The main goal of this proposal is the characterization of the mechanism of insulin mediator generation. Preliminary work shows that insulin stimulates the release of proteins anchored to the membrane by phosphatidylinositol-glycan (PI-glycan) following a time course which is consistent with the generation of a putative insulin mediator. Furthermore, we have shown that insulin stimulation induces a rapid appearance of this mediator in the extracellular medium. Based on these and other experiments using specific protease and cellular phospholipase C blockers, it is hypothesized that at least some of the mediators of insulin action originate from the PI-glycan anchored membrane proteins. A specific mechanism according to which two hydrolytic steps at the membrane surface are required for the generation of mediators is proposed. This mechanism also hypothesizes the existence of a mediator transport/uptake system. This project addresses experimentally the critical aspects of this model. The experiments are aimed to demonstrate that insulin promotes the cleavage of the PI-glycan anchor of certain membrane proteins, to investigate whether or not this cleavage is directly associated to the generation of insulin mediators and to identify at least some of the possible protein precursors. It is also proposed to investigate the specific mechanisms of cleavage using metabolic labeling techniques. Finally, on the basis of some very promising preliminary data, it is proposed to take advantage of the close structural similarities of the PI-glycan anchor of the variant surface antigens of trypanosomes with the PI-glycan anchor of mammalian proteins in order to probe the enzymes involved in the insulin-stimulated release of these proteins and the hypothesized mechanisms of mediator uptake or transport.
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