The long-term objective of the studies described in this proposal is to understand the mechanisms of biosynthesis and regulation of the mammalian facilitated diffusion glucose transporter (GT). These investigations are of medical significance because of the importance of the GT in the control of glucose homeostasis, the disruption of which is a consequence of several disease states, most notably the hyperglycemia and insulin-resistance observed in noninsulin-dependent diabetes. Moreover, the GT has proven to be an interesting model to study the mechanism of biosynthesis of multi-spanning membrane transport proteins. To achieve this objective, the specific aims of this proposal are: 1) To detemine the levels of control of GT activity in mouse 3T3 adipocytes in response to insulin, sulfonyl ureas, and modulation of glucose levels. Antibodies and a cDNA clone will be used to measure levels of GT activity, steady-state protein, rate of synthesis, mRNA, and gene transcription. 2) To transfect the human GT cDNA into 3T3 adipocytes and determine the subcellular distribution of the transfected protein in response to insulin. Site-directed mutagenesis of th cDNA clone prior to transfection will be used to identify putative translocation and targeting signals in the protein. 3) To clone and charaterize the human GT gene. 4) To use site-directed mutagenesis of a full-length GT cDNA clone in conjunction with fusion-protein constructions to identify and define informational amino acid sequences within the protein required for proper insertion into microsomal vesicles in in vivo translation/translocation systems.
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