INSULIN SIGNALING THROUGH GTP BINDING PROTEINS

During our investigation of insulin-stimulated Ras-dependent signal transduction, we have identified an important GTP binding protein involved in the insulin signaling pathway leading to the translocation of GLUT4-containing vesicles to the plasma membrane. We have obtained substantially evidence demonstrating that not only can activation of the trimeric GTP binding protein Gq and/or G11 induce GLUT4 translocation but that this is a necessary event in the insulin-stimulation of GLUT4 vesicle translocation. In order to further investigate the functional regulatory role of Gq/G11 in insulin signal transduction, we plan to determine the upstream mechanism(s) responsible for the insulin-stimulated activation of Gq/G11 in adipocytes. In parallel, we will work downstream to identify the targets of Gq/G11 activation directly leading to the translocation of GLUT4- containing cargo vesicles and their relationship with the insulin-stimulated signal transduction pathway mediated by the activation of the PI 3-kinase. In order to accomplish these goals, we propose two overall specific aims using both biochemical and cell biological approaches to determine the functional role of Gq/G11 in the insulin-dependent signal transduction pathway leading to GLUT4 translocation. This will include the use of adipocyte differentiating mouse embryo fibroblasts derived from Gqalpha null, G11alpha null and double Gqalpha/G11alpha null mice and by determining the insulin- stimulated upstream activation mechanisms and downstream effectors of Gq/G11. We will also use subcellular fraction to determine if Gq/G11 and PI 3-kinase dependent pathways recruit distinct population of intracellular GLUT4-containing compartments, the sum of which then accounts for the full extent of insulin stimulation. This studies will be performed in parallel with time lapse confocal fluorescent microscopy to examine the real time trafficking of GLUT4-containing vesicles to determine the specific steps in the translocation process regulated by Gq/G11 (trafficking, binding and fusion with the plasma membrane. Finally, we will then identify and characterize the downstream tyrosine phosphorylated targets of Gq/G11 activation, with particular emphasis on the approximately 65 kDa insulin-stimulated tyrosine phosphorylated protein that co- immunoprecipitates with Gq/Gli. Together, these studies will provide novel information with regard to the complex insulin signal transduction pathway and mechanisms directly involved in the regulation of GLUT4 vesicle exocytosis and fusion with the plasma membrane.