Marsh B J, Martin S, Melvin D R, Martin L B, Alm R A, Gould G W, James D E
Boulder Laboratory for Three-Dimensional Fine Structure, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA.
Am J Physiol. 1998 Sep;275(3):E412-22. doi: 10.1152/ajpendo.1998.275.3.E412.
The carboxy terminus of GLUT-4 contains a functional internalization motif (Leu-489Leu-490) that helps maintain its intracellular distribution in basal adipocytes. This motif is flanked by the major phosphorylation site in this protein (Ser-488), which may play a role in regulating GLUT-4 trafficking in adipocytes. In the present study, the targeting of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has been examined in stably transfected 3T3-L1 adipocytes. The trafficking of SAG was not significantly different from that of GLUT-4 in several respects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surface and was enriched in small intracellular vesicles. Second, SAG exhibited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its phosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containing the Golgi-derived gamma-adaptin subunit of AP-1 and that this localization is enhanced when Ser-488 is mutated to alanine. We conclude that the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may play a role in intracellular sorting at the trans-Golgi network but does not play a major role in the regulated movement of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes.
葡萄糖转运蛋白4（GLUT-4）的羧基末端含有一个功能性内化基序（亮氨酸-489-亮氨酸-490），有助于维持其在基础脂肪细胞内的分布。该基序两侧是该蛋白的主要磷酸化位点（丝氨酸-488），其可能在调节脂肪细胞中GLUT-4的转运中发挥作用。在本研究中，已在稳定转染的3T3-L1脂肪细胞中检测了丝氨酸-488突变为丙氨酸的GLUT-4（SAG）的靶向性。SAG的转运在几个方面与GLUT-4没有显著差异。首先，在没有胰岛素的情况下，SAG的分布与GLUT-4相似，即它在很大程度上被排除在细胞表面之外，并在小的细胞内囊泡中富集。其次，SAG表现出与GLUT-4相当的胰岛素依赖性向质膜的移动（4至5倍）。最后，以前已证明能刺激GLUT-4易位及其在丝氨酸-488处磷酸化的冈田酸，也同样刺激SAG向细胞表面的移动。使用免疫电子显微镜，我们已经表明GLUT-4定位于含有来自高尔基体的AP-1的γ-衔接蛋白亚基的细胞内囊泡中，并且当丝氨酸-488突变为丙氨酸时这种定位会增强。我们得出结论，GLUT-4中的羧基末端磷酸化位点（丝氨酸-488）可能在反式高尔基体网络的细胞内分选过程中发挥作用，但在3T3-L1脂肪细胞中GLUT-4向质膜的调节性移动中不发挥主要作用。
