The insulin receptor is a tyrosine-specific protein kinase, but its role in insulin action is unknown. Although many reports have described the kinase activity of purified receptors, only a few have described the phosphorylation events which occur in the intact cell. During the past several years, I have developed experimental systems to study the multisite phosphorylation of the insulin receptor in the intact cell. These techniques will be used to study the relationship between phosphorylation and function of the insulin receptor in primary cultures of isolated rat hepatocytes and a well differentiated, insulin-sensitive hepatoma cell line called Fao. When the experiments are limited by the concentration of insulin receptors in these cells, Chinese hamster ovary (CHO) cells, prepared by transfection to express about 106 human receptors per cell, will be used. Sequential immunoprecipitation with anti-phosphotyrosine (Alpha PY) and anti-insulin receptor antibodies will be used throughout this project to separate the subset of phosphotyrosine-containing receptors from the total pool of insulin receptors. The structural characteristics of each subset will be studied by SDS-PAGE, partial protelytic cleavage, and tryptic peptide mapping. The stoichiometry and cellular distribution of the phosphorylated subsets of the receptor will be determined. The function of the phosphoserine and phosphotyrosine forms of the receptor isolated from intact cells will be studied by in vitro kinase and insulin binding assays. Attempts will be made to determine the role of the individual phosphorylation sites. Potential substrates in the intact cell for the insulin receptor will be identified by immunoprecipitation by Alpha PY. Regulation of the insulin receptor by the calcium messenger system will be explored. The mechanism by which phorbol 12-myristate, 13-acetate (PMA) stimulates serine and threonine phosphorylation of the insulin receptor and inhibits insulin-stimulated tyrosine phosphorylation will be studied. Other agents and hormones which signal cellular events through (Ca2++), cAMP or protein kinase C will be compared to the effects of PMA. The site of phosphorylation will be identified and compared to those obtained by insulin or PMA stimulation. Protein kinase C will be purified from the Fao cell and its effect on the phosphorylation of the purified insulin receptor will be investigated. These experiments will help clarify the insulin signal mechanism.
Showing the most recent 10 out of 13 publications
