| The epidermal growth factor (EGF) receptor stimulates the activity of a number of cytoplasmic signaling cascades, resulting in a mitogenic response in most cell lines. This dissertation details studies that address the role of the kinase domain of the receptor in EGF receptor signaling by comparing EGF-stimulated responses of cells expressing wild-type EGF receptor and those of cells expressing either of two point mutants that lack significant kinase activity: D813A and K721R.; Investigations of wild-type and mutant EGF receptor function in Chinese hamster ovary cells, which express endogenous ErbB2, are described in Chapter II. Experiments were performed in which the EGF-stimulated responses of cells expressing wild-type and mutant EGF receptors were compared, including interactions of the receptors with Shc and Grb2, receptor internalization, ion uptake, [3H]-thymidine incorporation, and mitotic progression. These data suggest that, in the presence of ErbB2, the D813A and K721R mutants are comparably deficient compared to wild-type EGF receptor and that the two only subtly differ from one another in signaling capacity.; Chapter III describes the activity of wild-type EGF receptor, D813A and K721R when expressed in 32D cells, an interleukin-3 dependent cell line in which no endogenous ErbB proteins are expressed. These results suggest that the EGF receptor requires a functional kinase to stimulate phosphorylation of substrate proteins and mitosis. However, EGF receptor expression is shown to prevent apoptosis in the absence of interleukin-3 but in the presence of serum. While expression of K721R was able to prevent apoptosis in the absence of interleukin-3, expression of D813A was not. These data suggest a role of the EGF receptor in preventing apoptosis in the independent of interleukin-3 but requiring serum. These data also suggest that D813A and K721R differ in their abilities to prevent apoptosis in these cells. While the mechanisms underlying this phenomenon are not yet understood, additional strategies for investigating this phenomenon are proposed in Chapter IV. |
