Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Campus Isla Teja s/n, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.
Biochemistry. 2011 Oct 18;50(41):8834-45. doi: 10.1021/bi200748b. Epub 2011 Sep 27.
The facilitative hexose transporter GLUT1 activity is blocked by tyrosine kinase inhibitors that include natural products such as flavones and isoflavones and synthetic compounds such as tyrphostins, molecules that are structurally unrelated to the transported substrates [Vera, et al. (2001) Biochemistry, 40, 777-790]. Here we analyzed the interaction of GLUT1 with quercetin (a flavone), genistein (an isoflavone), and tyrphostin A47 and B46 to evaluate if they share one common or have several binding sites on the protein. Kinetic assays showed that genistein, quercetin, and tyrphostin B46 behave as competitive inhibitors of equilibrium exchange and zero-trans uptake transport and noncompetitive inhibitors of net sugar exit out of human red cells, suggesting that they interact with the external surface of the GLUT1 molecule. In contrast, tyrphostin A47 was a competitive inhibitor of equilibrium exchange and zero-trans exit transport and a noncompetitive inhibitor of net sugar entry into red cells, suggesting that it interacts with the cytoplasmic surface of the transporter. Genistein protected GLUT1 against iodide-elicited fluorescence quenching and also decreased the affinity of d-glucose for its external binding site, while quercetin and tyrphostins B46 and A47 promoted fluorescence quenching and did not affect the external d-glucose binding site. These findings are explained by a carrier that presents at least three binding sites for tyrosine kinase inhibitors, in which (i) genistein interacts with the transporter in a conformation that binds glucose on the external surface (outward-facing conformation), in a site which overlaps with the external binding site for d-glucose, (ii) quercetin and tyrphostin B46 interact with the GLUT1 conformation which binds glucose by the internal side of the membrane (inward-facing conformation), but to a site accessible from the external surface of the protein, and (iii) the binding site for tyrphostin A47 is accessible from the inner surface of GLUT1 by binding to the inward-facing conformation of the transporter. These data provide groundwork for a molecular understanding of how the tyrosine kinase inhibitors directly affect glucose transport in animal cells.
促进性己糖转运蛋白 GLUT1 的活性被包括天然产物（如黄酮类和异黄酮类）和合成化合物（如 tyrphostins）在内的酪氨酸激酶抑制剂所阻断，这些抑制剂与转运底物在结构上没有关系。[Vera 等人，（2001 年）生物化学，40，777-790]。在这里，我们分析了 GLUT1 与槲皮素（一种黄酮类）、染料木黄酮（一种异黄酮）和 tyrphostin A47 和 B46 的相互作用，以评估它们是否在蛋白质上共享一个共同的或有几个结合位点。动力学分析表明，染料木黄酮、槲皮素和 tyrphostin B46 作为平衡交换和零转运摄取的竞争性抑制剂，以及人红细胞中净糖外排的非竞争性抑制剂，表明它们与 GLUT1 分子的外表面相互作用。相比之下，tyrphostin A47 是平衡交换和零转运出口的竞争性抑制剂，以及净糖进入红细胞的非竞争性抑制剂，表明它与转运体的细胞质表面相互作用。染料木黄酮保护 GLUT1 免受碘化物诱导的荧光猝灭，也降低了 d-葡萄糖对其外部结合位点的亲和力，而槲皮素和 tyrphostins B46 和 A47 促进荧光猝灭，并不影响外部 d-葡萄糖结合位点。这些发现可以通过一种载体来解释，该载体至少有三个酪氨酸激酶抑制剂结合位点，其中（i）染料木黄酮与转运体相互作用，处于与外部表面（外向构象）结合葡萄糖的构象中，与 d-葡萄糖的外部结合位点重叠，（ii）槲皮素和 tyrphostin B46 与 GLUT1 构象相互作用，该构象通过膜的内部侧结合葡萄糖（内向构象），但结合位点可从蛋白质的外部表面进入，（iii）tyrphostin A47 的结合位点可通过与转运体的内向构象结合从 GLUT1 的内部表面进入。这些数据为直接影响动物细胞葡萄糖转运的酪氨酸激酶抑制剂如何作用提供了分子基础。
