Association of cytosolic Rab4 with GDI isoforms in insulin-sensitive 3T3-L1 adipocytes.
Association of cytosolic Rab4 with GDI isoforms in insulin-sensitive 3T3-L1 adipocytes.
复制标题
细胞质 Rab4 与胰岛素敏感 3T3-L1 脂肪细胞中 GDI 亚型的关联。
DOI：
10.1021/bi970202g
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Czech,MP
中科院分区：
文献类型：
--
作者：
Shisheva,A;Czech,MP
GLUT4葡萄糖转运体的细胞内池转移到脂肪和肌肉细胞表面被认为涉及到小的GTP结合蛋白，如Rab4蛋白。Rab蛋白在胞浆和胞内膜之间的循环是其功能所必需的，似乎受到GDP解离抑制剂(GDI)的调节，到目前为止已经克隆了三个GDI。先前的数据表明，当使用纯化的蛋白质时，Rab4类似地结合GDI(1和2)的两个异构体[Shisheva，A.，等人。(1994)Mol.Cell.比奥尔。14,3459−3468]。在本研究中，我们使用共沉淀技术分析了完整细胞中与GDI-1或GDI-2形成的复合体中的胞内Rab4。我们发现在胰岛素敏感的3T3-L1脂肪细胞和其他培养细胞中，Rab4同时与GDI-1和GDI-2形成稳定的胞浆复合体。培养的脂肪细胞经急性胰岛素处理后，胞浆中Rab4的水平显著升高，可与抗Rab4抗体进行定量免疫沉淀。令人惊讶的是，由于胰岛素的作用，胞浆中Rab4的增加主要与胞浆GDI-1有关。细胞内Rab4−Gdi-2复合体的水平几乎不受胰岛素的影响。在32P标记的3T3-L1脂肪细胞中未检测到Rab4和GDI-1的胰岛素依赖性磷酸化改变，提示另一种机制解释了Rab4与GDI-1结合的特异性。综上所述，这些数据表明，在胰岛素的作用下，存在选择性地形成Rab4−Gdi-1复合体，这在胰岛素的膜转运中发挥了作用。
Translocation of an intracellular pool of GLUT4 glucose transporters to the fat and muscle cell surface is thought to involve small GTP-binding proteins such as the Rab4 protein. The cycling of Rab proteins between cytosol and intracellular membranes necessary for their function appears to be regulated by GDP-dissociation inhibitors (GDI), three of which have been cloned thus far. Previous data suggest that Rab4 binds two of these isoforms of GDI (1 and 2) similarly when purified proteins are employed [Shisheva, A., et al. (1994)Mol.Cell. Biol. 14, 3459−3468]. In the present study, we have analyzed the cytosolic Rab4 in complexes with GDI-1 or GDI-2 in intact cells using a coprecipitation technique. We show here that in insulin-sensitive 3T3-L1 adipocytes and other cultured cells, Rab4 simultaneously forms stable cytosolic complexes with both GDI-1 and GDI-2. Acute insulin treatment of the cultured adipocytes significantly increases cytosolic levels of Rab4 which can be quantitatively immunoprecipitated with anti-Rab4 antibodies. Surprisingly, the increased cytosolic Rab4 due to insulin action is predominantly associated with cytosolic GDI-1. The levels of cytosolic Rab4−GDI-2 complexes were virtually unaltered by insulin. Insulin-dependent alterations of Rab4 and GDI-1 phosphorylation were not detected in32P-labeled 3T3-L1 adipocytes, suggesting another mechanism accounts for the specificity of Rab4 binding to GDI-1. Taken together, these data suggest there is selective formation of Rab4−GDI-1 complexes in response to insulin which plays a role in the action of insulin on membrane trafficking.