The long term goal of this proposal is to identify the molecular defect(s) underlying the insulin-resistance seen in human diabetes and obesity. More immediate goals are to understand the molecular mechanisms how the facilitative glucose transporters, a family in intrinsic transmembrane proteins, catalyze translocation of selected sugar molecules. We will use purified human erythrocyte glucose transporter (HEGT) and study its tertiar structure, particularly that of putative glucose channel. A hydropathy analysis of the amino acid sequence of this protein predicts that it contains a transmembrane domain made of twelve transmembrane segments, five of which are amphipathic and may line the purported aqueous channel. It also contains three intracellular and one extracellular nonmembrane domains We use this prediction as a working model in determining the channel structure. We will determine which of these twelve transmembrane segments are positioned in contact with the lipid bilayer, and which the aqueous channel. We will first establish the basic chemical methodology required for the studies. This will include separation and identification of the twelve transmembrane segments. To determine the native tertiary structure of these transmembrane segments. We will chemically crosslink and identify neighboring transmembrane segments, and identify the channel-forming segments and nonchannel-forming segments by measuring the accessibility of segments to transportable, covalently active glucose analogs (4ADG and 6ADG and lipophilic covalent probes, respectively, Based on these data, we will deduce the overall structure of the transmembrane domain. There are strong indication for the importance of intrinsic activity- regulation of glucose transporter function in glucose homeostasis in normal and diseased states. Understanding of this regulation of the intrinsic activity at the molecular level would be greatly assisted once we understan the tertiary structure of the glucose channel as proposed here.
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