The overall objective of this project is to understand the molecular mechanisms by which insulin regulates glucose transport, specifically the translocation of GLUT4 glucose transporter proteins from a specialized intracellular membrane compartment to the cell surface membrane. Current data indicate that this specialized GLUT4-sequestration compartment exhibits a characteristic density upon velocity gradient centrifugation and is partially depleted upon insulin stimulation. Thus, this membrane organelle appears to be the direct target of insulin action relevant to GLUT4 translocation. This project will address two critically important questions: 1). What structural elements direct GLUT4 transporter proteins to the insulin-sensitive intracellular membrane compartment in insulin-target cells? 2). How does insulin receptor signaling regulate this specialized compartment, causing GLUT4 translocation? Question 1 will be addressed by expression in 3T3-L1 adipocytes of exofacial HA-tagged glucose transporter chimerase containing selective mutations of motifs in the GLUT4 COOH-terminal region that may operate independent of the dileucine motif in directing intracellular retention of GLUT4. Experiments designed to address question 2 will exploit new exciting data from this laboratory suggesting that insulin causes specific targeting of PI 3-kinase activity to the GLUT4 sequestration compartment. This hypothesis will be tested rigorously in both 3T3-L1 adipocytes and primary rat fat cells using velocity gradient centrifugation for isolation of GLUT4-enriched membranes. Novel PI3-kinases expressed in 3T3-L1 adipocytes will be identified by molecular cloning (one already in hand) to determine whether multiple PI 3-kinase enzymes may be involved in the GLUT4 trafficking pathway. Cellular localizations and insulin-sensitivity of such novel PI 3-kinases will be determined. The applicant will also test the hypothesis that specific proteins are recruited to or released from GLUT4-enriched membranes acutely in response to insulin (perhaps in response to localized 3' phosphoinositide generation) using high resolution two-dimensional gel electrophoresis. One such protein which is rapidly increased in content in GLUT4 vesicles by insulin has already been identified in this laboratory and is an exciting new candidate for regulating the movement of GLUT4 to the plasma membrane. The objective is to isolate cDNA clones of such insulin-regulated proteins in GLUT4-enriched vesicles as a first step in evaluating their functions sin the trafficking of these membranes.
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