However, it has recently been demonstrated that in vitro, Munc18c can associate with the heterotrimeric SNARE core complex, with VAMP2 included (24Latham C.F. Lopez J.A. Hu S.H. Gee C.L. Westbury E. Blair D.H. Armishaw C.J. Alewood P.F. Bryant N.J. James D.E. Martin J.L. Traffic. 2006; 7: 1408-1419Crossref PubMed Scopus (100) Google Scholar). These data suggest that a cellular factor may be required to mediate the transient displacement of Munc18c. There is precedence for the existence of proteins that bind and impact SM-Syntaxin complexes. For example, the yeast protein Ypt1p, a Rab GTPase, binds the Sly1p-Sed5p complex, yeast SM-Syntaxin homologues (25Grabowski R. Gallwitz D. FEBS Lett. 1997; 411: 169-172Crossref PubMed Scopus (45) Google Scholar). In neurons, the SM protein Munc18-1 has several known binding proteins other than its cognate Syntaxin, several of which are C2 domain-containing proteins (26Tsuboi T. Fukuda M. Mol. Biol. Cell. 2006; 17: 2101-2112Crossref PubMed Scopus (62) Google Scholar, 27Verhage M. de Vries K.J. Roshol H. Burbach J.P. Gispen W.H. Sudhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 28Okamoto M. Sudhof T.C. J. Biol. Chem. 1997; 272: 31459-31464Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). One of these is Doc2β, which was shown to bind directly to Munc18-1 (27Verhage M. de Vries K.J. Roshol H. Burbach J.P. Gispen W.H. Sudhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Doc2β binds to a range of ligands including calcium, phospholipids, and intracellular proteins. Whereas a second isoform, Doc2α, is found primarily in brain/neuronal tissue, Doc2β is more widely expressed (27Verhage M. de Vries K.J. Roshol H. Burbach J.P. Gispen W.H. Sudhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 29Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. (Tokyo). 1996; 120: 671-676Crossref PubMed Scopus (45) Google Scholar, 30Duncan R.R. Shipston M.J. Chow R.H. Biochimie (Paris). 2000; 82: 421-426Crossref PubMed Scopus (40) Google Scholar, 31Sakaguchi G. Orita S. Maeda M. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 217: 1053-1061Crossref PubMed Scopus (62) Google Scholar, 32Orita S. Naito A. Sakaguchi G. Maeda M. Igarashi H. Sasaki T. Takai Y. J. Biol. Chem. 1997; 272: 16081-16084Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In this report we demonstrate that Doc2β is expressed in insulin-secreting beta cells as well as insulin-responsive adipocytes, in which it associates with Munc18c in a manner mutually exclusive of Munc18c's other known binding partner Syntaxin 4. Our results delineate binding domains of each protein sufficient to confer the interaction and further show that endogenous Munc18c-Doc2β association is functionally important for glucose-stimulated insulin secretion. Loss of association was coupled to decreased SNARE docking (Syntaxin 4 association with VAMP2 granules), whereas increased association or Doc2β expression was correlated with increased SNARE docking. In vitro studies revealed that Syntaxin 4 could displace Munc18c from preformed Munc18c-Doc2β complexes in a dose-dependent fashion, altogether supportive of a model in which Munc18c switches binding partners from Syntaxin 4 to Doc2β at the plasma membrane to promote exocytosis. Materials—Rabbit anti-Munc18c and anti-GLUT4 antibodies were generated as previously described (9Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The rabbit polyclonal anti-Syntaxin 4 and mouse monoclonal anti-VAMP2 antibodies were obtained from Chemicon (Temecula, CA) and Synaptic System (Gottingen, Germany), respectively. The rabbit polyclonal anti-Doc2 antibody was a kind gift from Dr. Matthijs Verhage (Vrije Universiteit, Netherlands). FLAG and clathrin antibodies were obtained from Sigma and BD Transduction Labs (Franklin Lakes, NJ), respectively. Rabbit and mouse anti-GFP antibodies were acquired from Abcam (Cambridge, MA) and Clontech Laboratories (Mountain View, CA), respectively. Rabbit polyclonal GFP and Syntaxin 1A antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). The monoclonal anti-Myc (9E10) antibody and protein G plus agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), respectively. MIN6 cells were a gift from Dr. John Hutton (University of Colorado Health Sciences Center). Goat anti-mouse and anti-rabbit horseradish peroxidase secondary antibodies and transfectin lipid reagent were acquired from Bio-Rad. Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA). Radioimmunoassay (RIA) grade bovine serum albumin and d-glucose were purchased from Sigma. Enhanced chemiluminescence reagent and Hyperfilm-MP were obtained from Amersham Biosciences. The human C-peptide and rat insulin RIA kits were purchased from Linco Research Inc (St. Charles, MO). Doc2β siRNA oligonucleotides were purchased from Ambion (Austin, TX): siDoc2 number 2-GGCAAAUAAGCUCAGAACAtt; siDoc2 number 1-UCAUCACACACGGAGAUCCtc. Plasmids—The pcDNA3.1-Doc2β-myc DNA construct was generated by subcloning a PCR-generated mouse Doc2β fragment into the XhoI and HindIII sites of the pcDNA3.1/myc-His(-) vector (Invitrogen). The pBluescriptIIKS(-)-Doc2β (a kind gift from Dr. Mitsunori Fukuda) was used as the template in a GC-rich PCR reaction system (Roche Applied Science) using the following primers: forward (5′-AGACTCGAGGCCTGCATGACCCTC) and reverse (5′-AGAAAGCTTGGTCGCTGAGTAC). GST-Doc2β fusion protein constructs pGEX-2T mouse Doc2β-C2A (amino acids 123–257), pGEX-2T mouse Doc2β-C2B (amino acids 257–375), pGEX-2T mouse Doc2β-C2AB (amino acids 123–375) were gifts from Dr. Mitsunori Fukuda. The pGEX-4T3-Doc2β plasmid was a gift from Dr. Alexander Groffen (Vrije Universiteit, The Netherlands). pET-28a(+)-His-Doc2β construct was made by subcloning a PCR-generated mouse Doc2β fragment into the BamHI and XhoI sites of the pET-28a(+) vector (Novagen, San Diego, CA). The Munc18c-GFP deletion constructs were generated as previously described (23Oh E. Thurmond D.C. J. Biol. Chem. 2006; 281: 17624-17634Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). An additional deletion construct, Munc18c-(173–255)-GFP was generated by subcloning a PCR-generated fragment of the 173–255 region into the BamHI and EcoRI sites of the pEGFP-N3 vector (Clontech), using the following primers: forward (5′-AGAGGATCCATGGAGGCAATGGCT), reverse (5′-AGAGAATTCTCATGCCTGAAAGGTCA). The pET-28a(+)-His-Munc18c construct was made by subcloning a PCR-generated full-length Munc18c fragment, using pcDNA3.1-Munc18c DNA as template (9Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), engineered with a 5′ NheI site and a 3′ EcoRI site for insertion into like sites present in the multiple cloning region of the pET28a(+) vector. The pAd5CMV-FLAG-Munc18c was generated by subcloning FLAG-Munc18c fragment excised from pcDNA3.1-FLAG-Munc18c (33Thurmond D.C. Pessin J.E. EMBO J. 2000; 19: 3565-3575Crossref PubMed Scopus (52) Google Scholar) using SpeI for insertion into SpeI-cut pAd5CMV vector. All constructs were verified by DNA sequencing. Cell Culture, Transient Transfection, and Secretion Assays—MIN6 beta cells were cultured in Dulbecco's modified Eagle's medium (DMEM with 25 mm glucose) supplemented with 15% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 292 μg/ml l-glutamine, and 50 μm β-mercaptoethanol as described previously (15Spurlin B.A. Thurmond D.C. Mol. Endocrinol. 2006; 20: 183-193Crossref PubMed Scopus (74) Google Scholar). MIN6 beta cells at 50–60% confluence were transfected with 40 μg of plasmid DNA per 10 cm2 dish using transfectin (Bio-Rad) to obtain ∼30–50% transfection efficiency. After 48 h of incubation, cells were washed twice with and incubated for 2 h in freshly prepared modified Krebs-Ringer bicarbonate buffer (MKRBB: 5 mm KCl, 120 mm NaCl, 15 mm Hepes pH 7.4, 24 mm NaHCO3, 1 mm MgCl2, 2 mm CaCl2, and 1 mg/ml BSA). Cells were stimulated with 20 mm glucose or 50 mm KCl for the times indicated in the figures. Cells were subsequently lysed in Nonidet P-40 lysis buffer (25 mm Tris, pH 7.4, 1% Nonidet P-40, 10% glycerol, 50 μm sodium fluoride, 10 mm sodium pyrophosphate, 137 mm sodium chloride, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin, and 5 μg/ml leupeptin), and lysates were cleared by microcentrifugation for 10 min at 4 °C for subsequent use in co-immunoprecipitation experiments. For measurement of human C-peptide release, MIN6 beta cells were transiently co-transfected with each plasmid plus human proinsulin cDNA (kind gift from Dr. Chris Newgard, Duke University), using transfectin with 2 μg of each DNA per 35-mm dish of cells at 50% confluence. 48 h following transfection, cells were preincubated for 2 h in MKRBB buffer and stimulated with 20 mm glucose for 1 h. MKRBB was collected for quantitation of human C-peptide released. Transfection of siRNA oligonucleotides into MIN6 cells was achieved using Lipofectamine 2000 (Invitrogen) with 100 nm oligonucleotides to obtain ∼70–80% transfection efficiency. A non-targeting RNA (scrambled siRNA, obtained from Ambion) was included as a control in parallel experiments. Transfected cells were maintained in supplemented DMEM for 48 h, starved in MKRBB and stimulated as described above, and insulin-secreted into the MKRBB quantitated by RIA. Cells were harvested in 1% Nonidet P-40 lysis buffer for detecting Doc2β depletion. CHO-K1 cells were purchased from the American Type Culture collection (Manassas, VA) and cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 292 μg/ml l-glutamine. At 80–90% confluence, cells were electroporated with 40 μg of DNA as previously described (9Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). After 48 h of incubation, cells were harvested in 1% Nonidet P-40 lysis buffer and lysates cleared by centrifugation at 14,000 × g for 10 min at 4 °C for subsequent use in co-immunoprecipitation experiments. Subcellular Fractionation—Subcellular fractions of beta cells were isolated as described previously (34Nevins A.K. Thurmond D.C. J. Biol. Chem. 2005; 280: 1944-1952Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Briefly, MIN6 beta cells at 80–90% confluence were harvested into 1 ml of homogenization buffer (20 mm Tris-HCl, pH 7.4, 0.5 mm EDTA, 0.5 mm EGTA, 250 mm sucrose, 1 mm dithiothreitol, and 1 mm sodium orthovanadate containing the protease inhibitors leupeptin (10 μg/ml), aprotinin (4 μg/ml), pepstatin (2 μg/ml), and phenylmethylsulfonyl fluoride (100 μm). Cells were disrupted by 10 strokes through a 27-gauge needle, and homogenates were centrifuged at 900 × g for 10 min. Postnuclear supernatants were centrifuged at 5,500 × g for 15 min, and the subsequent supernatant centrifuged at 25,000 × g for 20 min to obtain the secretory granule fraction in the pellet. The supernatant was further centrifuged at 100,000 × g for 1 h to obtain the cytosolic fraction. Plasma membrane fractions (PM) were obtained by mixing the postnuclear pellet with 1 ml of Buffer A (0.25 m sucrose, 1 mm MgCl2, and 10 mm Tris-HCl, pH 7.4) and 2 volumes of Buffer B (2 m sucrose, 1 mm MgCl2, and 10 mm Tris-HCl, pH 7.4). The mixture was overlaid with Buffer A and centrifuged at 113,000 × g for 1 h to obtain an interface containing the plasma membrane fraction. The interface was collected and diluted to 2 ml with homogenization buffer for centrifugation at 6,000 × g for 10 min, and the resulting pellet was collected as the plasma membrane fraction. All pellets were resuspended in 1% Nonidet P-40 lysis buffer to solubilize membrane proteins. Subcellular fractions of 3T3L1 adipocytes were obtained using the differential centrifugation method as described previously (9Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Briefly, 3T3L1 adipocytes were washed with and resuspended in HES buffer (20 mm HEPES pH 7.4, 1 mm EDTA, and 255 mm sucrose containing 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 5 μg/ml leupeptin). Lysates were sheared 10 times through a 22-gauge needle and centrifuged at 19,000 × g for 20 min at 4 °C. The low speed (HDM) fraction was obtained by centrifugation of the resulting supernatant at 41,000 × g for 20 min at 4 °C. The supernatant was removed and centrifuged at 180,000 × g for 75 min at 4 °C to generate the high speed (LDM) fraction. The plasma membrane fraction (PM) was obtained by resuspending the pellet from the initial 19,000 × g centrifugation in HES buffer followed by layering onto a 1.12 m sucrose cushion for centrifugation at 100,000 × g for 60 min. The plasma membrane layer was then removed from the cushion and centrifuged at 40,000 × g for 20 min, and that pellet then resuspended in HES buffer. Co-immunoprecipitation and Immunoblotting—MIN6 beta cells were preincubated in MKRBB for 2 h followed by glucose stimulation. Cells were subsequently lysed in Nonidet P-40 lysis buffer. MIN6 beta cell-cleared detergent homogenates (2–3 mg) were combined with rabbit anti-Munc18c antibody, rabbit anti-Syntaxin4 antibody, or rabbit anti-Doc2β antibody for 2 h at 4 °C followed by a second incubation with protein G Plus-agarose for 2 h. The resultant immunoprecipitates were subjected to 10% SDS-PAGE followed by transfer to PVDF membranes for immunoblotting. Munc18c, Syntaxin 4, and Doc2β antibodies were used at 1:5000, 1:500, and 1:1000 dilutions, respectively, and secondary antibodies conjugated to horseradish peroxidase were diluted at 1:5000 for visualization by chemiluminescence. Immunoprecipitations using CHO-K1 detergent-cleared cell lysates were performed similar to that of the MIN6 cell lysates. Recombinant Proteins and Interaction Assays—GST-Doc2β, GST-Doc2β-C2AB, GST-Doc2β-C2A, GST-Doc2β-C2B, and GST-Syntaxin 4 fusion proteins were expressed in Escherichia coli and purified by glutathione-agarose affinity chromatography as described previously (35Min J. Okada S. Kanzaki M. Elmendorf J.S. Coker K.J. Ceresa B.P. Syu L.J. Noda Y. Saltiel A.R. Pessin J.E. Mol. Cell. 1999; 3: 751-760Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Recombinant Syntaxin 4 and Doc2β proteins were obtained following thrombin cleavage of GST-Syntaxin 4, GST-Doc2β, respectively. Syntaxin 1A protein was purchased from Synaptic Systems. BSA control protein was purchased from Pierce. Recombinant His-tagged Munc18c was also expressed in E. coli and purified by Ni-NTA nickel-chelating resin (Invitrogen) under native conditions (50 mm NaH2PO4, 0.5 m NaCl, pH 8). Eluted protein was further dialyzed overnight in 50 mm Tris, pH 8, supplemented with 1 mm dithiothreitol. The interaction of GST-Doc2β with His-Munc18c was performed by incubating 2 μg of either GST-Doc2β-C2AB, GST-Doc2β-C2A, GST-Doc2β-C2B linked to Sepharose beads with 2 μg of recombinant His-Munc18c protein in Nonidet P-40 lysis buffer for 2 h at 4 °C. Following three washes with lysis buffer, proteins were eluted from the Sepharose beads and subjected to 10% SDS-PAGE followed by transfer to PVDF membrane for immunoblotting. Adenoviral Transduction of MIN6 Cells—MIN6 cells at 60% confluence were transduced with pAd5CMV-Munc18c CsCl-purified particles (generated by the University of Iowa Gene Targeting Vector Core, Iowa City, IA) for 2 h at 37 °C (MOI = 100). Transduced cells were then washed twice with phosphate-buffered saline and incubated for 48 h in complete medium at 37 °C, 5% CO2. Transduced cells were subsequently preincubated in MKRBB for 2 h and stimulated with 20 mm d-glucose for 5 min. Cleared detergent lysates were prepared as described above for the GST pull-down assay. Statistical Analysis—All data are expressed as mean ± S.E. Data were evaluated for statistical significance using the Student's t test. Doc2β and Munc18c Associate in MIN6 Beta Cells—Doc2β is considered to be a ubiquitously expressed protein, enriched in brain, heart, and lung tissues (27Verhage M. de Vries K.J. Roshol H. Burbach J.P. Gispen W.H. Sudhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 31Sakaguchi G. Orita S. Maeda M. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 217: 1053-1061Crossref PubMed Scopus (62) Google Scholar), but expression in adipocytes and islet cells has not yet been established. To address this, tissue and cultured cell lysates were used for immunoblotting to detect the presence of Doc2β protein using a previously validated antibody provided by Dr. Matthias Verhage. The Doc2β antibody recognized a single 45-kDa band in both human and mouse islet lysates, MIN6 beta cell lysate and recombinant purified full-length Doc2β protein (Fig. 1A). Moreover, of the three subcellular fractions of MIN6 beta cells examined, Doc2β was almost exclusively localized to the PM fraction (Fig. 1A, lane 4), and was nearly undetectable in granule and cytosolic fractions (data not shown). The purity of the fractions was validated by the presence of the PM protein Syntaxin 1A exclusively in the plasma membrane fraction (Fig. 1A) and by insulin content as we have documented previously (15Spurlin B.A. Thurmond D.C. Mol. Endocrinol. 2006; 20: 183-193Crossref PubMed Scopus (74) Google Scholar, 34Nevins A.K. Thurmond D.C. J. Biol. Chem. 2005; 280: 1944-1952Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In 3T3L1 adipocytes, Doc2β was also primarily localized to the PM fraction (Fig. 1B), with very little present in intracellular vesicle fractions (LDM and HDM). Subcellular fractions prepared from insulin-stimulated 3T3L1 adipocytes were validated by detection of GLUT4 protein in PM, LDM, and HDM fractions and detection of Syntaxin 4 protein principally in the PM fraction. RT-PCR was also used to confirm the presence of Doc2β mRNA in the MIN6 cells (data not shown). Thus, Doc2β and Munc18c both localize to the PM fraction in cell types where Munc18c-Syntaxin 4 complexes are known to be important for regulated exocytotic events. To next determine whether Doc2β could bind to Munc18c, Doc2β was expressed as a GST fusion protein in E. coli and attached to beads as bait for precipitating interacting proteins. To increase the abundance of Munc18c and enhance detection of a binding event, MIN6 lysates prepared from cells transduced to express recombinant FLAG-tagged Munc18c were initially used. Both anti-FLAG and anti-Munc18c antibodies detected Munc18c in precipitates with GST-Doc2β but not GST control (Fig. 2A). Coimmunoprecipitation was used as an independent approach to confirm this interaction. MIN6 cell lysates prepared from cells transfected to express recombinant Myc-tagged Doc2β were immunoprecipitated with anti-Myc antibody or with the vector control (pcDNA3.1-myc-his). As shown in Fig. 2B, Doc2β-myc was able to co-precipitate endogenous Munc18c, whereas the vector control had no effect. Finally, to determine if endogenous Doc2β-Munc" @default.
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• W2014228231 title "Doc2β Is a Novel Munc18c-interacting Partner and Positive Effector of Syntaxin 4-mediated Exocytosis" @default.
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