It is widely believed that oncogene products alter growth regulation of cells by interacting with the machinery controlling normal growth and metabolism. Although src was the first oncogene to be identified at the molecular level, there have been few detailed investigations of the effects of pp60v-src on the molecular machinery of growth control. During the previous grant period we investigated the effects of src on the EGF receptor, and determine that src expression causes a profound decrease in the number of EGF receptors, largely by inhibiting receptor biosynthesis. During the coming grant period we will analyze at the molecular level the basis for decreased EGF-receptor biosynthesis in src-transformed cells. We also will begin to focus on the insulin family of receptors, since insulin and/or IGFs are required for the growth of the fibroblastic cells which are targets for transformation by src, and because we have evidence that the functioning of these receptors may be altered in src-transformed cells. For these studies, we will take advantage of a large panel of src mutants which have been well defined both biologically and molecularly. Such mutants provide an exceptional opportunity to explore the relationships between oncogene structure, specific phosphorylations and biological phenotype.
Our specific aims are as follows: 1. Down-modulation of the EGF receptor in src-transformed cells. a. Regulation of EGF receptor gene expression. b. Degradation of the EGF receptor. 2. Structure and function of insulin and IGF receptors in src- transformed cells. a. Measurements of receptor amounts and ligand-binding affinities. b. Measurements of receptor kinase activity and phosphorylation state. c. Analysis of the mechanisms of src-induced changes in receptor structure and function. 3. Second messengers in src-transformed cells. a. Turnover of phosphatidyl inositol and the generation of related metabolites. b. Analysis of cAMP levels. c. Investigation of possible mediators of insulin action.
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