The reduction of the number of cell-surface insulin receptors by insulin contributes to insulin resistance. A current model for receptor regulation proposes that insulin shifts the distribution of surface receptor to internal sites, increasing receptor degradation in some cells. The hypoglycemic drug tolbutamide counteracts this process by unknown mechanisms increasing the level of surface receptor expressed by the cell. The proposal will continue an evaluation of the steps in receptor processing regulated by insulin and tolbutamide in 3T3-L1 adipocytes. The kinetics of surface receptor movement will be assessed by quantitating both receptor association with clathrin coated vesicles (coated pits) isolated with anticlathrin antibody, and its transit between surface and internal pools represented by its respective sensitivity or resistance to proteolysis. The effects of tolbutamide and insulin on the internal concentration and rate of degradation of labelled receptor will be measured and their relationship established. These studies and further analysis of photolabelled receptor metabolism should define the site of action of these agents and determine the role of receptor occupancy and dissociation in normal receptor regulation. The relationship of receptor location to phosphate and sialic acid content, and to modifications affecting pI and size will be monitored. Protein interacting with the receptor also will be identified by adsorbing labelled cell extracts to purified receptor bound to nitrocellulose or affinity supports, and by in situ crosslinkling of labelled cellular proteins to receptor in intact or permeabilized cells. Proteins interacting with the receptor in an insulin or tolbutamide regulated manner will be purified and further characterized. In both phases receptor will be isolated and quantitated by immunoprecipitation and gel electrophoresis. These studies should more directly define the kinetics and routes of receptor processing, identify the important receptor interactions or modifications involved, and their role in mediating the effects of insulin and tolbutamide on receptor processing. These studies should provide better understanding of the effects of some insulin resistant disease states and their treatment on insulin receptor metabolism and function.
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