The insulin receptor binds its ligand, insulin, at the plasma membrane of the cell. This binding event initiates two processes: 1) Autophosphorylation of the receptor protein on tyrosine residues. This autophosphorylation ultimately leads to the changes in cellular metabolism associated with insulin action. 2) Endocytosis of the insulin-receptor complex and the subsequent movement (routing or trafficking) of the receptor through the multiple compartments of the endosomal pathway. Both of these processes are affected by chronic treatment of the cultured cells with either insulin or glucocorticoids. The hypothesis to be tested in this project is that the key step by which these hormones regulate cellular insulin receptor number is by regulating the rate at which the receptor is inactivated and degraded. From previous work, the inactivation and degradation of the receptor occurs within the endosomal compartment. Therefore, the hypothesis will be tested through the following goals: 1) The various endosomal compartments through which the active insulin receptor moves in the basal or hormone-simulated states will be isolated by preparative isoelectric focusing. By metabolically labeling the receptor with 32 P-phosphate, changes in the autophosphorylation state of the receptor as it moves through the endosomal compartments will be quantified. 2) The mechanism(s) by which the insulin receptor protein is inactivated will be determined in the basal and hormone- stimulated states. Isoelectric focusing will be utilized to ascertain the endosomal compartment in which inactive receptor is activated. The enzymes responsible for these processes will be characterized and isolated. These studies will provide the first characterization off the physico-chemical status of a receptor protein as it moves through the various compartments of the endosomes, and will provide a mechanism by which a membrane-bound receptor is inactivated and removed from the cell.
