THe initial event in glucose-stimulated insulin secretion by pancreatic islet beta cells is the facilitated of glucose across the cell membrane via a glucose transporter (GT) protein. The GT protein family be subdivided, on the basis of functional transport kinetics and tissue-specific expression, into three GT the brain/erythrocyte GT, the islet/liver GT, and the adipocyte/muscle GT. Although the three GT share amino acid homology and a common predicted protein structure, there is little information regarding the structure-function relationship of the GT proteins or regarding which domains of each GT are responsible for the distinctive glucose transport kinetics of each GT species. In the proposed project, putative proteins domains encoded by the islet GT cDNA and adipocyte GT cDNA will be exchanged produce chimeric islet-adipocyte GT cDNAs. The chimeric GT cDNAs will be expressed in Xenopus oocytes and the Km and Vmax for glucose influx and efflux, the Km and Vmax under equilibrium exchange and the binding constants for cytochalasin B determined. These kinetic properties differ markedly between the islet GT and the adipocyte GT. Comparison of the kinetics of glucose transport between the chimeric and the unmodified islet and adipocyte GTs will identify protein domains responsible for the distinctive kinetics of each GT species. Based on this information, precise mapping of the amino acid residue(s) responsible for the distinctive kinetic properties will be achieved by site-directed mutagenesis. In parallel approach to study the structure-function of the islet GT, the islet GT from an insulin-producing islet line with abnormal glucose transport kinetics despite normal levels of the islet GT RNA will be characterized by expression in oocytes, by determination of the nucleotide sequence and by immunoblotting with an islet GT specific antiserum. This will investigate the role of either a mutation in the islet GT cDNA or an islet cellular factor that suppresses the function of the islet GT and will provide insight into the function the normal islet GT protein. The proposed project will utilize recent advances in GT molecular biology to elucidate the domains the islet GT protein responsible for its unique functional characteristics and to identify structure-function relationships of the islet and adipocyte GT proteins. These results will provide new insights into the molecular mechanisms of glucose transport in islet cells.
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