Tumor promoters accelerate the process of cellular transformation through alteration of normal cellular growth and differentiation processes. One approach that has yielded important information regarding the regulation of growth factor receptors and the mechanisms by which tumor promoters act is to study the interaction of tumor promoters with receptor systems. Thapsigargin is a protein kinase C-independent tumor promoter that has recently been shown to alter epidermal growth factor (EGF) receptor binding and kinase activity. Evidence suggests that the response of the receptor is due to activation by thapsigargin of a kinase that phosphorylates select sites on the EGF receptor that differ from those phosphorylated by protein kinase C. We now propose to identify these sites and to directly test their role in the action of thapsigargin through site-directed mutagenesis studies of the EGF receptor. We also plan to investigate the mechanism by which thapsigargin stimulates kinase activation. These studies provide a new approach to understanding the regulation of cellular receptors by exogenous agents, and will lead to the elucidation of new signal transduction pathways in the cell.