The interaction of growth factors with their receptors plays a central role in the pathophysiology of the enormously prevalent and disabling conditions of diabetes mellitus and malignancy. Gaining insight into the intricacies of the sometimes overlapping, sometimes distinct signalling pathways of IGF-I and insulin is of pivotal importance to understanding both receptor biology and the disease states arising from abnormal hormone-receptor interplay. In this application, I propose to study the hormone-receptor interactions of IGF-I and insulin as they bind to receptors and activate the cellular machinery for mitogenesis and metabolism. The application is divided into three sections: 1. Hybrid Receptors and Ligand-Receptor Interaction: The binding characteristics and the function of hybrid IGF-I:insulin receptors will be studied using the techniques of immunoprecipitation, Western blotting, and autophosphorylation. It will be determined whether one receptor (IGF-I, insulin or hybrid receptor) can bind two molecules of insulin or two molecules of IGF-I or both insulin and IGF-I at the same time. 2. IGF-I Receptor Structure-Function Relationships: Site-directed mutagenesis will be applied to the IGF-I receptor to define receptor domains responsible for specific biological actions of the receptor. Mutant IGF-I receptors will be constructed with the goal of gaining new insight into the signalling mechanisms of the receptor. 3. IGF-I Receptor Signalling: Experiments are proposed to investigate the mechanism by which the IGF-I receptor utilizes tyrosine kinase autophosphorylation to activate the cellular machinery. Immunoblotting techniques will be used to find proteins associated with the IGF-I receptor, specifically looking for proteins with SH2 domains. Immobilized SH2 domain fusion proteins will be used to study the interaction between the SH2 proteins, the IGF-I receptor and mutant receptors.
