Sorting mechanismof glucose transporters in mammalian cells.

人分化的肠上皮细胞系Caco-2是研究顶膜蛋白和基底膜蛋白极化靶向机制的有前途的模型。利用表达载体将大鼠GLUT5和兔GLUT1基因稳定地导入Caco-2细胞。免疫组织化学研究表明，GLUT5蛋白表达定位于顶膜，而GLUT1主要表达于生长在可渗透支架上的细胞的基底膜。接下来，为了研究决定葡萄糖转运蛋白顶端与基底侧向分选的结构域，我们制备了几个GLUT1-GLUT5嵌合cDNA，并将它们导入Caco-2细胞。GLUT1(N-末端-第6跨膜区(TM6))-GLUT5(细胞内环(IL)-C-末端)嵌合体仅见于顶膜，而GLUT1(N-末端-IL)-GLUT5(TM7-C末端)和GLUT1(N-末端-TM12)-GLUT5(C-末端结构域)嵌合体主要在基底膜上观察到，定位与GLUT1相似。基于这些结果，C-末端结构域并不决定GLUT1和GLUT5的异构体特异性靶向，相反，在Caco-2细胞中，葡萄糖转运蛋白的细胞内环似乎在顶端-基侧分选信号中发挥关键作用。

Caco-2, a human differentiated intestinal epithelial cell line, is a promising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transgected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells with an expression vector. Immunohistochemical study revealed that the GLUT5 protein expressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on permeable support. Next, to investigate the domain responsible for determining apical vs basolateral sorting in glucose transporters, we prepared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Caco-2 cells. A GLUT1 (N-terminus-6th transmembrane domain (TM6)) -GLUT5 (intracellular loop (IL) -C-terminus) chimera was observed exclusively at the apical membrane, while GLUT1 (N-terminus-IL) -GLUT5 (TM7-C-terminus) and GLUT1 (N-terminus-TM12) -GLUT5 (C-terminal domain) chimeras were observed mainly at the basolateral membrane, a localization similar to that of GLUT1. Based on these results, the C-terminal domain does not determine isoform-specific targeting of GLUT1 and GLUT5.Rather, it is the intracellular loop in glucose transporters that appears to play a pivotal role in apical-basolateral sorting signals in Caco-2 cells.