title: Structure-function analysis of liver-type (GLUT2) and brain-type (GLUT3) glucose transporters: expression of chimeric transporters in Xenopus oocytes suggests an important role for putative transmembrane helix 7 in determining substrate selectivity.
abstract: The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (K(m) values for deoxyglucose transport of 11.2 +/- 1.1 and 1.4 +/- 0.06 mM, respectively) and patterns of substrate transport (GLUT2 is capable of D-fructose transport, GLUT3 is not) [Gould, G. W., Thomas, H. M., Jess, T. J., & Bell, G. I. (1991) Biochemistry 30, 5139-5145]. We have generated a range of chimeric glucose transporters composed of regions of GLUT2 and GLUT3 with a view to identifying the regions of the transporter which are involved in substrate recognition and binding. The functional characteristics of these chimeras were determined by expression in Xenopus oocytes after microinjection of cRNA. Replacement of the region from the start of putative transmembrane helix 7 to the C-terminus of GLUT3 with the corresponding region from GLUT2 results in a chimera with the ability to transport fructose and exhibits a K(m) for 2-deoxyglucose transport of close to that observed for wild-type GLUT2 (8.3 +/- 0.3 mM compared to 11.2 +/- 1.1 mM). Replacement of the region in GLUT3 from the end of helix 7 to the C-terminus with the corresponding region from GLUT2 resulted in a species which was unable to transport fructose and whose K(m) for 2-deoxyglucose was indistinguishable from wild-type GLUT3. We have determined the affinity for 2-deoxyglucose, D-fructose, and D-galactose of these and other chimeras. In addition, the Ki for maltose, a competitive inhibitor of 2-deoxyglucose transport, which binds to the exofacial sugar binding site was determined for these chimeras. The results obtained support a model in which the seventh putative transmembrane-spanning helix is intimately involved in the selection of transported substrate and in which this region plays an important role in determining the K(m) for 2-deoxyglucose. Additional data is presented which suggests that a region between the end of putative transmembrane helix 7 and the end of helix 10, together with sequences in the N-terminal half of the protein may also participate in substrate recognition and transport catalysis.
authors.affiliation: Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland.
authors.forename: M I
authors.initials: MI
authors.lastname: Arbuckle
authors.forename: S
authors.initials: S
authors.lastname: Kane
authors.forename: L M
authors.initials: LM
authors.lastname: Porter
authors.forename: M J
authors.initials: MJ
authors.lastname: Seatter
authors.forename: G W
authors.initials: GW
authors.lastname: Gould
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005260:Female; D051273:Glucose Transporter Type 2; D051274:Glucose Transporter Type 3; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D017931:DNA Primers; D051273:Glucose Transporter Type 2; D051274:Glucose Transporter Type 3; D009004:Monosaccharide Transport Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C495687:SLC2A3 protein, human