The mechanism of action of insulin and epidermal growth factor was studied by genetic and biochemical means. Particular emphasis was placed on the ability of these factors to induce DNA synthesis and the relationship of endocytosis to that ability. Insulin was crosslinked to the active fragment A of diphtheria toxin. This conjugate specifically killed cultured mouse cells through an insulin receptor-mediated process. The conjugate was used to select genetic variants resistant to its cytotoxic effect. Six resistant variants were isolated, 2 of which retained very low insulin receptor activity. When these two variants were further analyzed both displayed altered cell shape and growth properties. The CI-3 variant also was shown to have a deficient lysosomal system and failed to efficiently degrade epidermal growth factor. This variant was, however, fully responsive to the mitogenic action of EGF. This suggested that lysosomal processing is unimportant in the production of a mitogenic stimulus by EGF. EGF was found to be endocytosed by fibroblasts through 2 separate pathways. One pathway involves an unidentified organelle and correlated with increased degradation of the ligand. The other pathway involves a Golgi-like component and is correlated with a lack of degradation and uptake into a dense, non-lysosomal organelle. Uptake of EGF into this non-lysosomal component, which we named mitosomes, correlated with the ability of EGF to induce DNA synthesis. From these results, a model was constructed for the coupling of endocytosis, uptake into mitosomes and the stimulation of DNA synthesis.

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published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy