Various pathological conditions in man, such as type II Diabetes Mellitus, are characterized by a failure of the cell to respond to insulin. Insulin is a major anabolic hormone, which stimulates the cellular uptake of nutrients and diverse other substances necessary for cellular function. This stimulatory effect is mediated in part through a rapid increase in the cell surface number of specialized transport proteins and of high affinity receptors for extracellular macromolecules such as transferrin, alpha 2 macroglobulin and insulin-like growth factor II. The long term goal of this project is to elucidate the molecular mechanisms that are involved in the stimulation by insulin of the cell surface expression of receptors for extracellular macromolecules. Previous data have shown that the process of endocytosis of high affinity receptors for extracellular macromolecules is regulated by insulin. Preliminary data presented in this proposal, have provided evidence to indicate that the clathrin coated structures in the plasma membrane which mediate the endocytosis of these receptors may be an important target for insulin action. In this work, cellular fractionation techniques, methods for in-vitro reconstitution of clathrin coated pit formation, and monoclonal antibodies to coated membrane components will be used to a) further analyze which of the steps involved in the assembly and disassembly of clathrin coated pits is sensitive to insulin action, b) to test the hypothesis that casein kinase 2 is one of the key elements involved in this process, and c) to test the hypothesis that the effects of insulin on clathrin coated structures and on receptor endocytosis are mediated through casein kinase 2.
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