Structural domains that contribute to substrate specificity in facilitated glucose transporters are distinct from those involved in kinetic function: studies with GLUT-1/GLUT-2 chimeras.
GLUT-2 differs from other members of the facilitated glucose transporter family because it transports a wider range of substrates and exhibits a higher Km for transport of glucose analogs such as 2-deoxyglucose (2-DOG). In order to investigate the structural determinants of the unique substrate specificity and kinetic function of GLUT-2, recombinant adenoviruses were used to express native, mutant, and chimeric glucose transporters in the kidney cell line CV-1, yielding the following key observations. (1) A chimera consisting of GLUT-1 with the C-terminal tail of GLUT-2 had a Km for 2-DOG of 9.9 +/- 1.5 that was intermediate between that of native GLUT-1 (3.7 +/- 0.4) and native GLUT-2 (26.3 +/- 3.3). In contrast to the effect of the GLUT-2 C terminus on Km for 2-DOG, this substitution did not confer enhanced uptake of three alternative substrates (fructose, arabinose, or streptozotocin) which are transported efficiently by native GLUT-2 but not by GLUT-1. (2) A chimera consisting of GLUT-2 with the N-terminal 87 amino acids of GLUT-1 exhibited no change in Km for 2-DOG relative to native GLUT-2 but exhibited a significant reduction in capacity for transport of the three alternative substrates. (3) Mutation of asparagine 62 in GLUT-2 to glutamine produced a transporter lacking its N-linked oligosaccharide that exhibited a 2.5-fold increase in Km for 2-DOG but equally efficient transport of the three alternative substrates relative to native GLUT-2. These data provide insight into structural domains that affect substrate specificity in facilitated glucose transporters and demonstrate that they are distinct from elements involved in glucose transport kinetics.
Noel
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LE
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Newgard
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CB
D019325:3-O-Methylglucose / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D003847:Deoxyglucose / Q000378:metabolism; D051272:Glucose Transporter Type 1; D051273:Glucose Transporter Type 2; D000882:Haplorhini; D006801:Humans; D007668:Kidney; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D051272:Glucose Transporter Type 1; D051273:Glucose Transporter Type 2; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C495677:SLC2A1 protein, human; D019325:3-O-Methylglucose; D003847:Deoxyglucose