Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin.
GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.
Katagiri
H
H
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Asano
T
T
Ishihara
H
H
Lin
J L
JL
Inukai
K
K
Shanahan
M F
MF
Tsukuda
K
K
Kikuchi
M
M
Yazaki
Y
Y
Oka
Y
Y
D019325:3-O-Methylglucose; D000345:Affinity Labels; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000378:metabolism*; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D003847:Deoxyglucose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D014162:Transfection; D014364:Tryptophan
D000345:Affinity Labels; D051272:Glucose Transporter Type 1; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D019325:3-O-Methylglucose; D005576:Colforsin; D014364:Tryptophan; D004247:DNA; D003847:Deoxyglucose; D005947:Glucose