This proposal is a cont1nuing effort to elucidate the molecular mechanism of the human erythrocyte glucose transporter function. The long term objects are to understand the nature of the defect underlying insulin-resistive states seen in Type II diabetes and obesity. More immediate objectives are to determine tertiary structure of the transporter and the conformational dynamics associated with the transport function. The amino acid sequence of this protein is known, which predicts that the protein is made of twelve transmembrane segments and four nontransmembrane hydrophilic segments. Based on these predictions, the following experiments are proposed. First, these transmembrane and nontransmembrane segments will be isolated and identified by protease-digestion. HPLC-separation and determination of characterlstic amino acid contents predicted by the model. The three dimensional arrangement of these segments will be determined by labelling them with crosslinking reagents, lipophilic covalent probes, and tritiated water protons. Possible changes in these three dimensional arrangements upon the removal of the nontransmembrane, hydrophilic segments will be monitored by the same procedures. The changes will also be monitored based on circula, dichroism, Fourier transform infrared spectroscopy, radiation inactivation target size and fluorescence energy transfer measurements. The substrate binding sites in the substrate recognition pockets and in the translocatlon pathway in the protein will be studied by tagging them with photoreactive substrate analogs. Side-specific, nontransportable analogs and transportable analogs will be used to differentiate these sites. Cytochalasin B binding pocket will be characterized by identifying its binding site. Those transmembrane or nontransmembrane hydrophilic segments that are affected by the substrates or inhibitor-induced conformational perturbation will be determined by labelling and identifying the amino acid residues whose reaction to certain alkylating reagents are known to inactivate the protein function and are sensitive to the substrate and inhibitors. Four different alkylating reagents will be used for this purpose. Lastly, as a long term goal, crystallization of the purified transporter protein will be attempted for future x-ray diffraction studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK013376-18A1
Application #
3225027
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-07-01
Project End
1990-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
18
Fiscal Year
1987
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
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Hah, Jong Sik; Ryu, Jiwon; Lee, Wan et al. (2002) The hepatocyte glucose-6-phosphatase subcomponent T3: its relationship to GLUT2. Biochim Biophys Acta 1564:198-206
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Lachaal, M; Spangler, R A; Jung, C Y (2001) Adenosine and adenosine triphosphate modulate the substrate binding affinity of glucose transporter GLUT1 in vitro. Biochim Biophys Acta 1511:123-33
Lee, W; Ryu, J; Hah, J et al. (2000) Association of carboxyl esterase with facilitative glucose transporter isoform 4 (GLUT4) intracellular compartments in rat adipocytes and its possible role in insulin-induced GLUT4 recruitment. J Biol Chem 275:10041-6
Lee, W; Ryu, J; Spangler, R A et al. (2000) Modulation of GLUT4 and GLUT1 recycling by insulin in rat adipocytes: kinetic analysis based on the involvement of multiple intracellular compartments. Biochemistry 39:9358-66
Lee, W; Ryu, J; Souto, R P et al. (1999) Separation and partial characterization of three distinct intracellular GLUT4 compartments in rat adipocytes. Subcellular fractionation without homogenization. J Biol Chem 274:37755-62
Shi, Y; Samuel, S J; Lee, W et al. (1999) Cloning of an L-3-hydroxyacyl-CoA dehydrogenase that interacts with the GLUT4 C-terminus. Arch Biochem Biophys 363:323-32
Zeng, H; Parthasarathy, R; Rampal, A L et al. (1996) Proposed structure of putative glucose channel in GLUT1 facilitative glucose transporter. Biophys J 70:14-21

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