Mori H, Hashiramoto M, Clark A E, Yang J, Muraoka A, Tamori Y, Kasuga M, Holman G D
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
J Biol Chem. 1994 Apr 15;269(15):11578-83.
Tyrosines 292 and 293 in the mammalian glucose transporter GLUT1 have been substituted by either isoleucine or phenylalanine. Chinese hamster ovary clones that were transfected with Tyr-292-->Ile, Tyr-292-->Phe, Tyr-293-->Ile, and Tyr-293-->Phe constructs of GLUT1 were shown, by Western blotting and cell surface carbohydrate labeling, to have expression levels that were comparable with the wild type. The Vmax for 2-deoxy-D-glucose transport was markedly reduced only as a result of the Tyr-293-->Ile mutation. The ability of the Tyr-293-->Ile mutated GLUT1 to bind the exofacial ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) and the endofacial ligand cytochalasin B were assessed by photolabeling procedures. The ability to bind the bis-mannose compound was unimpaired, whereas the ability to bind cytochalasin B was totally abolished, and the level of labeling was lower than in the nontransfected clone. Affinities of the wild-type and Tyr-293-->Ile GLUT1 for D-glucose, the exofacial ligands (ATB-BMPA and 4,6-O-ethylidene-D-glucose), and the endofacial ligand (cytochalasin B) were assessed by the ability of these agents to displace the radioactive ATB-BMPA photolabel. These data indicated that the Tyr-293-->Ile substitution produced no change in the affinity for D-glucose, a relatively small enhancement in the affinity for exofacial ligands, but a large approximately 300-fold reduction in affinity for cytochalasin B, suggesting that the mutated GLUT1 is locked in an outward facing conformation. The observation that the Tyr-293-->Ile mutant transporter can bind nontransported C4 and C6 substituted hexose analogues but cannot catalyze transport is interpreted as indicating that Tyr-293 is involved in closing the exofacial site around C4 and C6 of D-glucose in the transport catalysis process.
哺乳动物葡萄糖转运蛋白GLUT1中的酪氨酸292和293已被异亮氨酸或苯丙氨酸取代。通过蛋白质印迹法和细胞表面碳水化合物标记显示，用GLUT1的Tyr-292→Ile、Tyr-292→Phe、Tyr-293→Ile和Tyr-293→Phe构建体转染的中国仓鼠卵巢克隆，其表达水平与野生型相当。只有Tyr-293→Ile突变导致2-脱氧-D-葡萄糖转运的Vmax显著降低。通过光标记程序评估Tyr-293→Ile突变的GLUT1结合外表面配体2-N-4-(1-叠氮基-2,2,2-三氟乙基)苯甲酰基-1,3-双-(D-甘露糖-4-氧基)-2-丙胺(ATB-BMPA)和内表面配体细胞松弛素B的能力。结合双甘露糖化合物的能力未受影响，而结合细胞松弛素B的能力完全丧失，且标记水平低于未转染的克隆。通过这些试剂取代放射性ATB-BMPA光标记的能力，评估野生型和Tyr-293→Ile GLUT1对D-葡萄糖、外表面配体(ATB-BMPA和4,6-O-亚乙基-D-葡萄糖)和内表面配体(细胞松弛素B)的亲和力。这些数据表明，Tyr-293→Ile取代对D-葡萄糖的亲和力没有变化，对外表面配体的亲和力有相对较小的增强，但对细胞松弛素B的亲和力大幅降低约300倍，这表明突变的GLUT1锁定在向外的构象中。Tyr-293→Ile突变体转运蛋白能结合未转运的C4和C6取代的己糖类似物但不能催化转运这一观察结果被解释为表明Tyr-293在转运催化过程中参与封闭D-葡萄糖C4和C6周围的外表面位点。
