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Review
. 2018 Nov 20;475(22):3511-3534.
doi: 10.1042/BCJ20170677.

Chemical biology probes of mammalian GLUT structure and function

Geoffrey D Holman  1
Affiliations
Review

Chemical biology probes of mammalian GLUT structure and function

Geoffrey D Holman. Biochem J. .

Abstract

The structure and function of glucose transporters of the mammalian GLUT family of proteins has been studied over many decades, and the proteins have fascinated numerous research groups over this time. This interest is related to the importance of the GLUTs as archetypical membrane transport facilitators, as key limiters of the supply of glucose to cell metabolism, as targets of cell insulin and exercise signalling and of regulated membrane traffic, and as potential drug targets to combat cancer and metabolic diseases such as type 2 diabetes and obesity. This review focusses on the use of chemical biology approaches and sugar analogue probes to study these important proteins.

Keywords: GLUT proteins; GLUT4 translocation; insulin action.

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Conflict of interest statement

The Author declares that there are no competing interests associated with this manuscript.

Figures

Figure 1.
Figure 1.. Use of glucose analogues to explore the GLUT-binding site structure.
(a) Hydroxyls may H-bond to GLUT side chains through electronegative membrane groups to the H of the glucose OH, or from a protonated membrane group to the O of the glucose OH. A fluorine group can only H-bond with a protonated membrane group. Alky substitutions (for example, methyl and propyl) can probe the spatial limitations around the binding site cleft. (b) Hydrogen bonds occur to C1β-O, C3-O, C4-OH as indicated. H-bonding to C6-O occurs with an additional hydrophobic interaction with a membrane group which was suggested to be a tyrosine or tryptophan. Figure from Figure 4 of ref. [25]. (c) If R=R′=H, both conformations of the membrane are possible, and transport occurs. If R=propyl and R′=H, binding can occur only with conformation A. If R=H and R′=propyl, binding can occur only with conformation B. The approach to the binding site alternates between outward-facing and inward-facing clefts which polarise the sugar with C1-OH leading and C4/C6-OH trailing. Figure from Figure 1 of ref. [26].
Figure 2.
Figure 2.. Bis-mannose analogues.
Bis-mannose compounds were produced by linking two d-mannose moieties through the C4-OH position, as analogue studies had revealed space around this region of the binding cleft in GLUT1 and GLUT4 and an enhancement in affinity from hydrophobic substitutions around C4/C6. (a) A hexane linker has been used; the Ki for interaction GLUT4 was 1.23 mM. (b) The hexane-linked d-mannose has been half reduced which opens the ring; the affinity for interaction with GLUT4 is reduced by 2-fold. In (c), a propylamine-linked bis-mannose has been substituted with a photoreactive group to produce ATB-BMPA. The associated increase in hydrophobicity leads to increased affinity; Ki for interaction with GLUT4 was 250 µM. Tritium was introduced into the linking propylamine.
Figure 3.
Figure 3.. Role of TM7B and TM10A residues of GLUTs in alternating binding site occlusion.
Only the C-terminal sections are shown as viewed from the centres of the proteins. TM7 is highlighted in green, TM10 in yellow. (a) GLUT3 is in a partially outside-open state with maltose (from PDB: 4ZWB). (b) GLUT3 is in a partially outside-open state with glucose (from PDB: 4ZW9). In (c), GLUT1 is in an inside-open state with nonyl-β-d-glucoside (from PDB: 4PYB). These structures are described in detail in ref. [56]. In all three cases, the central glucose moiety is in the same position with C1-OH leading inward and C4/C6-OH trailing. In (a), the trailing glucose moiety of maltose is above the central glucose. In (c), the alkyl chain extends below the central glucose and trails out of the inside-open binding site. The sugar polarity is therefore preserved in the outside-open and the inside-open conformations and is consistent with the predictions based on glucose analogue studies (Figure 1c). Both TM7 and 10 have a linked break in their helix. This allows regions TM7B and TM10A to simultaneously rotate to switch between the outside-open (b) and inside-open (c) conformations. Tyrosine 292, tyrosine 293 and tryptophan 388 (GLUT1 numbering; GLUT3 numbering is −2 at these positions) are highlighted as space-filling structures to illustrate their movement between the two conformations and their role in site occlusion.
Figure 4.
Figure 4.. Translocation of GLUT in response to insulin action.
The bis-hexose photolabels have been used to investigate the movement of GLUT4 from the cell surface to intracellular compartments and back again. (a) The figure is based on the translocation hypothesis of refs [17,64]. The tritiated version of the bis-mannose photolabel, ATB-BMPA, was used to show that GLUT4 (red) continually recycled even in the presence of insulin and that insulin increased the rate of exocytosis from its sequestered reservoir compartment. Modelling suggested that endosome recycling occurred but was not altered by insulin action. GLUT4 can tag biotinylated photolabel (green in b) and rates of trafficking can be determined by quenching the surface signal with extracellular avidin addition (blue in c) and then detecting the remaining internal GLUT4. This avoids use of subcellular fractionation to separate the plasma membrane from intracellular membranes.
Figure 5.
Figure 5.. Biotinylated bis-hexoses.
A biotin substitution onto the bridge between two hexoses can be introduced without any loss of affinity compared with the non-biotinylated ATB-BMPA. This is consistent with the exofacial binding cleft being deep and open enough to accommodate the large substituent. The bis-glucose (b) compound has only slightly higher affinity than the bis-mannose compound (a).
Figure 6.
Figure 6.. Cell-surface photolabelling of human muscle.
(a) Type 2 diabetic subjects have consistently lower cell-surface GLUT4 as detected by photoaffinity labelling with Bio-LC-ATB-BMPA. The GLUT4 is tagged with biotin, separated by SDS–PAGE, and blotted with GLUT4 antibody and the signal is quantified (b). Insulin, hypoxia and a combined treatment all increase cell-surface GLUT4 above basal levels. These stimulations are reduced in type 2 diabetes (black bars in b), but hypoxia and a combined treatment give partial rescue of the impaired insulin response. Data from ref. [76].
Figure 7.
Figure 7.. Extension of the linker in the biotinylated photolabel GP15 is necessary for interaction with avidin in intact cells.
(a) GP15 is a glucose analogue substituted at C4-O with a photolabelling diazirine substituent and a very long polyoxyethylene spacer (of ∼70 Å). This spacer is necessary for interaction with avidin in intact cells (b). (b) Human erythrocytes have been labelled with GP15, a fluorescently labelled avidin was added to intact cells and fluorescence was detected by confocal microscopy. Data from ref. [82]. The necessity for a long spacer is presumably associated with the deep outside cleft of the GLUTs that is now evident in the crystal structures (Figure 3).
Figure 8.
Figure 8.. Insulin but not AMPK stimulation increases GLUT4 exocytosis in muscle.
Exocytosis was determined using the biotin pulse, avidin chase protocol (Figure 4b,c) in which cell surface-biotinylated GLUT4 is internalised and then any cell-surface signal is quenched with avidin. This is followed in rat cardiac (a) and mouse epitrochlearis muscle (b). The exocytosis (loss of internal GLUT4, left panels) is quantified and rate constants are determined (right panels). Insulin stimulates the exocytosis rate constant above basal levels but AMPK activators, oligomycin in cardiomyocytes and AICAR in skeletal muscle do not. These data indicate that the insulin and AMPK stimulatory pathways (which both stimulate the levels of cell-surface GLUT4) do not converge at this step of GLUT4 translocation. Data are from refs [84,85].
Figure 9.
Figure 9.. Components of a GLUT4 membrane fusion assay.
The fusion assay is based on FRET between two, separately tagged, membrane compartments (a). The GLUT4 vesicle compartment is tagged with GP15 (b) onto which a fluorescent Dylight-streptavidin tetramer is conjugated. A fluorescent long chain-biotin europium Bio-Eu(TMT) (c) is enclosed within plasma membrane liposomes (formed from isolated plasma membrane and exogenous lipid). The components are incubated with cytoplasm that facilitates fusion that allows formation of a streptavidin complex that brings the two fluorescent molecules into close enough proximity for FRET (a). The complex is excited at 340 nm and the emission of the FRET signal is measured at 670 nm.
Figure 10.
Figure 10.. Insulin stimulation of GLUT4 vesicle fusion requires an insulin-activated plasma membrane.
(a) A time-course assay reveals that insulin stimulates the fusion process in a completely cell-free system to an extent that fully capitulates the process of GLUT4 exocytosis in cells. The advantage of the cell-free approach is that components can be mixed in combinations that do not occur in cells. This allows a narrowing down of the critical components of the insulin-stimulated fusion. These component mixing experiments (b) reveal that insulin-activated plasma membrane can stimulate with the cytoplasm from the basal state fusion, whereas insulin-activated cytoplasm with basal plasma membrane is insufficient to stimulate fusion. In all cases, the GLUT4 vesicles are from the basal state. Data are from ref. [87].
Figure 11.
Figure 11.. A new reagent for investigation of small G-proteins.
A new G-protein labelling reagent Bio-ATB-GTP (a) has been developed for studying the extent of insulin activation of these proteins. The substitution onto GTP was through the ribose hydroxyls as the crystal structures of G-proteins (b) revealed that these were more exposed on the surface of the protein that the guanine base or the ribose phosphates.
Figure 12.
Figure 12.. Approaches used for detection of insulin-activated Rab proteins.
(a) A targeted approach for the determination of the extent of insulin activation of G-proteins of interest is used. Membranes from rat adipocytes are labelled with Bio-ATB-GTP, solubilised and precipitated on immobilised streptavidin, resolved on SDS–PAGE gels and then blotted with target-specific antibodies. Background signal is determined by competition with GTP (+G) and an unlabelled membrane sample (UL). TC10, RalA and Rab11 (top row of panels) have significantly higher labelling in membranes from insulin-treated cells (I) than basal cells (B), but Rab8, Rab10 and Rab14 (bottom row of panels) do not. In (b), the labelled membranes are resolved on 2D gels and G-proteins detected by streptavidin peroxidase and identified by mass spectroscopy. Both Rab 3b (1) and Rab11a (2) have higher levels of labelling in membranes from insulin-stimulated compared with basal cells. Data (apart from the Rabs 8, 10 and 14 blots) are from ref. [102].
Figure 13.
Figure 13.. Analogues used to study the fructose transporter GLUT5.
Fructose (a) can adopt α and β of fructofuranose and fructopyranose in solution (in a ratio αP:βP:αF:βF <1%:75%:4%:21%). GLUT5 interacts with both forms as the closed-ring structures (b–d) are good inhibitors of transport with Ki values comparable with the Km values of d-fructose. The C2-β-O-methyl fructofuranoside and fructopyranoside both have ∼5-fold higher affinity than the corresponding C2-α-O-methyl compounds. The closed-ring 2,5-anhydro-d-mannitol (2-deoxy-d-fructose) (b) is a useful compound for synthesis of d-fructose biotinylated fructose photolabels FP1, FP2 (e,f). As in the GLUT1–4 photolabelling compounds, the spacer distance between the fructose and benzoyl-diazirine group is critical for affinity enhancement; compare fructose (a) with FP1 (e) and FP2 (f). Data are from refs [20,120].
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