A complex of Rab13 with MICAL-L2 and α-actinin-4 is essential for insulin-dependent GLUT4 exocytosis.
A complex of Rab13 with MICAL-L2 and α-actinin-4 is essential for insulin-dependent GLUT4 exocytosis.
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DOI：
10.1091/mbc.e15-05-0319
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发表时间：
2016-01-01
影响因子：
3.3
通讯作者：
Klip A
中科院分区：
文献类型：
--
作者：
Sun Y;Jaldin-Fincati J;Liu Z;Bilan PJ;Klip A
Rab 13是肌肉中胰岛素调节的GLUT 4-囊泡胞吐所必需的。生物化学和成像分析提供了证据，表明激活的Rab 13与支架蛋白MICAL-L2结合，与Rab 13和α-辅肌动蛋白-4形成复合物。通过GLUT 4与α-辅肌动蛋白-4相互作用，GLUT 4囊泡被募集到肌肉质膜。胰岛素通过将葡萄糖转运蛋白4（GLUT 4）募集到质膜来促进骨骼肌对葡萄糖的吸收。Rab GTP酶是动员胞内囊泡的分子开关，Rab 13是肌肉细胞中胰岛素调节的GLUT 4-囊泡胞外转位所必需的。我们发现Rab 13在这个过程中与支架蛋白MICAL-L2结合。RNA干扰介导的MICAL-L2或截短的MICAL-L2（MICAL-L2-CT）敲低损害胰岛素刺激的GLUT 4易位。胰岛素增加Rab 13与MICAL-L2的结合，通过共聚焦荧光和结构照明显微镜下的下拉和共定位进行评估。使用TIRF显微镜在细胞周边也观察到缔合。胰岛素进一步增加MICAL-L2与α-辅肌动蛋白-4（ACTN 4）的结合，ACTN 4是一种参与GLUT 4易位的蛋白质。Rab 13、MICAL-L2和ACTN 4形成胰岛素依赖性复合物，通过下拉和共聚焦荧光成像进行评估。值得注意的是，GLUT 4与响应胰岛素的复合物相关，需要MICAL-L2中的ACTN 4结合结构域。这通过用MICAL-L2的不同片段的下拉以及共聚焦和结构化照明显微镜来证明。最后，MICAL-L2-CT的表达消除了Rab 13与ACTN 4或Rab 13与GLUT 4的胰岛素依赖性共定位。我们的研究结果表明，MICAL-L2是胰岛素激活的Rab 13的效应子，Rab 13通过ACTN 4与GLUT 4连接，将GLUT 4囊泡定位在肌细胞外周，使其与膜融合。
Rab13 is necessary for insulin-regulated GLUT4-vesicle exocytosis in muscle. Biochemical and imaging analyses provide evidence that activated Rab13 engages a scaffold protein MICAL-L2 to form a complex with Rab13 and α-actinin-4. Through GLUT4 interaction with α-actinin-4, GLUT4 vesicles are recruited to the muscle plasma membrane. Insulin promotes glucose uptake into skeletal muscle through recruitment of glucose transporter 4 (GLUT4) to the plasma membrane. Rab GTPases are molecular switches mobilizing intracellular vesicles, and Rab13 is necessary for insulin-regulated GLUT4–vesicle exocytic translocation in muscle cells. We show that Rab13 engages the scaffold protein MICAL-L2 in this process. RNA interference–mediated knockdown of MICAL-L2 or truncated MICAL-L2 (MICAL-L2-CT) impaired insulin-stimulated GLUT4 translocation. Insulin increased Rab13 binding to MICAL-L2, assessed by pull down and colocalization under confocal fluorescence and structured illumination microscopies. Association was also visualized at the cell periphery using TIRF microscopy. Insulin further increased binding of MICAL-L2 to α-actinin-4 (ACTN4), a protein involved in GLUT4 translocation. Rab13, MICAL-L2, and ACTN4 formed an insulin-dependent complex assessed by pull down and confocal fluorescence imaging. Of note, GLUT4 associated with the complex in response to insulin, requiring the ACTN4-binding domain in MICAL-L2. This was demonstrated by pull down with distinct fragments of MICAL-L2 and confocal and structured illumination microscopies. Finally, expression of MICAL-L2-CT abrogated the insulin-dependent colocalization of Rab13 with ACTN4 or Rab13 with GLUT4. Our findings suggest that MICAL-L2 is an effector of insulin-activated Rab13, which links to GLUT4 through ACTN4, localizing GLUT4 vesicles at the muscle cell periphery to enable their fusion with the membrane.