Characterization of SNARE protein expression in beta cell lines and pancreatic islets.
Characterization of SNARE protein expression in beta cell lines and pancreatic islets.
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DOI：
10.1210/endo.137.4.8625909
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发表时间：
1996-04
期刊：
影响因子：
4.8
通讯作者：
M. Wheeler;L. Sheu;M. Ghai;A. Bouquillon;G. Grondin;U. Weller;A. Beaudoin;M. Bennett;W. Trimble;H. Gaisano
中科院分区：
文献类型：
--
作者：
M. Wheeler;L. Sheu;M. Ghai;A. Bouquillon;G. Grondin;U. Weller;A. Beaudoin;M. Bennett;W. Trimble;H. Gaisano
本研究使用胰岛β细胞和胰岛细胞系来验证这一假说，即控制神经细胞胞吐的分子机制可能被β细胞用来调节胰岛素的分泌。利用针对一系列与突触囊泡融合和胞吐有关的突触蛋白(SNARE)的特异性抗血清，我们已经鉴定了几种SNARs在胰岛β细胞系、βTC6-f7和HIT-T15以及胰岛中的表达。在βTC6-f7和HIT-T15细胞和胰岛中均检测到v-SNARE囊泡相关膜蛋白(VAMP)-2，但未检测到VAMP-1免疫反应蛋白。在这些胰岛来源的细胞系中，这个18 kDa的蛋白迁移到被破伤风毒素(TeTx)切割的大鼠脑突触小泡VAMP-2中。免疫荧光共聚焦显微镜和电子显微镜将VAMP-2定位于含有胰岛素分泌颗粒膜的细胞质一侧。在链溶素O通透性的HIT-T15细胞中，TeTx可抑制钙离子诱导的胰岛素释放83+/-4.3%，这与VAMP-2的裂解有很好的相关性。这些β细胞系还表达第二个囊泡(V)-SNARE，cell ubrevin。Western blotting还检测到神经元靶标(T)-膜陷阱、SNAP-25和Synaxin亚型1-4。与脑部异构体观察到的情况一样，β细胞25-kDa SNAP-25蛋白和合成素亚型1-3分别被肉毒杆菌A和C毒素特异性切割。通过免疫荧光显微镜观察，这些潜在的t-陷阱主要定位于β细胞系和胰岛β细胞的质膜。为了确定免疫反应性突触素-2和-3亚型的特异性，并探索这些β细胞表达可能的钙离子敏感分子突触素III的可能性，对这些β细胞系进行了RT-PCR。这些研究证实，BetaTC6-F7细胞表达Synaxin-2异构体2和2‘，但不表达2’‘，并表达Synaxin-3。DNA序列分析表明，大鼠和小鼠β细胞合成素2，2‘和突触素III在核苷酸和预测氨基酸水平上高度保守(95-98%)。VAMP-2、NSEC/MUNC-18、SNAP-25和Synaxin家族蛋白以及突触素III在胰岛细胞和β细胞系中的存在为神经元和β细胞具有相似的钙调节胞吐的分子机制提供了证据。HIT-T15细胞VAMP-2的蛋白水解性裂解抑制了钙离子诱导的胰岛素分泌，支持这一假说，即这些蛋白在控制胰岛素胞吐中起着不可或缺的作用。
Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2'' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.