MOLECULAR ANALYSIS OF STRUCTURE-FUNCTION RELATIONSHIP OF GLUCOSE TRANSPORTER AND INSULIN ACTION

研究了葡萄糖转座异构体的生物学和生化特性。用表达载体将大鼠GLUT5 cDNA转染到中国仓鼠卵巢细胞。GLUT5仅针对质膜，并表现出对果糖的转运活性，而不是葡萄糖，其亲和力远高于另一种具有果糖转运活性的葡萄糖转运蛋白GLUT2。有趣的是，GLUT5没有被细胞松弛素B光标记，而相似量的GLUT1在相同的实验条件下被充分光标记。此外，为了阐明GLUT1和/或GLUT5中负责葡萄糖/果糖运输的结构域，我们表达了几种嵌合的GLUT1/GLUT5蛋白，并检测了它们的葡萄糖运输和/或果糖运输活性。细胞内的中间环以及从第7到第12个跨膜结构域的区域在GLUT1的葡萄糖转运中起着至关重要的作用，而用GLUT5的相应区域替换n端一半或细胞内c端区域对葡萄糖转运活性没有显著影响。相反，从n端穿过第6跨膜结构域的n端一半和细胞内c端区域都是GLUT5的果糖运输所必需的。因此，GLUT5是一种专门的果糖转运蛋白，在己糖转运蛋白中，果糖转运所需的结构比葡萄糖转运所需的结构更为严格。

The biological and biochemical properties of glucose transpoter isoforms have been investigated. The rat GLUT5 cDNA was transfected into Chinese hamster ovary cells with an expression vector. GLUT5 was exclusively targeted to the plasma membrane and exhibited a transport activity, not for glucose, but for fructose with much higher affinity than GLUT2, the other glucose transporter with fructose transport activity. Interestingly, GLUT5 was not photolabeled with cytochalasin B,while a similar amount of GLUT1 was adequately photolabeled under the same experimental conditions. In addition, to elucidate the domains responsible for transport of glucose/fructose in GLUT1 and/or GLUT5, several chimeric GLUT1/GLUT5 proteins were expressed and their glucose transport and/or fructose transport activities were examined. The intracellular middle loop as well as the region encompassing from the 7th through the 12th membrane spanning domain were observed to have a crucial role in glucose transport of GLUT1, while replacement of the N-terminal half or the intracellular C-terminal region with the corresponding region of GLUT5 did not induce a marked effect on glucose transport activity. In contrast, both the N-terminal half from the N-terminus through the 6th membrane spanning domain and the intracellular C-terminal region were mandatory for fructose transport of GLUT5. Thus, GLUT5 is a transporter exclusively for fructose and the structure required for fructose transport is more stringent than that for glucose transport in hexose transporter proteins.