Sorting mechanismof glucose transporters in mammalian cells.

Caco-2是一种分化的人肠上皮细胞系，是研究顶端和基底外侧膜蛋白极化靶向机制的有前途的模型。我们用表达载体将大鼠GLUT 5 cDNA和兔GLUT 1 cDNA稳定转染Caco-2细胞。免疫组化研究表明，GLUT 5蛋白表达定位于顶膜和GLUT 1表达主要存在于基底外侧膜上的细胞生长的可渗透的支持。接下来，为了研究负责决定葡萄糖转运蛋白中顶侧与基底侧分选的结构域，我们制备了几种GLUT 1-GLUT 5嵌合cDNA并将其转染到Caco-2细胞中。仅在顶膜处观察到GLUT 1（N-末端-第6跨膜结构域（TM 6））-GLUT 5（细胞内环（IL）-C-末端）嵌合体，而GLUT 1（N-末端-IL）-GLUT 5（TM 7-C-末端）和GLUT 1（N-末端-TM 12）-GLUT 5（C-末端结构域）嵌合体主要在基底外侧膜处观察到，其定位与GLUT 1相似。基于这些结果，C端结构域并不决定GLUT 1和GLUT 5的异构体特异性靶向，而是葡萄糖转运蛋白的胞内环在Caco-2细胞的顶-底外侧分选信号中起关键作用。

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.