Role of Ceveolae on Translocation of Glucose Transporter

我将GFP融合蛋白制成具有野生型Caveolin-1（人）的全长，该蛋白是构成小窝的蛋白质，并转染到3 T3-L1细胞中，我通过抗生素稳定表达的细胞选择了该融合蛋白。我能够得到稳定表达GFP融合蛋白的细胞，使其具有该蛋白的N末端部分缺失型。另外，Caveolin-3和Caveolin-1的N端缺失型DNA在Caveolin-1的支架结构域的一部分的两个氨基酸处发生突变。关于葡萄糖转运蛋白，我比较了我转染GLUT 4-GFP融合型的3 T3-L1细胞在引导脂肪细胞分化后对胰岛素刺激的反应。作为抑制小窝形成的因子，我瞬时转染了N末端部分缺失型（DGV）的小窝蛋白-3。作为抑制网格蛋白形成的因子，我转染了AP 180 DNA的C末端部分。我用胰岛素刺激这两个细胞。结果，GLUT 4向细胞膜的易位受到两种蛋白质表达的抑制。由此得出结论，在葡萄糖转运蛋白向细胞膜的移位中，网格蛋白包被的囊泡和小窝的存在都受到抑制。我检查了水通道蛋白-2的移位和再循环，以将其与GLUT 4的移位进行比较。据我所知，水通道蛋白-2的易位是由鞘糖脂缺陷引起的筏功能限制所阻碍的。此外，回收后，水通道蛋白与小窝蛋白-1检索内体。当我转染caveolin-1时，我明白了什么阻碍了这个细胞的返回，因为caveolin-1在支架结构域的一部分的两个氨基酸位置上发生了变化，抑制了caveolae的形成。

I made GFP fusion protein to have full length of wild type Caveolin-1(human) which was the protein which constituted caveolae toward and transfected into a 3T3-L1 cell and I got selected this by antibiotics stably expressed cells. I was able to get GFP fusion proteins stably expressed cells to have N terminal portion deleted type of this protein. In addition, N terminal deletion type of Caveolin-3 and Caveolin-1 DNA in two places of amino acids mutation of apart of scaffolding domain of Caveolin-1.About a glucose transporter, I compared reaction for insulin stimulation after having guided fat cell differentiation with the 3T3-L1 cell which I transfected a GLUT4-GFP fusion type. As the factor which restrained the formation of caveolae, I transiently tranfected a N terminal portion deleted type (DGV) of Caveolin-3. As the factor which restrained the clathrin formation, I tranfected a C terminal part of AP180 DNA. I stimulated these two cells by insulin. As a result, a translocation of GLUT4 to a cell membrane was restrained by both protein expressions. This got a conclusion that both existence of clathrin coated vesicles and caveolae was restrained in a translocation to a cell membrane of a glucose transporter.I examined a translocation and recycling of aquaporin-2 to compare it with a translocation of GLUT4. I understood that a translocation of aquaporin-2 was obstructed by functional restraint of raft caused by a defect of glycosphingolipid. Furthermore, after recycling, aquaporin retrieved endosomes with Caveolin-1. I understood what was obstructed return to this cell when I transfected caveolin-1 which had variation into two places of amino acids of a part of scaffolding domain which restrained the formation of caveolae.