Identification of epidermal growth factor receptors in a hyperproducing human epidermoid carcinoma cell line.

The human epidermoid carcinoma cell line A431 has an usually high number of specific saturable receptor sites for the mitogenic polypeptide hormone epidermal growth factor (EGF). When A431 cells were treated with unlabeled EGF in the culture medium, approximately 50% of the EGF specific binding activity was lost within a few hours (receptor down regulation). A comparison of the surface proteins of untreated and down-regulated cells by surface iodination and gel electrophoresis revealed the EGF receptor of this cell line to be a protein of apparent IV, = 175,000. In contrast to observations with normal mouse and human fibroblasts, the EGF receptor on A431 cells is one of the most heavily labeled proteins following surface-specific iodination. The EGF receptor on A431 cells was also identified by “direct labeling,” a process in which a portion of the cell-bound radiolabeled EGF becomes attached to receptor in a form not dissociable by boiling in aqueous sodium dodecyl sulfate solution containing &mercaptoethanol. Gel electrophoresis of solubilized A431 celi proteins direct-labeled by incubation with high specific activity ‘251-EGF revealed a major radioactive band of apparent M, = 180,000. The small difference in mobility between receptor labeled by surface iodination and direct labeling can be explained by increased molecular weight due to attachment of EGF to its receptor. The loss of EGF binding activity by cells during down regulation closely paralleled the decrease in receptor subject to direct labeling and the decrease in the 175,000-dalton band labeled by surface-specific radioiodination.