Role of glucokinase localization in regulating activity

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DESCRIPTION (provided by applicant): The glucose phosphorylating enzyme, glucokinase (GK),acts as the glucose sensor in glucose stimulated insulin release from the pancreatic B-islet cells. Although the enzymatic characteristics of GK have been characterized extensively, the mechanism of GK activation has remained elusive. Recently, electron micrographs of GK distribution in pancreatic islets have revealed that GK is located on insulin secretory granules. Given that GK activity in other tissues is regulated through changes in localization, it is proposed that GK association with insulin secretory granules may be directly related to GK activity. In order to test this hypothesis, the dynamics of GK association with insulin granules will be assessed in cells expressing a fluorescently tagged GK chimera. Quantitative bleaching studies will be used to characterize the association of GK with insulin granules and specifically examine the role of glucose-GK interactions in triggering GK redistribution to the cytoplasm. The protein domains in GK that participate in targeting OK to insulin granules will be identified by deletion mutagenesis. Identification of the GK targeting domain will allow specific disruption of GK targeting by site-directed mutagenesis and examination of the importance of GK translocation to glucose metabolism in the B-cell. These studies will further our understanding of GK regulation in the pancreatic islet and the role of GK compartmentalization in glucose stimulated insulin secretion.