The overall objective of this project is to elucidate the molecular details of the cell surface receptors for insulin and the insulin-like growth factors, and to relate these structures to their biological functions. Experiments performed over the previous grant period have allowed the successful affinity labeling of these receptor structures and the determination of the general subunit compositions and stoichiometries of the receptor complexes. The proposed studies in this application seek to define the molecular basis of the striking similarity in the receptor structures for insulin and insulin-like growth factor-I. These receptors will be purified using a newly developed preparative dodecyl sulfate gel procedure to purify microgram quantities of pure Alpha and Beta subunits from both the insulin and IGF-I receptors. These receptor subunits will be biosynthetically labeled by incubations of cells in the presence of high specific activity amino acids. Microsequencing of cynanogen bromide and trypsin fragments of the receptors will be performed to evaluate possible amino acid sequence homologies between these receptor structures. In addition, cell mutants will be selected for in a H-35 hepatoma cell line which we have shown responds exquisitely to low concentrations of insulin. Such mutants defective in their response to insulin will be characterized for the cellular locus which is defective (receptor or coupling mechanism). Parallel experiments will be performed which will attempt to covalently crosslink the insulin receptor to putative coupling proteins in the cell surface membrane. The approach has been very successful for affinity labeling the receptor itself and may prove to be useful for identifying other cell components involved in insulin action. Other experiments will attempt to study the effect of physiological concentrations of insulin on the receptor for insulin-like growth factor-II. Experiments in our laboratory have indicated a ten-fold increase in the affinity of this latter receptor for its ligand in response to insulin. Our experiments will attempt to combine membrane chemistry technology with affinity labeling and binding methodology to identify components that may be acting to transduce the effect of insulin receptor on IGF-II receptor.
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