Role of conserved arginine and glutamate residues on the cytosolic surface of glucose transporters for transporter function.
Role of conserved arginine and glutamate residues on the cytosolic surface of glucose transporters for transporter function.
复制标题
葡萄糖转运蛋白胞质表面上保守的精氨酸和谷氨酸残基对转运蛋白功能的作用。
作者：
A. Schürmann;H. Doege;H. Ohnimus;V. Monser;and Andreas Buchs;H. Joost
通过定点诱变和 COS-7 细胞中构建体的表达，研究了 GLUT4 细胞质表面的保守精氨酸和谷氨酸残基对转运蛋白功能的作用。在转染细胞的膜中测定重建的葡萄糖转运活性、细胞松弛素 B 结合和用外表面标记 2-N4-(1-azi-2,2,2-三氟乙基)苯甲酰基-1, 3-双(d-甘露糖氧基)-2-丙胺 (ATB-BMPA) 进行的光标记，并校正表达转运蛋白的免疫反应性。 Arg 92（R92L 氨基酸残基根据 GLUT1 中的相应残基编号）或 Arg 333/334 (RR333/4LA) 的交换降低或抑制了转运活性，对 ATB-BMPA 的光标记和细胞松弛素 B 结合没有影响或影响很小。这表明这些残基的缺乏选择性地干扰运输过程中底物诱导的载体构象变化。 Glu 146 (E146D) 或 Arg 153 (R153L) 的交换显着降低了转运活性、ATB-BMPA 光标记和细胞松弛素 B 结合。突变体 E329Q、E393D 和 R400L 中的转运活性和 ATB-BMPA 标记被消除，而细胞松弛素 B 的结合正常。因此，Glu 329、Glu 393 和 Arg 400 的交换似乎将转运蛋白阻止在向内的构象中。结论是，葡萄糖转运蛋白 GLUT4 细胞质表面的保守精氨酸和谷氨酸残基对其适当的构象至关重要，并且带电残基的相互作用介导了向外和向内状态之间的振荡。
The role of conserved arginine and glutamic acid residues at the cytoplasmic surface of the GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose transport activity, cytochalasin B binding, and photolabeling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis(d-mannosyloxy)-2-propylamine (ATB-BMPA) was assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Exchange of Arg 92 (R92L amino acid residues are numbered according to the corresponding residues in the GLUT1) or Arg 333/334 (RR333/4LA) reduced or suppressed transport activity with no or very little effect on photolabeling with ATB-BMPA and cytochalasin B binding. It is suggested that the lack of these residues selectively disturbes the substrate-induced conformational change of the carrier during transport. Exchange of Glu 146 (E146D) or Arg 153 (R153L) markedly reduced transport activity, ATB-BMPA photolabeling, and cytochalasin B binding. Transport activity and ATB-BMPA labeling were abolished in the mutants E329Q, E393D, and R400L, whereas binding of cytochalasin B was normal. Thus, exchange of Glu 329, Glu 393, and Arg 400 appears to arrest the transporter in an inward facing conformation. It is concluded that the conserved arginine and glutamate residues at the cytoplasmic surface of the glucose transporter GLUT4 are essential for its appropriate conformation, and that it is the interaction of charged residues which mediates the oscillation between outward and inward facing states.