Expression and function of epidermal growth factor (EGF) receptor is modulated by viral transformation. Normal rat kidney cells possess 300,000 EGF receptors per cell. Upon transformation by Kirsten murine sarcoma virus, cells lose their ability to bind EGF. In cells transformed by a temperature-sensitive mutant virus, the loss of EGF binding also correlates with the transformed phenotype. The mechanism leading to the loss of EGF binding in transformed cells will be approached by studying (1) the state of phosphorylation of EGF receptor, (2) the possible interaction of C-kinase with the EGF receptor in transformed cells and (3) the possible alteration in the carbohydrate moiety in the EGF receptor of transformed cells. To study the difference in the state of receptor phosphorylation in normal and transformed cells, membranes are treated with alkaline phosphatase to remove phosphate groups. Dephosphorylated membranes are assayed for their EGF binding activity, tyrosine kinase activity, and autophosphorylating activity. The possibility of receptor phosphorylation by C-kinase is carried out by labeling cells in vivo with [32P]Pi or in vitro by phosphorylating membranes with [gamma-32P]-ATP followed by immunoprecipitation with EGF receptor antibody. If C-kinase phosphorylates EGF receptor in transformed cells, additional phosphopeptides should be observed. Membrane-bound and cytosolic C-kinase activity in normal and transformed cells will also be assessed. Alteration of sugar residues in the receptor in transforming cells is examined by analyzing the size and oligosaccharide composition of [3H]-mannose labeled cells. The same strategy will be used to investigate the modulation of receptors by temperature in cells transformed by a temperature-sensitive mutant virus. Information obtained from this study will help to elucidate the roles of phosphorylation, glycosylation and interaction with C-kinase in the function of EGF receptor.
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