The goal of this proposal is to use morphological and biochemical approaches to analyze insulin receptor regulation. Ultrastructural studies on insulin-sensitive cells have shown significant cell-specific heterogeneity in the organization, distribution and mobility of insulin receptors. These morphological observations correlate with cell-specific differences found in biochemical binding studies on the same cells. Recent evidence suggests that cell-to-cell variability may exist in the processes involved in insulin receptor internalization and recycling. The specific aims of this proposal are: (1) to qualitatively and quantitatively determine and compare insulin receptor organization and distribution on different cell types. Relationships, if any, will be established between occupied insulin receptor and specific cell surface structures and components. (2) to analyze the post-occupancy mobility of insulin receptors on cells that demonstrate occupancy-induced changes in receptor organization or distribution. Various chemical reagents will be used in attempts to block receptor microaggregation in order to determine the mechanisms involved and relationships to insulin action. (3) to determine the routes and subcellular structures involved in the internalization, degradation or processing, and recycling of insulin and insulin receptors. In addition to using monomeric ferritin-insulin for morphological studies, immunocytochemistry using both anti-insulin receptor antibody and biotinyl-labeled insulin binding to insulin receptors followed by antibody or avidin conjugated colloidal gold particles and/or peroxidase will be used to demonstrate the insulin receptors in intact as well as permeabilized cells. This laboratory is in a unique position to accomplish this proposal because of our proven capability to perform both quantitative morphological and correlative biochemical studies. These studies will contribute to our understanding of insulin action and of possible sites for post-binding defects in insulin resistant states.