Insulin stimulates multiple signaling pathways controlling mitogenic and metabolic outputs. In fat and muscle, at least one of these signaling pathways leads to the redistribution of GLUT4 glucose transporters. Movement of GLUT4 to the plasma membrane is required for insulin to enhance glucose uptake. The insulin signaling pathway(s) involved in this process impinge on a protein complex containing GLUT4 and TUG, a protein that retains GLUT4 intracellularly in unstimulated cells. Insulin stimulates the dissociation of TUG and GLUT4 to redistribute GLUT4 and augment glucose uptake. ? ? We hypothesize that TUG is the first identified component of a large protein complex that is formed on GLUT4, and that this complex is stably assembled in the absence of insulin. Insulin acts to alter the posttranslational modification of one or more components of this complex, causing its disassembly and mobilizing the glucose transporter. A detailed characterization of this protein complex and of how insulin acts on it will be essential to understanding insulin's metabolic actions. ? ? Here, we propose to use proteomic approaches to analyze the protein complex containing TUG and GLUT4.
In Aim 1, we will study the composition of this complex in unstimulated cells. We will use several biochemical methods to purify proteins present in the complex, and will identify these using tandem mass spectrometry.
In Aim 2, we will use proteomic techniques to study how insulin alters the posttranslational modification of proteins in the complex, as well as how it alters the overall composition of the protein complex. We anticipate that the data we obtain will contribute to understanding of the molecular mechanisms by which insulin regulates glucose homeostasis. Additionally, this work may have broader implications for insulin signaling and for protein targeting. ? ?
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