Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2
Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2
复制标题
DOI：
10.1042/bj3170945
复制
发表时间：
1996-08-01
影响因子：
4.1
通讯作者：
Ward, CW
中科院分区：
文献类型：
--
作者：
Jagadish, MN;Fernandez, CS;Ward, CW
SNAP-25（突触体相关蛋白25）、syntaxin和synaptobrevin是三种SNARE[可溶性NSF附着蛋白受体（其中NSF = n -乙基丙烯酰亚胺敏感融合蛋白）]蛋白，它们形成核心复合物，参与突触囊泡对接并随后与靶膜融合。本研究旨在了解人类胰岛素反应组织中携带葡萄糖转运蛋白的囊泡与质膜融合的机制。它描述了编码SNAP-25 A和B异构体、syntaxin 4和synaptobrevins（也称为载体相关膜蛋白）的cDNA分子从两种主要的人类胰岛素反应组织，骨骼肌和脂肪中分离和表征。SNAP-25的DNA和推断的氨基酸序列与先前报道的人类神经SNAP-25 A和B亚型完全一致。我们的研究结果表明，在胰岛素反应组织和体外培养的3T3-L1细胞中都存在这两种异构体，但表明基因表达的差异模式：异构体a是脂肪组织中的主要物种，而异构体B是骨骼肌中的主要物种。使用抗SNAP-25单克隆抗体，通过免疫荧光显微镜证实了3T3-L1细胞中SNAP-25蛋白的存在。使用同一单克隆抗体的免疫沉淀实验也显示，大鼠附睾脂肪垫质膜组分中存在SNAP-25蛋白。骨骼肌的syntaxin 4编码区与先前报道的胎盘cDNA序列有5个核苷酸差异，其中两个导致氨基酸变化：Asp-174变为Glu， Val-269变为Ala。来自骨骼肌的synaptobrevin 1 cDNA与来自神经组织的相应克隆相比，包含两个核苷酸差异，其中一个是沉默的，另一个导致氨基酸Thr-102变为Ala。脂肪蛋白的cDNA序列与人神经组织的synaptobrevin 1相同。此外，我们通过PCR扩增和Southern杂交分析证实了脂肪中存在syntaxin 4，骨骼肌中存在synaptobrevin 2。利用酵母双杂交系统，在syntaxin 4的全长胞质结构域和synaptobrevin 2之间观察到相互作用，synaptobrevin 2是一种囊泡膜SNARE，之前被其他人证明与携带GLUT4葡萄糖转运蛋白的囊泡有关，但未发现与synaptobrevin 1相互作用。利用从脂肪细胞分离的低密度微粒体的流式细胞术证实了syntaxin 4与携带GLUT4蛋白的囊泡亚群的结合；而SNAP-25本身与这些囊泡的结合很差，syntaxin 4-SNAP-25复合物具有很强的相互作用。
SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE [soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)] proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane. The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues. It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat. The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms. Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle. The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody. Immuno-precipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads. The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala. The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala. The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues. Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis. Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1. Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.