Cloudman S91 melanoma cells have an unusual response to insulin in that their proliferation is inhibited upon exposure to the hormone in culture. We have taken advantage of these results and are isolating a variety of mutant cell lines which are resistant to the growth inhibition by insulin. We are attempting to select for temperature-sensitive mutants in hopes that any biochemical lesions we identify will also be temperature-sensitive. We propose to analyze these mutants by combining genetic and biochemical techniques. Particular emphasis will be placed on the identification of cell lines having receptors for insulin which differ from the receptors in wild type cells. Insulin receptors on mutant and wild type cells will be characterized from a number of standpoints including affinity constants, number of receptors, pH optima, kinetics of the binding reaction, and cooperativity of binding sites. The specificity of the receptors will be analyzed by the ability of unlabelled insulins from different species, insulin derivatives, and insulin-like growth factors to compete with 125I-insulin for binding sites. Also cellular degradation of insulin and internalization of insulin will be investigated. In addition we will assay the effects of insulin on glucose transport and fatty acid synthesis in mutant and wild type cells to determine whether there is any correlation between the control of these metabolic pathways and the control of proliferation. Finally, we will perform genetic analyses with the techniques of somatic cell hybridization to investigate the number and nature of the genes involved in the insulin response. We feel that these approaches should yield important information on the basis of action of insulin.
NIH ExPorter
NIH ExPorter