Diabetes arises from an impairment in insulin synthesis, secretion, or action. One major action of insulin is to stimulate glucose uptake by skeletal muscle and adipose tissue. Insulin-stimulated glucose uptake by cells is mediated by translocation of the glucose transporter protein, Glut4, from intracellular vesicular pools to the cell surface. The Rab4 GTPase has been implicated in insulin-induced Glut4 translocation in several studies. The Rab family of Ras-like GTPases regulate cellular endocytosis and exocytosis, and throughout their catalytic cycles, interact with numerous regulatory proteins. The overall goals of this proposal are to clone and characterize Rab4- interacting proteins in 3T3-L1 adipocytes, and to determine their role in insulin-stimulated translocation of Glut4. We will use a yeast two- hybrid interaction screen to clone Rab4-interacting proteins from a murine adipocyte cDNA library. We will determine the cellular localization and tissue distribution, binding specificities, binding determinants, and activities of these Rab4-interacting proteins using standard molecular techniques, to understand their role in the catalytic cycle of the Rab4 GTPase. Lastly, we will use recombinant adenovirus expression vectors to overexpress these Rab4-interacting proteins in 3T3-L1 adipocytes to determine the effect on insulin-stimulated Glut4 translocation, transferrin receptor recycling, and glucose transport. Understanding the role of Rab4-interacting proteins in insulin- stimulated Glut4 translocation will further the understanding of how insulin mediates metabolic and mitogenic changes in cells and may provide targets for the treatment and control of diabetes and insulin- resistance states.
