Abstract
The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.
Highlights
The structure-function relationship of the HepG2/ isoforms [1].All five are predicted to possess nearly identical erythrocyte-type glucose transporter(GLUTl)has two-dimensional membrane topography and are likely to have beenstudiedby in vitro site-directedmutagenesis. similar tertiary structure
F the 3 residues mutated in this studyo,nly glutamine282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the protein [18].Interestingly, we have found that these residues can be divided into those that are importantfor binding the inside site-specific ligand cytochalasin B and those that are outsidesite-specific ligands ATB-BMPA and 4,6-0- important for binding the outside site-specific ligands ATB
BMPA’ and 4,6-O-ethylidene-~-glucosWe.e report here the effects of substitution at glutamine 282 and asparagine 288, which is predicted to occur in transmembrane helix 7 (TM7), and of asparagine 317, which is predicted to occur in transThe facilitated uptake of glucose into mammalian cells is membrane helix 8 (TM8), and have examined the relative mediated by at least fivehomologousglucose transporter importance of these residues for transportand inner and
Summary
Overexpression o i HumanGLUTlin Chinese Hamster Ouary Cells-CHO-K1 cells, which have low endogenous glucose transport activity (Zl), were maintained in Ham's F-12 medium containing 10% fetal calf serum, transfected with pRC/CMV-GTl(WT), Q282L, N2881, and N317I by the calcium phosphate method, and selected by their resistance tc a 600 pg/ml concentration of the neomycin derivative G418 (GIBCO). The cells expressing these glucose transporters were identified by Western blot analysis of the cell lysate using an anti-peptide antibody against the COOH-terminal domain of human GLUTl as described below.
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