The facilitative hexose transporter GLUT1 is a multifunctional protein that transports hexoses and dehydroascorbic acid, the oxidized form of vitamin C, and interacts with several molecules structurally unrelated to the transported substrates. Here we analyzed in detail the interaction of GLUT1 with a group of tyrosine kinase inhibitors that include natural products of the family of flavones and isoflavones and synthetic compounds such as the tyrphostins. These compounds inhibited, in a dose-dependent manner, the transport of hexoses and dehydroascorbic acid in human myeloid HL-60 cells, in transfected Chinese hamster ovary cells overexpressing GLUT1, and in normal human erythrocytes, and blocked the glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts. Kinetic analysis of transport data indicated that only tyrosine kinase inhibitors with specificity for ATP binding sites inhibited the transport activity of GLUT1 in a competitive manner. In contrast, those inhibitors that are competitive with tyrosine but not with ATP failed to inhibit hexose uptake or did so in a noncompetitive manner. These results, together with recent evidence demonstrating that GLUT1 is a nucleotide binding protein, support the concept that the inhibitory effect on transport is related to the direct interaction of the inhibitors with GLUT1. We conclude that predicted nucleotide-binding motifs present in GLUT1 are important for the interaction of the tyrosine kinase inhibitors with the transporter and may participate directly in the binding transport of substrates by GLUT1.
Genistein is a dietary-derived plant product that inhibits the activity of protein-tyrosine kinases. We show here that it is a potent inhibitor of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, genistein inhibited the transport of dehydroascorbic acid, deoxyglucose, and methylglucose in a dose-dependent manner. Transport was not affected by daidzein, an inactive genistein analog that does not inhibit protein-tyrosine kinase activity, or by the general protein kinase inhibitor staurosporine. Genistein inhibited the uptake of deoxyglucose and dehydroascorbic acid in Chinese hamster ovary (CHO) cells overexpressing GLUT1 in a similar dose-dependent manner. Genistein also inhibited the uptake of deoxyglucose in human erythrocytes indicating that its effect on glucose transporter function is cell-independent. The inhibitory action of genistein on transport was instantaneous, with no additional effect observed in cells preincubated with it for various periods of time. Genistein did not alter the uptake of leucine by HL-60 cells, indicating that its inhibitory effect was specific for the glucose transporters. The inhibitory effect of genistein was of the competitive type, with a K i of approximately 12 M for inhibition of the transport of both methylglucose and deoxyglucose. Binding studies showed that genistein inhibited glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts in a competitive manner, with a K i of 7 M. These data indicate that genistein inhibits the transport of dehydroascorbic acid and hexoses by directly interacting with the hexose transporter GLUT1 and interfering with its transport activity, rather than as a consequence of its known ability to inhibit protein-tyrosine kinases. These observations indicate that some of the many effects of genistein on cellular physiology may be related to its ability to disrupt the normal cellular flux of substrates through GLUT1, a hexose transporter universally expressed in cells, and is responsible for the basal uptake of glucose.
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