Glucose transport proteins (GLUTs) catalyze equilibrative, cellular sugar transport, are essential for carbohydrate homeostasis in humans, contain 12 membrane-spanning alpha helices (TMs) which form a translocation pathway (amphipathic TMs 1, 2, 4, 5, 7, 8, 10 and 11) stabilized by a scaffold (hydrophobic TMs 3, 6, 9 and 12) and adopt 3 conformational states: the inward, the occluded and the outward orientations. Gaps in our understanding of the GLUTs are: 1) How substrates bind;2) How substrates are translocated;3) How GLUTs oligomerize. We address these gaps and ask how each process is altered in a human disease - GLUT1 deficiency syndrome (GLUT1-DS).
Specific Aim 1 tests the hypothesis that substrates bind in the GLUT translocation pathway. GLUT1 transports glucose but not fructose while GLUT5 transports fructose not glucose. Mutagenesis studies suggest that TMs 2, 7 and 11 contribute to GLUT5 fructose binding and TMs 5, 7, 8, 10 and 11 to GLUT1 glucose binding. Using homology scanning mutagenesis, we ask if substitution of specific GLUT5 translocation TMs into GLUT1 converts GLUT1 from a glucose to a fructose transporter and vice versa and we ask which residues are critical for binding. Mass spectrometry of purified human GLUT1 will reveal which GLUT1 residues are modified by photo-affinity substrates/antagonists and which are substrate- protected against hydrogen-deuterium exchange (H/DX)? We then ask if critical substrate binding residues are altered in GLUT1-DS.
Specific Aim 2 tests the hypothesis that transport involves translocation pathway conformational changes controlled by scaffold TM6 - pathway TM1 interactions. GLUT1 hydrogens (80%) undergo conformation-sensitive exchange with solvent tritium or deuterium but which amino acids exchange is unknown. We will identify them by H/DX-mass spectrometry of purified human GLUT1 trapped in inward, occluded and outward orientations. GLUT1-GLUT4 homology scanning mutagenesis shows that TM6 controls the rate of conformational change between inward and outward GLUT1 and GLUT4 orientations by interacting with TM1. We ask if this is a hallmark of all GLUTs, we investigate which TM6 residues interact with TM1 by tryptophan scanning and dicysteine cross-linking mutagenesis and ask if these residues are altered in GLUT1-DS.
Specific Aim 3 tests the hypothesis that GLUT dimerization and tetramerization determine transport efficiency and are mediated by specific translocation and scaffold TMs. GLUT1 forms homo- dimers and homo-tetramers whereas GLUTs 2-4 form homo-dimers. The GLUTs do not hetero-oligomerize. Substituting GLUT1 scaffold TM9 into GLUT3 allows GLUT3 to tetramerize and to form heterocomplexes with GLUT1. GLUT3 TM9 causes GLUT1 to dissociate into dimers. Pathway TMs 5, 8, 2 and 11 may be responsible for isoform specific dimerization. We propose to test this hypothesis directly and outline experiments to investigate how GLUT activity is affected by oligomeric state and whether mutations that cause GLUT1-DS also affect GLUT1 quaternary structure and/or the effects of quaternary structure on function.

Public Health Relevance

Glycopenia (tissue glucose shortage) can have genetic, endocrine and pharmacologic causes, results in seizures, neurologic deficits, coma and, if uncorrected, impairs development. This project investigates the GLUT family of human glucose transport proteins which is responsible for body glucose distribution following its absorption from the gut and kidneys. We ask how these transporters function in human health and disease by using a combination of biochemical, genetic and computational approaches. What we learn will lead to interventions for clinical glycopenia, the development of specific inhibitors of glucose transport by tumors, screening of HIV protease inhibitors that avoid insulin resistance and will also enhance our understanding of the much wider family of transport proteins that distribute substances other than glucose.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01DK044888-22
Application #
8733151
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Sechi, Salvatore
Project Start
Project End
Budget Start
Budget End
Support Year
22
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Worcester
State
MA
Country
United States
Zip Code
01655
Sage, Jay M; Carruthers, Anthony (2014) Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex. Am J Physiol Cell Physiol 306:C910-7
De Zutter, Julie K; Levine, Kara B; Deng, Di et al. (2013) Sequence determinants of GLUT1 oligomerization: analysis by homology-scanning mutagenesis. J Biol Chem 288:20734-44
Robichaud, Trista; Appleyard, Antony N; Herbert, Richard B et al. (2011) Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site. Biochemistry 50:3137-48
Mangia, Silvia; DiNuzzo, Mauro; Giove, Federico et al. (2011) Response to 'comment on recent modeling studies of astrocyte-neuron metabolic interactions': much ado about nothing. J Cereb Blood Flow Metab 31:1346-53
Cura, Anthony J; Carruthers, Anthony (2010) Acute modulation of sugar transport in brain capillary endothelial cell cultures during activation of the metabolic stress pathway. J Biol Chem 285:15430-9
Carruthers, Anthony; DeZutter, Julie; Ganguly, Amit et al. (2009) Will the original glucose transporter isoform please stand up! Am J Physiol Endocrinol Metab 297:E836-48
Leitch, Jeffry M; Carruthers, Anthony (2009) alpha- and beta-monosaccharide transport in human erythrocytes. Am J Physiol Cell Physiol 296:C151-61
Mangia, Silvia; Simpson, Ian A; Vannucci, Susan J et al. (2009) The in vivo neuron-to-astrocyte lactate shuttle in human brain: evidence from modeling of measured lactate levels during visual stimulation. J Neurochem 109 Suppl 1:55-62
Blodgett, David M; Graybill, Christopher; Carruthers, Anthony (2008) Analysis of glucose transporter topology and structural dynamics. J Biol Chem 283:36416-24
Simpson, Ian A; Carruthers, Anthony; Vannucci, Susan J (2007) Supply and demand in cerebral energy metabolism: the role of nutrient transporters. J Cereb Blood Flow Metab 27:1766-91

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