Kao A W, Ceresa B P, Santeler S R, Pessin J E
Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242-1109, USA.
J Biol Chem. 1998 Sep 25;273(39):25450-7. doi: 10.1074/jbc.273.39.25450.
To examine the role of clathrin-coated vesicle endocytosis in insulin receptor signaling and GLUT4 trafficking, we used recombinant adenovirus to express a dominant interfering mutant of dynamin (K44A/dynamin) in 3T3L1 adipocytes. Functional expression of K44A/dynamin, as measured by inhibition of transferrin receptor internalization, did not affect insulin-stimulated insulin receptor autophosphorylation, Shc tyrosine phosphorylation, or mitogen-activated protein kinase activation. Although the tyrosine phosphorylation of insulin receptor substrate-1 was slightly reduced, correlating with a 25% decrease in insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, insulin-stimulated Akt kinase activation was unaffected. In contrast, expression of K44A/dynamin resulted in the cell-surface accumulation of GLUT4 under basal conditions and an inhibition of GLUT4 endocytosis without affecting insulin-stimulated GLUT4 exocytosis. These data demonstrate that disruption of clathrin-mediated endocytosis does not significantly perturb insulin receptor signal transduction pathways. Furthermore, K44A/dynamin expression causes an accumulation of GLUT4 at the cell surface, suggesting that GLUT4 vesicles exist in at least two distinct intracellular compartments, one that undergoes continuous recycling and a second that is responsive to insulin.
为了研究网格蛋白包被小泡内吞作用在胰岛素受体信号传导和葡萄糖转运蛋白4（GLUT4）转运中的作用，我们使用重组腺病毒在3T3L1脂肪细胞中表达动力蛋白的显性干扰突变体（K44A/动力蛋白）。通过抑制转铁蛋白受体内化来衡量，K44A/动力蛋白的功能性表达并不影响胰岛素刺激的胰岛素受体自身磷酸化、Shc酪氨酸磷酸化或丝裂原活化蛋白激酶激活。尽管胰岛素受体底物-1的酪氨酸磷酸化略有降低，这与胰岛素受体底物-1相关的磷脂酰肌醇3激酶活性降低25%相关，但胰岛素刺激的Akt激酶激活并未受到影响。相反，K44A/动力蛋白的表达导致基础条件下GLUT4在细胞表面积累，并抑制GLUT4内吞作用，而不影响胰岛素刺激的GLUT4胞吐作用。这些数据表明，网格蛋白介导的内吞作用的破坏不会显著干扰胰岛素受体信号转导途径。此外，K44A/动力蛋白的表达导致GLUT4在细胞表面积累，这表明GLUT4小泡存在于至少两个不同的细胞内区室中，一个进行持续循环，另一个对胰岛素有反应。
