Structure, function and regulation of mammalian glucose transporters of the SLC2 family

Abstract

The SLC2 genes code for a family of GLUT proteins that are part of the major facilitator superfamily (MFS) of membrane transporters. Crystal structures have recently revealed how the unique protein fold of these proteins enables the catalysis of transport. The proteins have 12 transmembrane spans built from a replicated trimer substructure. This enables 4 trimer substructures to move relative to each other, and thereby alternately opening and closing a cleft to either the internal or the external side of the membrane. The physiological substrate for the GLUTs is usually a hexose but substrates for GLUTs can include urate, dehydro-ascorbate and myo-inositol. The GLUT proteins have varied physiological functions that are related to their principal substrates, the cell type in which the GLUTs are expressed and the extent to which the proteins are associated with subcellular compartments. Some of the GLUT proteins translocate between subcellular compartments and this facilitates the control of their function over long- and short-time scales. The control of GLUT function is necessary for a regulated supply of metabolites (mainly glucose) to tissues. Pathophysiological abnormalities in GLUT proteins are responsible for, or associated with, clinical problems including type 2 diabetes and cancer and a range of tissue disorders, related to tissue-specific GLUT protein profiles. The availability of GLUT crystal structures has facilitated the search for inhibitors and substrates and that are specific for each GLUT and that can be used therapeutically. Recent studies are starting to unravel the drug targetable properties of each of the GLUT proteins.

Keywords: ATP depletion; GLUT proteins; GLUT1; GLUT2; GLUT3; GLUT4; GLUT5; Glucose transport; Hypoxia; Insulin; Membrane transport; Regulated transport.

Fig 1

Compartmentalisation and retention of GLUT4 in insulin-target tissues. In basal cells, most of the cellular GLUT4 is sorted into intracellular compartments (EE: early endosomes; SE sorting endosomes; TGN: trans-Golgi network; Golgi: Golgi stacks; ERGIC; endoplasmic reticulum- Golgi intermediate compartment; ER endoplasmic reticulum; lysosomes: lysosomes including multi-vesicular body (MVB) pre-lysosomal compartments; GSV/IRV GLUT4 storage vesicles/insulin-responsive vesicles. Several processing and sorting steps (blue text) are involved in maintained intracellular GLUT4. These include EE retrieval of GLUT4 from the plasma membrane via CHC17 clathrin-coated vesicles. The SE compartments segregate GLUT4 either for recycling or for lysosomal degradation via MBV compartments that lead to lysosomes (this appears to be dependent on ubiquitinoylation of GLUT4 or GLUT4 associated proteins). In the absence of ubiquitinoylation, or following deubiquitinylation by USP25, vesicle recoating with clathrin (CHC22 in humans) occurs in a process involving sortilin and GGA2 and with retrograde transfer to the TGN and GSV/IRV. GLUT4 is also trafficked from ER to ERGIC. It is retained in ERGIC but is available for release (possibly directly to GSV/IRV). The candidate proteins for this further processing and sorting are clathrin-coating CHC22 in humans, the Golgi tether p115, the insulin-responsive amino-peptidase IRAP, TUG and the enzyme tankarase, UBC9 (reviewed in detail in ref [26, 120]). Insulin signalling through tyrosine phosphorylation leads to activation of SNARE proteins (VAMP2 on GLUT4 vesicles and the SNAP23, Syntaxin4, Munc18c complex at the plasma membrane). These activations facilitate fusion of GLUT4 vesicles with the plasma membrane. Insulin signalling through serine kinase cascades leads to phosphorylation and inactivation of the Rab-GAP activities of TBC1D1 and TBC1D4. This is associated with GTP loading of the Rabs on GLUT4 vesicles (mainly Rab10-GTP) and these GTP-Rabs direct the recruitment of the vesicles to the plasma membrane, ref [120] for review. This figure is adapted from the GLUT4 traffic map described in [32] and kindly supplied by the Brodsky group https://www.ucl.ac.uk/research/domains/food-metabolism-and-society/events/glut4-traffic-map-workshop

Fig. 2

Crystal structures of GLUT proteins define the structural basis for alternate exposure of binding site clefts to either outside or inside solutions. Discrete conformations for the GLUTs are identified as OPO (open-outside without substrate; rat GLUT5, pdb:4ybq), OPO-S (open-outside with substrate; human GLUT3 with maltose, pdb:4zwc), OPI-S (open-inside with substrate; human GLUT1 with nonyl-glucoside, 4pyp) and OPI (open-inside without substrate; bovine GLUT5, pdb:4yb9). The structural fold has four inverted trimer repeats with TM1-3 and TM7-9 showing inverted repeat similarity to TM4-6 and TM10-12, respectively. The first TM helix of each of the four trimers is shown as cartoon representation: TM1 (blue); TM4 (green); TM7 (yellow); TM10 (orange), while the rest of the protein is shown as transparent ribbon. The upper half of TM7 (TM7b) and the lower half of TM10 (TM10b) are particularly important for occluding the binding site from the external and internal solutions, respectively. The occlusion OPO to OPI is associated with hydrophobic residues in TM7b moving closer to TM1 while hydrophobic residues in TM10b move away from TM4. The reverse occurs in the OPI to OPO conformational changes. TM7b hydrophobic residues 291, 292, 293 and 294 and TM10b residues 386 and 387 in GLUT1 (with equivalent residues in GLUT3 and GLUT5) are shown with space filling to illustrate this occlusion. In addition, salt bridges between residues at the cytosolic ends of the TMs are formed to bunch these TM ends, and the C-terminal ICH5 region, closer together in the OPO conformations. The location of the substrate glucose moiety that is revealed from both the GLUT3 structures with maltose (OPO-S, and GLUT1 with nonyl-glucose (OPI-S) is the same as in the GLUT3 structure with glucose (not shown, pdb:4zw9). In all cases, the glucose in the central site is polarised with C1-O projecting toward the internal solution with C4-O trailing. The structures shown are constructed using the VMD software and using the pdb files reported and described by the Yan group ([55, 56] for GLUT1, 3) and the Drew group ([158] for GLUT5)

Fig. 3

Substrates and inhibitors reveal differing specificity requirements for the GLUTs and may aid the therapeutic targeting of individual GLUTs that are implicated in disease. In a substrate and inhibitor, interactions with the GLUTs are associated with H-bonding (involving either electron donating or withdrawing groups) that can be examined using analogues that are H-bond accepting only (fluorine substitution for –OH). Spatial limitations to binding can be explored using O-alkyl groups. In b, β-methyl-D-glucoside has very low affinity for the outside site of GLUT1 suggesting a close approach to C1-O, while a 4-O-propyl group (c) is well tolerated at the outside site. The outside site can accommodate quite bulky substitutions at C4-OH with disaccharides such as maltose (e and Fig. 2 in OPO-S), and bis-glucose propylamine BGPA (f) derivatives being well tolerated. In f, the R group on the phenyl-diazirine photoreactive moiety can be a very large spacer arm with biotin. In contrast to these spatial restraints at the outside site, C1-O substitutions as in β-O-propyl-glucoside (d) or β-O-nonyl-glucoside (Fig. 2 in OPI-S) are well tolerated at the inward-facing site. Both fructofuranose and fructopyranose forms of fructose are transported by GLUTs such as GLUT5. The closed ring forms, including 2-5-anhydro-D-mannitol (h) and the β-methyl-fructofuranosides and β-methyl-fructopyranosides (j and k), are good substrates and inhibitors. Several new derivatives, including fluorescent and photolabeling compounds, based on 2-5-anhydro-D-mannitol have been described. The introduction of an H-bond accepting fluoro group at C3 of 2-5-anhydro-D-mannitol (i) reduces affinity for GLUT5 but increases affinity for GLUT1 suggesting tuning analogues for a specific GLUT is possible. The GLUT family is not restricted to glucose or fructose as substrate. GLUT9 is a transporter for urate (l). Dehydro-ascorbate in solution as a hydrate (m) is transported well by several GLUTs, and particularly GLUT10. GLUT13 transports myo-inositol (n) with good specificity. Now that GLUT structures are available, therapeutic targeting of individual GLUTs is becoming possible. In silico docking-aided screening of compound libraries has led to the identification of Bay 876 (o) as a high affinity inhibitor of GLUT1 (and not other class 1 GLUTs) and MSNBA (p) as a specific inhibitor of GLUT5 with negligible affinity for class 1 GLUTs