Insulin regulates the membrane arrival, fusion, and C-terminal unmasking of glucose transporter-4 via distinct phosphoinositides
Insulin regulates the membrane arrival, fusion, and C-terminal unmasking of glucose transporter-4 via distinct phosphoinositides
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DOI：
10.1074/jbc.m500501200
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发表时间：
2005-08-05
影响因子：
4.8
通讯作者：
Klip, A
中科院分区：
文献类型：
--
作者：
Ishiki, M;Randhawa, VK;Klip, A
胰岛素通过葡萄糖转运蛋白4(GLUT4)转位到细胞膜来增加肌肉对葡萄糖的摄取，但GLUT4交通中的调节事件尚不清楚。在这里，我们利用表达GLUT4myc的L6肌肉细胞，重点研究了IA类磷脂酰肌醇(PI)3-激酶和特定的磷脂酰肌醇在GLUT4到达和与膜融合的步骤中的作用。为此，我们检测了myc表位在细胞表面或囊泡内空间的可用性，以及面向胞浆的C末端表位在它们衍生的细胞和膜草坪中的可用性。我们观察到以下情况：(A)Wortmannin和LY294002在抑制IA类PI 3-Kinase的浓度下减少了C末端的增益，但并未减弱C末端的增益，但myc表位无法检测，除非草坪或细胞被通透，这表明GLUT4myc存在于停靠的、未融合的小泡中。因此，用免疫电子显微镜观察经Wortmannin和胰岛素处理的细胞膜中含有GLUT4myc的囊泡，但不能单独检测到胰岛素或Wortmannin。(B)与myc表位相比，胰岛素对C末端的免疫可利用性更高，这表明C末端已被揭开。向完整细胞输送磷脂酰肌醇3，4，5-三磷酸(PI(3，4，5)P3)显著增加草坪相关myc信号，而不增加C端。相反，磷脂酰肌醇3-磷酸(PI3P)增加了C末端表位的检测，而没有任何myc收益。我们认为，胰岛素通过不同的磷脂酰肌醇调节GLUT4膜的到达、融合和C末端的去屏蔽。PI(3，4，5)P3导致到达和融合而不去掩蔽，而PI3P导致到达和去掩蔽而不融合。
Insulin increases glucose uptake into muscle via glucose transporter-4 ( GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol ( PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed the following: ( a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. ( b) Insulin caused greater immunological availability of the C terminus than myc epitopes, suggesting that C terminus unmasking had occurred. Delivering phosphatidylinositol 3,4,5-trisphosphate ( PI( 3,4,5) P3) to intact cells significantly increased lawn-associated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate ( PI3P) increased the detection of C terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion, and C terminus unmasking, through distinct phosphoinositides. PI( 3,4,5) P3 causes arrival and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.