REGULATION OF GLUCOSE TRANSPORTER TARGETING AND ACTIVITY

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Not all of the molecular events are understood which provide proper regulation of glucose transport across the plasma membrane of cells in healthy individuals, or disrupt regulation in type II diabetics. GLUT1 and GLUT4 are two members of a homologous family of sodium independent glucose transporters which can be characterized as low and high response transporters, respectively, primarily by their relative ability to redistribute to the plasma membrane in response to insulin. Differences in kinetic parameters for glucose transport, membrane targeting in basal and insulin stimulated states, and susceptibility to degradation are important properties which distinguish these two transporters. There are 5 major domains of non-identity which could confer unique structural and hence the unique functional properties which characterize each transporter. They include the: 1)N-terminus, 2)C-terminus, 3) large extracellular loop, 4)large intracellular loop, and 5)transmembrane domains 2/3. The primary goal of this proposal is to compare the properties of chimeric transporters formed by interchanging each of these domains between GLUT1 and GLUT4. If the domain transferred confers one or more of the unique distribution, kinetic, or stability properties upon the native transporter, then that property should be transferred from the native donor transporter to the recipient chimeric transporter. A recently isolated transporter cDNA, designated GLUT4B, encodes a variant transporter in which transmembrane domain XII and the C-terminal domain of GLUT4 are replaced by a unique 33 amino acid sequence. The properties of GLUT4B are unknown and therefore an additional goal of this proposal is to characterize and compare GLUT4B to GLUT4 to assess how the structural differences of GLUT4B might alter transporter function. To accomplish these goals native and chimeric transporters will be expressed in Xenopus oocytes by microinjecting in vitro transcribed transporter message, and the kinetic constants for transport determined by measuring 3-O-methylglucose flux. Chimeric transporters will also be expressed in insulin responsive 3T3-L1 adipocytes via a retroviral vector, and the rates of transporter degradation compared by pulse chase labelling 3T3-L1 adipocytes with 35S-methionine and immunoprecipitating each labelled chimeric transporter with domain specific antibodies. The membrane distribution of native and chimeric transporters will be determined in both oocytes and 3T3-L1 adipocytes by using membrane fractionation techniques documented for each cell type and quantifying the solubilized transporters with domain specific antibodies. The effect of insulin on the membrane distribution of chimeric transporters will be measured in 3T3-L1 adipocytes. Identifying which domains confer one or more unique functions to GLUT1,GLUT4, or GLUT4B, as well as understanding which interchanged domains in chimeric transporters alter or destroy transporter function will further define structural regions important to the normal function of each transporter. These studies hopefully will provide groundwork for future studies designed to identify regulatory proteins interacting with these domains and evaluate whether mutations in these proteins or transporter domains might account for some forms of insulin resistance in type II diabetes.