Insulin is a major hormone involved in overall glucose homeostasis, and abnormalities of insulin action are central to the pathogenesis of a variety of disease states. Consequently, detailed knowledge of the basic events underlying insulin's ability to interact with cells will lead to a better understanding of the mechanisms of disease states in which insulin action is abnormal. Therefore, the overall goals of this research proposal are to study the relationships between insulin binding to its cell surface receptors and the events which occur following this initial step. These events include: 1) activation of the glucose transport system, 2) internalization with subsequent lysosomal processing and hormone degradation, and 3) receptor regulation. To achieve these goals, several approaches will be taken. We will use a newly discovered non-transformed human mutant fibroblast line, which displays a defect in coupling between insulin receptors and the glucose transport system, in order to elucidate the cellular mechanisms of this defect and to learn more about the basic events in insulin action. Additionally, we have recently identified a diabetic patient who secretes a biologically inactive insulin molecule. We plan to use this mutant insulin species to study the various events following insulin's initial interactions with cells, with specific focus on assessing the mechanisms whereby this mutant insulin antagonizes the biologic effects of normal insulin. Furthermore, we will explore the concept of insulin receptor heterogeneity and attempt to quantitate the relationships between the endocytotic-lysosomal pathway, insulin degradation, activation of glucose transport, and receptor regulation. In these experiments, the anti-insulin receptor antibody will be used as an important probe. To further understand insulin action, we will examine the role of intracellular energy in the coupling between insulin receptor complexes and various insulin effector systems. Lastly, the relationships between deactivation of glucose transport and dissociation of insulin receptors will be examined. The studies outlined in this proposal should provide needed information which will further our understanding of the cellular actions of this important hormone.