Peck Grantley R, Ye Siying, Pham Vi, Fernando Ruani N, Macaulay S Lance, Chai Siew Yeen, Albiston Anthony L
Department of Medicine, Howard Florey Institute, University of Melbourne, Parkville, Victoria 3052, Australia.
Mol Endocrinol. 2006 Oct;20(10):2576-83. doi: 10.1210/me.2005-0476. Epub 2006 Jun 8.
Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.
胰岛素调节氨肽酶（IRAP）是葡萄糖转运蛋白4（GLUT4）储存囊泡（GSV）的一种标志物，是已知唯一与GLUT4一同转运的蛋白质。在基础状态下，GSV从组成型循环的内体系统中隔离出来，进入一个对胰岛素有反应的细胞内池。胰岛素诱导GSV从这个池中快速转运到细胞表面，导致IRAP和GLUT4并入质膜。我们试图鉴定与IRAP相互作用的蛋白质，以进一步了解这种GSV转运过程。本研究描述了我们对IRAP氨基末端与Akt底物AS160（160 kDa的Akt底物）之间新型相互作用的鉴定。通过对过表达蛋白和内源性蛋白进行共免疫沉淀，证实了这种相互作用的有效性。此外，共聚焦显微镜显示了这些蛋白质的共定位。另外，我们证明AS160的IRAP结合结构域位于其第二个磷酸酪氨酸结合结构域内，并且这种相互作用不受AS160磷酸化的调节。我们推测，AS160通过与IRAP相互作用定位于含有GLUT4的囊泡，在那里它抑制其附近Rab底物的活性，有效地将囊泡束缚在细胞内。
