Capilla Encarnación, Suzuki Naoko, Pessin Jeffrey E, Hou June Chunqiu
Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794-8651, USA.
Mol Endocrinol. 2007 Dec;21(12):3087-99. doi: 10.1210/me.2006-0476. Epub 2007 Aug 30.
Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized gamma-ear-containing Arf-binding protein (GGA) dependent, whereas insulin-stimulated translocation is regulated by Akt substrate of 160 kDa (AS160). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminus and the large intracellular loop of GLUT4 into GLUT1 (chimera 1-441) fully recapitulated the basal state retention, insulin-stimulated translocation, and GGA and AS160 sensitivity of wild-type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when coexpressed with both wild-type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by coexpression of dominant-interfering GGA. In addition, coexpression with a dominant-interfering AS160 (AS160-4P) abolished insulin-stimulated GLUT4-WT but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminus and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane; however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.
新合成的葡萄糖转运蛋白4（GLUT4）通过一个依赖于高尔基体定位的含γ耳的Arf结合蛋白（GGA）的过程进入胰岛素反应性储存区室，而胰岛素刺激的转位则由160 kDa的Akt底物（AS160）调节。在本研究中，我们使用了多种GLUT4/GLUT1嵌合体，分析了GLUT4中对于GGA和AS160调节GLUT4转运至关重要的特定基序。将GLUT4的氨基末端和大的细胞内环替换为GLUT1（嵌合体1-441）完全重现了野生型GLUT4（GLUT4-WT）的基础状态保留、胰岛素刺激的转位以及对GGA和AS160的敏感性。当与野生型GGA和AS160共表达时，GLUT4点突变（GLUT4-F5A）导致GLUT4在基础状态下细胞内保留的丧失。然而，与GLUT4-WT类似，共表达显性干扰GGA可显著抑制GLUT4-F5A的胰岛素刺激的质膜定位。此外，与显性干扰AS160（AS160-4P）共表达可消除胰岛素刺激的GLUT4-WT的转位，但不影响GLUT4-F5A的转位。GLUT4的内吞作用和细胞内隔离也需要GLUT4的氨基末端和大的细胞质环。此外，FQQI和SLL基序都参与了从质膜的初始内吞作用；然而，一旦内化，与FQQI基序不同，SLL基序不负责GLUT4细胞内循环回到特殊区室。总之，我们已经证明GLUT4氨基末端内的FQQI基序对于GLUT4的内吞作用和AS160依赖的细胞内保留是必不可少的，但对于GGA依赖的将GLUT4分选到胰岛素反应性储存区室不是必需的。
