Thirteen "glucose transporters" (GLUTs 1-12 &HMIT) catalyze equilibrative sugar transport in humans. The GLUTs are members of a wider family of Major Facilitator Superfamily (MFS) transporters that catalyze transport of a diverse array of molecules but share a common architecture of 12 membrane-spanning helices (TMs) with cytoplasmic N- and C-termini. The GLUTs display distinctive substrate specificities and sensitivity to inhibition by small molecules and a range of transport behaviors including uniport, symport, antiport and oligomerization-dependent cooperativity. This proposal describes our continuing efforts to understand GLUT function in health and disease by determining the molecular basis of GLUT-mediated substrate transport. To do this, we must resolve: 1) Determinants of substrate specificity and their locations within GLUT architecture;2) How some transporters catalyze uniport while others catalyze symport or antiport; 3) Which transporter elements promote transporter oligomerization and cooperativity. The GLUTs are well-suited to such studies being amenable to biochemical, molecular and kinetic analysis. This proposal exploits the structural similarities of the GLUT family of proteins to answer these questions. Our studies of GLUT1/GLUT3 chimerae show that GLUT1 TM9 is essential for GLUT1 oligomerization.
Specific Aim 1 tests the hypothesis that TM9 presents a dimerization surface to adjacent GLUT1 subunits by investigating the ability of wt-TM9 and TM9 dimerization surface mutants to promote chimerae oligomerization by using novel co-immunoprecipitation, chemical crosslinking and TOXCAT assays. We also ask whether other GLUTs form homo- and hetero- oligomers. Recognizing that GLUT5 is a cytochalasn B (CB) insensitive fructose transporter that cannot transport 2-deoxy-D-glucose (2DOG) and GLUT1 is a CB-inhibited 2DOG transporter that does not transport fructose, Specific Aim 2 tests the hypothesis that specific clusters of GLUT sequence form exo- and endofacial substrate binding sites. GLUT1/GLUT5 chimerae will be assayed for GLUT5-sequence dependent loss of CB- inhibtion, loss of 2DOG transport and gain of fructose transport. Reciprocal gain of function studies with GLUT5/GLUT1 chimerae will verify the results. A novel, scanning factor Xa proteolysis technique will also define GLUT exo- and endofacial ligand binding sites. GLUTs 1 and 3 show "trans-acceleration" - accelerated sugar uptake in cells preloaded with sugar. GLUTs 2 and 4 do not.
Specific Aim 3 tests the hypothesis that specific clusters of GLUT sequence determine antiport (trans-acceleration) and symport functions by scanning GLUT1/GLUT4 or GLUT1/GLUT2 chimerae mutagenesis. Chimerae will be assayed for GLUT2/4-sequence dependent loss of trans-acceleration and reciprocal gain of function studies with GLUT4/GLUT1 chimerae used to verify the results. Scanning GLUT1/HMIT chimerae will be used to expose HMIT domains required for H+:myo-inositol symport. Our results will be mapped onto GLUT1 structure to provide new insights into GLUT structure and function and will have broad significance to the MFS proteins that mediate organismal energy homeostasis, solute equilibrium and drug delivery/resistance.

Public Health Relevance

Glycopenia (tissue glucose shortage) can have genetic, endocrine and pharmacologic origins, results in seizures, focal neurologic deficits, coma and, if uncorrected, impairs development. This proposal continues our efforts to understand how the family of human glucose transport proteins allows organs and cells to absorb glucose and other sugars from the blood. The insights we gain from these studies will impact our understanding of the wider family of Major Facilitator Superfamily transport proteins. Our long-term goal is to translate these insights into practical intervention in clinical glycopenia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK044888-20
Application #
8266393
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Sechi, Salvatore
Project Start
1992-05-01
Project End
2013-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
20
Fiscal Year
2012
Total Cost
$282,147
Indirect Cost
$110,629
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
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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