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Isoproterenol Stimulates Transient SNAP23–VAMP2 Interaction in Rat

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Isoproterenol Stimulates Transient SNAP23–VAMP2 Interaction in Rat View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 587 (2013) 583–589 journal homepage: www.FEBSLetters.org Isoproterenol stimulates transient SNAP23–VAMP2 interaction in rat parotid glands ⇑ Taishin Takuma a, , Akiko Shitara a, Toshiya Arakawa a, Miki Okayama b, Itaru Mizoguchi b, Yoshifumi Tajima c a Department of Biochemistry, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-0293, Japan b Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-0293, Japan c Department of Oral Pathology, School of Dentistry, Meikai University, Sakado, Saitama 350-0283, Japan article info abstract Article history: The exocytosis of salivary proteins is mainly regulated by cAMP, although soluble N-ethylmalei- Received 11 December 2012 mide-sensitive factor attachment protein receptors (SNAREs), which mediate cAMP-dependent exo- Revised 10 January 2013 cytic membrane fusion, have remained unidentified. Here we examined the effect of isoproterenol Accepted 20 January 2013 (ISO) and cytochalasin D (CyD) on the level of SNARE complexes in rat parotid glands. When SNARE Available online 1 February 2013 complexes were immunoprecipitated by anti-SNAP23, the coprecipitation of VAMP2 was signifi- Edited by Gianni Cesareni cantly increased in response to ISO and/or CyD, although the coprecipitation of VAMP8 or syntaxin 4 was scarcely augmented. These results suggest that the SNAP23–VAMP2 interaction plays a key role in cAMP-mediated exocytosis from parotid glands. Keywords: Parotid gland Exocytosis Structured summary of protein interactions: Soluble N-ethylmaleimide-sensitive factor Snap23 physically interacts with Vamp8, Vamp3 and Syn-4 by anti bait coimmunoprecipitation (View attachment protein receptor (SNARE) Interaction: 1, 2, 3) VAMP2 Vamp2 physically interacts with Syn-4, Syn-3 and Snap23 by anti bait coimmunoprecipitation (View SNAP23 interaction) Cytochalasin D Syn-3 physically interacts with Snap23 by anti bait coimmunoprecipitation (View interaction) Vamp2 physically interacts with Snap23 and Syn-4 by anti bait coimmunoprecipitation (View interac- tion) Snap23 physically interacts with Vamp8, Syn-4 and Vamp2 by anti bait coimmunoprecipitation (View interaction: 1, 2) Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction However, the mechanism of cAMP-induced exocytic membrane fu- sion is totally unknown. The salivary glands are unique exocrine glands that secrete var- The regulated exocytosis is a spatio-temporal enhancement of ious salivary proteins mainly through a cyclic AMP-mediated reg- membrane fusion between the plasma membrane and the mem- ulatory pathway [1,2]. Exocytosis of salivary proteins, including brane of secretory granules that are produced and pooled in ad- amylase and mucins, is evoked by various agonists that elevate vance. Intracellular membrane fusion is widely recognized to be the intracellular cAMP level in intact acinar cells [1,2], and also mediated by soluble N-ethylmaleimide-sensitive factor attachment by exogenously added cAMP analogs [3,4] and by the catalytic sub- protein receptors (SNAREs) and a variety of their associated proteins unit of cAMP-dependent protein kinase (PKA) [5] in permeabilized [7,8]. In general a set of cognate SNARE proteins, which reside either acinar cells. Cyclic AMP in the salivary glands triggers 2 separate on the membrane of transporting vesicles (v-SNAREs) or on target signaling pathways, one involving PKA and the other Epac [6]; membranes (t-SNAREs), makes a four-helix-bundle (3Q + 1R) and the PKA pathway is so far recognized to be the main route. structure that is composed of 3 Q-SNARE motifs (Qa, Qb, Qc) on 2 or 3 t-SNAREs and 1 R-SNARE motif on a v-SNARE, when the two Abbreviations: SNARE, soluble N-ethylmaleimide-sensitive factor attachment membranes dock and fuse. Gene knockout studies have firmly protein receptor; ISO, isoproterenol; CyD, cytochalasin D; GFP, green fluorescent established that several SNAREs are essential for certain regulated protein; VAMP, vesicle-associated membrane protein secretory pathways, especially in the case of synaptic exocytosis ⇑ Corresponding author. Fax: +81 1332 3 1391. for neurotransmitter release [9–13]. Salivary glands contain most E-mail address: [email protected] (T. Takuma). 0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.01.039 584 T. Takuma et al. / FEBS Letters 587 (2013) 583–589 SNARE proteins [14–16], although the quantity of each SNARE in the and/or ISO, the tissue suspensions were first divided into four glands is much less than that in the brain. Previous studies using parts, and then incubated in the presence or absence of 10 lM clostridial neurotoxins and specific antibodies against Rabs and CyD for 5 min followed by incubation with or without 10 lM ISO Rab-associated proteins, those of which were introduced into for 25 min, both at 37 °C. permeabilized parotid acinar cells, suggested that some proteins, including VAMP2 and Rab27B, are involved in the exocytic process 2.3. Immunoprecipitation and immunoblotting [14,17]. Furthermore, gene knockout studies clearly established that VAMP8 is one of major v-SNAREs in exocrine glands, including the After the incubation, the parotid minces were pelleted and pancreas, salivary, and lacrimal glands; although salivary protein homogenized in a motor-driven Teflon-glass homogenizer with secretion stimulated by pilocarpine is incompletely blocked in 1 ml lysis buffer containing 1% Nonidet P-40, 10 mM Tris–HCl (pH VAMP8-null mice [12,13]. These findings suggest that several 7.4), 150 mM NaCl, 10 mM NaF, 5 mM EDTA, and protease inhibitor combinations of SNARE proteins may produce SNARE complexes cocktail (Complete Mini, Rosche). The homogenates were centri- that mediate exocytic membrane fusion in salivary glands. fuged at 15000 rpm for 15 min at 4 °C, and the resulting superna- We previously isolated a binary t-SNARE complex composed of tants were used for subsequent immunoprecipitation. The SNAP23 and syntaxin 4 from the lysate of rat parotid acinar cells, supernatants were pre-cleared by rotation at 4 °C for 30 min with but failed to detect SNARE complexes that contained both v- and 30 ll of anti-mouse or anti-rabbit IgG-agarose beads (Sigma) to re- t-SNAREs [18]. Then plausible ternary SNARE complexes contain- move non-specific binding to the agarose beads, and centrifuged at ing syntaxin 4, SNAP23, and VAMP2 were isolated, when FLAG- 5000 rpm for 10 s; then the resulting supernatants were further ro- tagged and GFP-tagged SNARE proteins were overexpressed in tated at 4 °C for 3 h with 2 lg of monoclonal anti-VAMP2 antibody COS-7 cells [19]; although the interaction of SNARE proteins is (Synaptic Systems) or polyclonal anti-SNAP23 antibody (abcam) known to be highly promiscuous under artificial experimental con- and 30 ll of anti-mouse or anti-rabbit IgG-agarose beads (Sigma). ditions [7,8]. In a recent study we succeeded to immunoprecipitate The agarose beads were washed five times with PBS containing endogenous SNARE complexes containing both v- and t-SNAREs 1% Triton X-100 and two times with PBS alone. SNARE complexes from HeLa cells [20]. Thus in this study we tried again to detect were eluted from the beads with 50 ll of SDS sample cocktail with- SNARE complexes in rat parotid glands and successfully demon- out boiling, and then the complexes were boiled for 5 min accord- strated that isoproterenol (ISO) and/or cytochalasin D (CyD) treat- ing to the instructions of the Rabbit IgG TrueBlot kit (eBioscience). ments significantly increased the transient interaction between The supernatants (2.5–5 ll for input) and aliquots of isolated SNAP23 and VAMP2. SNARE complexes, which were equivalent to 125–250 ll superna- tants, were applied to a 5–20% gradient SDS–PAGE gel (ATTO, To- kyo), resolved at 30 mA for 50 min, and then transferred to a PVDF 2. Materials and methods membrane (Immobilon-P, Millipore) at 100 mA per mini-gel (90 Â 73 Â 1 mm) for 60 min in a semi-dry blotter (ATTO) with 2.1. Materials 0.1 M Tris—0.192 M glycine buffer containing 5% methanol. The membrane was blocked with Block Ace (Snow Brand Milk Products, Rabbit polyclonal antibodies against SNAREs were purchased Co., Tokyo) or 5% skim milk in PBS at room temperature for 2 h or from Synaptic Systems (Goettingen, Germany). In addition, anti- overnight at 4 °C, and then incubated for 1 h at room temperature SNAP23 (rabbit polyclonal; ab3340) and anti-syntaxin 3 (rabbit with primary antibodies properly diluted in PBS containing 0.05% polyclonal; S5547) were obtained from Abcam and Sigma, respec- Tween-20 (PBST) and 20% Block Ace. Next the membrane was tively. Rabbit IgG TrueBlot was from eBioscience (San Diego, CA, washed three times with 50 ml PBST for 15 min each time, and USA). was further incubated for 1 h with secondary antibodies specific for mouse IgG or rabbit IgG (TruBlot, eBioscience) according to the 2.2. Preparation of parotid minces and treatment with NEM, CyD, and manufacturer’s protocol. It was again washed three times with PBST ISO as above, and HRP activity of the secondary antibodies was visual- ized with the Immobilon Western (Millipore). Chemiluminescent Male Wistar strain rats were anesthetized with ether and then images were captured and analyzed by an ATTO Cool Saver (ATTO). sacrificed by heart puncture. The experiments with rats had re- ceived the approval of the Animal Care and Use Committee of 2.4. Determination of amylase release the Health Sciences University of Hokkaido and were conducted in accordance with the Guideline for the Care and Use of Labora- Parotid tissue suspensions (1 ml) were preincubated for 5 min tory Animals of the Health Sciences University of Hokkaido.
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