Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street Worcester, Massachusetts 01605, United States.
Biochemistry. 2011 Apr 19;50(15):3137-48. doi: 10.1021/bi1020327. Epub 2011 Mar 25.
Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport [ Cloherty, E. K., Levine, K. B., and Carruthers, A. ((2001)) The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40 ((51)) 15549-15561]. This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogues, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but noncompetitively inhibits sugar uptake into cells lacking sugar at 4 °C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [(3)H]-CB binding to red cell membranes depleted of peripheral proteins at 4 °C. Others produce a moderate stimulation of [(3)H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogues modestly stimulate [(3)H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure, and we evaluate the major determinants of ligand binding affinity and cooperativity.
细胞松弛素 B（CB）和佛司可林（FSK）通过与 GLUT1 内侧面糖结合位点结合或接近该结合位点来抑制红细胞中 GLUT1 介导的糖转运。矛盾的是，每种抑制剂的极低浓度都会适度刺激糖转运[Cloherty, E. K., Levine, K. B., and Carruthers, A. ((2001)) 红细胞葡萄糖转运蛋白呈现多个核苷酸敏感的糖出口位点。生物化学 40((51))15549-15561]。这一结果与以下假说一致，即葡萄糖转运蛋白包含多个相互作用的内侧面结合位点，可与 CB 和 FSK 结合。本研究直接检验了这一假说，并通过筛选细胞松弛素和佛司可林类似物文库，探讨了内侧面结合位点配体的哪些结构特征决定了结合位点的亲和力和协同性。与 CB 一样，FSK 竞争性抑制 3-O-甲基葡萄糖交换转运（细胞内含糖时的糖摄取），但非竞争性抑制 4°C 时无糖细胞的糖摄取。这反驳了 FSK 结合 GLUT1 内侧面和外侧面糖结合位点的假说。一些佛司可林衍生物和细胞松弛素抑制在 4°C 下耗尽外周蛋白的红细胞膜上的平衡[3H]-CB 结合。其他类似物在低浓度下引入时会适度刺激[3H]-CB 结合，但随着浓度的增加则抑制结合。然而，其他类似物在应用的所有抑制剂浓度下适度刺激[3H]-CB 结合。这些发现可以用至少具有两个相互作用的内侧面 CB 或 FSK 结合位点的载体来解释。我们在 GLUT1 介导的糖转运和 GLUT1 四级结构模型的背景下讨论了这一结果，并评估了配体结合亲和力和协同性的主要决定因素。
