INTRACELLULAR SIGNALING BY THE INSULIN RECEPTOR KINASE

University Of Iowa, Iowa City IA

Investigators

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Abstract

DESCRIPTION: The overall goals of this research proposal are to identify and characterize the insulin signal transduction pathways leading to the functional regulation of the protein components directly involved in the intracellular trafficking of GLUT4-containing vesicles. Recently, this investigator and others have demonstrated that syntaxin 4 functions as a plasma membrane t-SNARE whereas VAMP2 functions as a vesicle v-SNARE for insulin-stimulated GLUT4 translocation. In addition to syntaxin 4 and VAMP2, several accessory proteins appear to be necessary for t-SNARE function. In particular, the syntaxin 4 binding proteins Synip and Munc18c have been postulated to have essential functions in insulin-stimulated GLUT4 translocation. However, the precise molecular roles and the mechanism(s) by which insulin regulates these interactions are completely unknown. The preliminary data indicates that Munc18c is required for GLUT4-containing cargo vesicle plasma membrane fusion but is not required for the intracellular trafficking to the plasma membrane. In contrast Synip function is important in the insulin-stimulated binding of GLUT4-containing vesicles to the plasma membrane. To further examine the insulin regulation of SNARE protein interactions mediating GLUT4 translocation and the molecular consequences of these interactions, the investigators will perform a detailed analysis of GLUT4 vesicle trafficking to, binding and fusion with the plasma membrane. In addition, the investigators will use homologous recombination to determine the physiologic role of these proteins in the various steps in GLUT4 translocation. To accomplish these objectives, the investigators specifically determine the cellular stoichiometry of syntaxin 4 bound to Synip, Munc18c and VAMP2. In parallel, the investigators will use time lapse confocal fluorescent microscopy to examine the functional role of Munc18c and Synip in the regulation of GLUT4 vesicle trafficking, plasma membrane binding and fusion. These translocation steps will then be quantitatively assessed by a comparison with glucose uptake, cell surface labeling, subcellular fractionation, plasma membrane sheet isolation and immunoelectron microscopy. To generate a genetic models useful for understanding the physiological role of these protein in vivo, the investigators will generate syntaxin 4, Munc18c, VAM0P2 and Synip knock-out mice. In addition, these mice will be used to prepare mouse embryo fibroblast cell lines that can be transfected with various cDNAs and subsequently differentiated into an adipocyte phenotype. Using this system, the investigators will examine the insulin-stimulated translocation of GLUT4 in specific null backgrounds and upon re-introduction of various mutants of the ablated protein. In this manner, the investigators will establish several of the key biochemical and cell biological pathways regulating the insulin-stimulated trafficking/tethering/docking/fusion of GLUT4-containing vesicles.