Marsh B J, Alm R A, McIntosh S R, James D E
Centre for Molecular and Cellular Biology, University of Queensland, St. Lucia, Australia.
J Cell Biol. 1995 Sep;130(5):1081-91. doi: 10.1083/jcb.130.5.1081.
Insulin stimulates glucose transport in muscle and adipose tissue by triggering the movement of the glucose transporter GLUT-4 from an intracellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated cells. Two distinct targeting motifs in the amino and carboxy termini of GLUT-4 have been previously identified by expressing chimeras comprised of portions of GLUT-4 and GLUT-1, a transporter isoform that is constitutively targeted to the cell surface, in heterologous cells. These motifs-FQQI in the NH2 terminus and LL in the COOH terminus-resemble endocytic signals that have been described in other proteins. In the present study we have investigated the roles of these motifs in GLUT-4 targeting in insulin-sensitive cells. Epitope-tagged GLUT-4 constructs engineered to differentiate between endogenous and transfected GLUT-4 were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fractionation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F5 to A5) caused GLUT-4 to constitutively accumulate at the cell surface regardless of expression level. Mutation of the dileucine motif (L489L490 to A489A490) caused an increase in cell surface distribution only at higher levels of expression, but the overall cells surface distribution of this mutant was less than that of the amino-terminal mutants. Both NH2- and COOH-terminal mutants retained insulin-dependent movement from an intracellular to a cell surface locale, suggesting that neither of these motifs is involved in the insulin-dependent redistribution of GLUT-4. We conclude that the phenylalanine-based NH2-terminal and the dileucine-based COOH-terminal motifs play important and distinct roles in GLUT-4 targeting in 3T3-L1 adipocytes.
胰岛素通过触发葡萄糖转运蛋白GLUT-4从细胞内区室转移到细胞表面，刺激肌肉和脂肪组织中的葡萄糖转运。这一过程的基础是在未受刺激的细胞中GLUT-4的细胞内隔离。先前通过在异源细胞中表达由GLUT-4和GLUT-1（一种组成型靶向细胞表面的转运异构体）的部分组成的嵌合体，已在GLUT-4的氨基和羧基末端鉴定出两个不同的靶向基序。这些基序——氨基末端的FQQI和羧基末端的LL——类似于在其他蛋白质中描述的内吞信号。在本研究中，我们研究了这些基序在胰岛素敏感细胞中GLUT-4靶向中的作用。经工程改造以区分内源性和转染的GLUT-4的表位标记GLUT-4构建体在3T3-L1脂肪细胞中稳定表达。通过亚细胞分级分离法，在存在或不存在胰岛素的情况下孵育的细胞中评估靶向作用。表位标记的GLUT-4的靶向作用与内源性GLUT-4无法区分。FQQI基序（F5突变为A5）的突变导致GLUT-4无论表达水平如何都在细胞表面组成性积累。双亮氨酸基序（L489L490突变为A489A490）的突变仅在较高表达水平时导致细胞表面分布增加，但该突变体的整体细胞表面分布低于氨基末端突变体。氨基末端和羧基末端突变体均保留了胰岛素依赖性从细胞内到细胞表面区域的移动，这表明这些基序均不参与GLUT-4的胰岛素依赖性再分布。我们得出结论，基于苯丙氨酸的氨基末端基序和基于双亮氨酸的羧基末端基序在3T3-L1脂肪细胞中GLUT-4靶向中发挥重要且不同的作用。
