**3.3. Structural features & substrate specificity**

The protein sequences of the 14 isoforms protein sequences are between 14-63% identical and 30-79% conserved. Common to all isoforms of the GLUT protein family are the predicted 12 transmembrane helices that are based on the initial hydropathy plot for GLUT1 (Figure 2B). The 12-helix model has been supported by studies applying glycosylation scanning, the use of epitope-tags placed within the extrafacial loops as well as antibodies directed against the predicted extra-and intracellular loops and the N-and C-terminal parts of the proteins. All family members are highly glycosylated membrane proteins harbouring an N-linked glyco‐ sylation site. For Class I and II family members this is positioned in the first exofacial loop between transmembrane helices 1 and 2, while class III family members contain a shorter extracellular loop 1 and harbor the glycosylation site within the larger loop 9 [3].

Sequence comparisons between all isoforms identified conserved residues that have been termed sugar transporter signatures [3]. These include conserved glycine residues in helices 1, 2, 4, 5, 7, 8 and 10, indicating a critical role in the structure of these helices. In particular helix 7 appears to be important for substrate binding from the exofacial site.

Nonetheless, the primary sequences of the various isoforms do not allow prediction of their substrate specificity or kinetics of transport. However, on the basis of mutational analyses, several residues and motifs have been demonstrated to participate in the substrate recognition by GLUT1 as well as other isoforms. GLUT1, 3, and 4, which transport glucose but not fructose, have the QLS sequence in helix 7.

GLUT2 and 5, which both transport fructose, have a HVA or MGG in this position. Structure function analysis of GLUT2 and 3 chimeras expressed in *Xenopus laevis* oocytes demonstrated that GLUT3 can be converted to a glucose/fructose transporter with GLUT2 transport kinetics when the amino acid sequence of GLUT2 from the beginning of helix *7* to the COOH terminus is inserted into GLUT3. This demonstrates the impact of helix7 on substrate specificity and kinetics of transport. A specific feature of all Class II transporters is their ability to transport fructose, with GLUT5 representing the *bona fide* fructose transporter. Again, helix7 is important for exofacial substrate recognition, since transport of fructose by class II isoforms has been linked to the presence of a NXV/NXI motif. Mutating the isoleucine position significantly reduced fructose transport, while glucose uptake was unaltered. Interestingly, Class II isoforms were unable to transport 2-deoxy-D-glucose (2-DG) or galactose. A valine to isoleu‐ cine mutation in GLUT2 (V165I) within helix 5 abolished 2-DG transport, and introduction of the same point mutation in GLUT1 also resulted in a comparable reduction of 2-DG transport. For GLUT1, the topology and relative orientation of the 12 transmembrane helices with the outward-facing substrate-binding sites have been been proposed by two models. More than 50% of the complete polypeptide sequence has been analyzed by cysteine scanning mutagen‐ esis using the substituted cysteine accessibility method (SCAM) allowing a detailed prediction of the exofacial substrate-binding site and the folding of the human GLUT1. A three-dimen‐ sional model for GLUT1 has been developed based on structural information from crystallized members of the major facilitator superfamily, glycerol-3-phosphate transporter and lactose permease. Binding of glucose, forskolin, and phloretin was predicted in close proximity to the exofacial vestibule in this model. While a second binding site for forskolin and phloretin was predicted at the intracellular portion of GLUT1, cytochalasin B has been docked only at one particular endofacial position of the protein.
