Molecular manipulations to probe the relationship between structure and function of the glucose transporter

最近的cDNA克隆揭示了一个结构相关的蛋白家族的存在，催化促进葡萄糖转运通过质膜。然而，尽管在结构上有明显的相似性，但在组织分布和亚细胞分布以及葡萄糖运输的特征上，不同亚型之间存在差异。我们关注的是c端结构域，因为它的大小和氨基酸序列在葡萄糖转运蛋白异构体中是不同的。GLUT2的Km和Vmax值远高于GLUT1。为了研究细胞内c端结构域在葡萄糖转运中的作用，我们在中国仓鼠卵巢细胞中表达了突变的GLUT1，通过工程修饰的嵌合cDNA将其细胞内c端结构域替换为GLUT2。细胞松弛素B对GLUT2蛋白的亲和力要低得多，它以类似于GLUT1的方式与这种嵌合蛋白结合。相比之下，在10 mM 2-脱氧-d -葡萄糖浓度下，与野生型GLUT1相比，该嵌合葡萄糖转运蛋白的转运活性更强。对2-脱氧-d -葡萄糖摄取的动力学研究表明，与野生型GLUT1相比，该嵌合葡萄糖转运体的Km增加3.8倍，Vmax增加4.3倍。因此，细胞内c端结构域的替换赋予了葡萄糖转运体上的glut2样特性。这些结果强烈表明，细胞内c端结构域的多样性有助于葡萄糖在同工异构体之间的转运特性的多样性。

Recent cDNA cloning has disclosed the presence of a structurally related family of proteins that catalize facilitated glucose transporter across the plasma membranes. However, in spite of the marked similarity in structure, the tissue distribution and subcellular distribution, as well as characteristics of glucose transport, are different among isoforms. We have focused on a C-terminal domain, since it is diverse in size and amino acid sequence among glucose transporter isoforms. GLUT2 has far higher Km and Vmax values compared with GLUT1. To investigate the role pf the intracellular C-terminal domain in glucose transport, we expressed in Chinese hamster ovary cells the mutated GLUT1 whose intracellular C-terminal domain was replaced with that of GLUT2 by means of engineering the chimeric cDNA.Cytochalasin B, for which GLUT2 protein has much lower affinity, bound to this chimeric protein in a fashion similar to GLUT1. In contrast, greater transport activity was observed in this chimeric glucose transporter compared with the wild-type GLUT1 at 10 mM 2-deoxy-D-glucose concentration. The kinetic studies on 2-deoxy-D-glucose uptake revealed a 3.8-fold increase in Km and a 4.3-fold increase in Vmax in this chimeric glucose transporter compared with the wild-type GLUT1. Thus, replacement of the intracellular C-terminal domain confers the GLUT2-like property on the glucose transporter. These results strongly suggest that the diversity of intracellular C-terminal domain contributes to the diversity of glucose transport characteristics among isoforms.