The ultimate goal of this investigation is to define at a molecular level, the mechanisms by which insulin and glucocorticoids affect the dynamic cellular processing of the insulin receptor. Chronic treatment of cultured fibroblasts with insulin alone or dexamethasone alone has opposite effects on the steady-state insulin receptor level: insulin decreases or """"""""down-regulates"""""""" the cellular insulin receptor level, and dexamethasone increases or """"""""up-regulates"""""""" the receptor level. While the effects of these drugs are opposite in nature, both agents appear to act at a common locus: the rate of inactivation of receptor. The working hypothesis is, therefore, that the key step in which hormones regulate cellular insulin receptor level (and thereby, cellular responsiveness to insulin) is by regulating not receptor synthesis, but the rate at which receptor is inactivated (i.e. rendered incapable of binding insulin). This application proposes to define, on a molecular level, the component events which constitute this key regulatory process. Through the combined use of biochemical, immunochemical and genetic approaches, it will be established how insulin affects these cellular events to promote down-regulation and how glucocorticoids act upon these processes to induce up-regulation. In order to achieve these goals, the following specific aims will be met: 1) The changes in the physico-chemical status of the insulin receptor upon its internalization, inactivation and eventual degradation will be established using both immunochemical and affinity lebeling techniques. 2) The modification(s) of the receptor protein catalyzed by the enzymes responsible for the translocation, inactivation and degradation will be determined. 3) The mechanism(s) by which insulin and glucocorticoids act to down- and up-regulate, respectively, the insulin receptor expression will be established by examining their actions to alter the events found to constitute the translocation, inactivation and degradation processes.
