Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells.

Jacobsson, Gunilla; Bean, Andrew J.; Scheller, Richard H.; Juntti‐Berggren, Lisa; Deeney, Jude T.; Berggren, Per Olof; Meister, Björn

doi:10.1073/pnas.91.26.12487

Cited by 189 publications

(141 citation statements)

References 30 publications

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“…Based on our protein quantification of SNARE complexes, we suggest that transition from the syntaxin1/SNAP-25 heterodimer to the ternary SNARE complex simply requires engagement of synaptobrevin. The localization of syntaxin1 and SNAP-25 on the chromaffin cell plasma membrane, in the absence of synaptobrevin, agrees well with the SNARE distribution in neurons and pancreatic secretory cells (15,25). The segregation of secretory granules from the plasma membrane, in chromaffin cells, is thought to be due to the cortical actin network (13,26).…”

Section: Discussionsupporting

confidence: 66%

“…In particular, studies of the three neuronal SNARE proteins, synaptobrevin2 from secretory vesicles and the plasma membrane syntaxin1 and SNAP-25, contributed significantly to our understanding of the last step of vesicle exocytosis. These three proteins are also involved in exocytosis from many endocrine cells throughout the body, including adrenaline-secreting chromaffin cells and insulin-secreting pancreatic ␤-cells (13,15). It is now generally accepted that the three SNARE proteins form a parallel four-helix bundle that can drive the fusion of two apposing membranes (3).…”

Section: Discussionmentioning

confidence: 99%

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High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

Rickman

Meunier

Binz

et al. 2004

Journal of Biological Chemistry

105

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The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.Hormonal and neurotransmitter release occurs when vesicles fuse with the plasma membrane in a calcium-dependent manner. The three SNARE 1 proteins, namely synaptobrevin2 (also known as VAMP2) on the vesicular membrane and syntaxin1 and SNAP-25 on the target plasma membrane, are essential for the last step of vesicular exocytosis, the fusion of membranes (1, 2). These three proteins form a highly stable ternary complex that likely drives the fusion of the two apposing membranes (3). Botulinum toxins, which specifically cleave SNARE proteins prior to the formation of the ternary SNARE complex, block vesicle exocytosis in both neurons and endocrine cells (2, 4). The plasma membrane protein SNAP-25 is a molecular target for botulinum toxin E (5). This toxin removes the C-terminal 26 amino acids from the SNAP-25 molecule and has been used previously to dissect stages of calcium-triggered exocytosis (6, 7). Although these studies demonstrated that the ternary SNARE complex forms at a late stage of vesicle exocytosis, the molecular events preceding formation of this complex remain largely unknown.Several contradicting theories have been put forward with regard to the molecular pathway leading to SNARE complex assembly. One study suggested that SNAREs, although active, do not pre-assemble in resting cells into any intermediate complex but would form the ternary complex upon activation of exocytosis (8). It has also been proposed that SNAP-25 engages vesicular synaptobrevin in a priming step and that binding of syntaxin1 would be the pivotal event leading to membrane fusion (9). An alternative theory states that syntaxin1 and SNAP-25 pre-assemble into a stable heterodimer on the plasma membrane, and only then does vesicular synaptobrevin engage the two target SNAREs (10, 11). Importantly, it is not clear whether any SNARE intermediates actually exist in intact secretory cells as a prelude to SNARE-mediated fusion.To address these outstanding issues, we analyzed SNARE binary re...

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Section: Discussionsupporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

Rickman

Meunier

Binz

et al. 2004

Journal of Biological Chemistry

105

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“…First, syntaxin 4 was reported previously not to be involved in the regulation of insulin secretion based upon the overexpression of the full-length syntaxin 4 protein (14,30,31). However, the full-length syntaxin 4 protein does not function as a dominant-interfering mutant in other cell systems, and currently, there are no specific endotoxins for the syntaxin 4 isoform.…”

Section: Discussionmentioning

confidence: 99%

Syntaxin 4 and Synip (Syntaxin 4 Interacting Protein) Regulate Insulin Secretion in the Pancreatic β HC-9 Cell

Saito

Okada

Yamada

et al. 2003

Journal of Biological Chemistry

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Although syntaxin 1 is generally thought to function as the primary target-N-ethylmaleimide-sensitive factor attachment protein receptor required for pancreatic ␤ cell insulin secretion, we have observed that overexpression of a dominant-interfering syntaxin 4 mutant (syntaxin 4/⌬TM) attenuated glucose-stimulated insulin secretion in ␤HC-9 cells. Furthermore, these cells express the selective syntaxin 4-binding protein Synip (syntaxin 4 interacting protein), and Synip was specifically co-immunoprecipitated with syntaxin 4 but not syntaxin 1. Overexpression of the full-length Synip protein (Synip/wild type) inhibited VAMP2 association with syntaxin 4 and decreased glucose-stimulated insulin secretion. This did not occur with a Synip mutant (Synip/ ⌬EF) that was incapable of binding syntaxin 4. Consistent with a functional role of syntaxin 4 in this process, expression of syntaxin 4/⌬TM also inhibited glucosestimulated insulin secretion. Furthermore, analysis of first and second phase insulin secretion demonstrated that syntaxin 4/⌬TM mainly suppressed the second phase of insulin secretion. In contrast, overexpression of Synip resulted in an inhibition of both the first and second phase of glucose-stimulated insulin secretion. These data demonstrate that syntaxin 4 plays a functional role on insulin release and granule fusion in ␤ cells and that this process is regulated by the syntaxin 4-specific binding protein Synip.It is well established that glucose-stimulated insulin secretion occurs through a complex metabolic network in which the increased flux of glucose in pancreatic ␤ cells elevates the intracellular ATP/ADP ratio (1-5). The increased ATP/ADP ratio results in the closure of the ATP-sensitive potassium channel (K ATP channels) and subsequent cellular depolarization (6, 7). In turn, depolarization causes the activation of the L-type voltage-dependent calcium channels that result in the influx of extracellular calcium. This increased intracellular calcium then serves as a trigger for the initial fusion of insulincontaining dense core granules with the plasma membrane (8, 9).Although the calcium trigger mechanism, trafficking, and fusion of insulin granules have not been fully elucidated, recent studies have suggested that the majority of granules that initially fuse with the plasma membrane are localized beneath the plasma membrane in a bound (docked) state (10 -12). This pool of insulin granules has been referred as the readily releasable pool, and it appears to account for the first phase of stimulusmediated insulin secretion (13). This initial release process requires the functional interactions of the t-SNARE 1 proteins, syntaxin 1 and SNAP25, with the v-SNARE protein, VAMP2. For example, the use of SNARE mutants or various SNARE protein-specific proteolytic toxins prevent regulated insulin secretion (14 -17). However, since these endotoxins do not proteolyze complexed SNARE proteins, it is thought that the plasma membrane-bound (docked) insulin granules first undergo a priming step (core SNARE complex...

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“…These components, including N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment protein (SNAP), and membrane-associated SNAP receptors (SNAREs) such as syntaxin-1 and SNAP-25, are also expressed in pancreatic beta cells [3,4,5], which play an important role in insulin exocytosis [6,7,8,9]. We [10] and others [11,12] have demonstrated that the expression of SNARE proteins is decreased in diabetic animal models.…”

Section: Introductionmentioning

confidence: 97%

Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analysed by total internal reflection fluorescence microscopy

et al. 2004

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Aims/hypothesis. The interaction of syntaxin-1 and SNAP-25 with insulin exocytosis was examined using the diabetic Goto-Kakizaki (GK) rat and a total internal reflection fluorescence (TIRF) imaging system. Methods. Primary rat pancreatic beta cells were immunostained with anti-syntaxin-1A, anti-SNAP-25 and anti-insulin antibodies, and then observed by TIRF microscopy. The real-time image of GFP-labelled insulin granules motion was monitored by TIRF.Results. The number of syntaxin-1A and SNAP-25 clusters, and the number of docked insulin granules on the plasma membrane were reduced in GK beta cells. When GK rats were treated with daily insulin injection for 2 weeks, the number of syntaxin-1 and SNAP-25 clusters was restored, along with the number of docked insulin granules. The infection of GK beta cells with Adex1CA SNAP-25 increased the number of docked insulin granules. TIRF imaging analysis demonstrated that the decreased number of fusion events from previously docked insulin granules in GK beta cells was restored when the number of docked insulin granules increased by insulin treatment or Adex1CA SNAP-25 infection. Conclusions/interpretation. There was a close correlation between the number of syntaxin-1 and SNAP-25 clusters and the number of docked insulin granules, which is associated with the fusion of insulin granules.

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Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells.

Cited by 189 publications

References 30 publications

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

Syntaxin 4 and Synip (Syntaxin 4 Interacting Protein) Regulate Insulin Secretion in the Pancreatic β HC-9 Cell

Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analysed by total internal reflection fluorescence microscopy

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