INVOLVEMENT OF HIGH-AFFINITY BINDING-SITE FOR EGF RECEPTOR IN FORMATION OF ROUNDING IN A-431 EPIDERMOID CARCINOMA-CELLS

The introduction of a bacterial aminoglycoside phosphotransferase gene (neo gene) into A-431 cells was found to result in disappearance of h igh-affinity binding sites of the epidermal growth factor receptor (EG FR), probably by affecting the phosphorylation level of the receptors. Using A-431 cells and their neo gene-transfectants, we studied the re lation between ''rounding'' and the high-affinity sites for EGF; and w e also examined the role of protein kinase C (PKC) and A (PKA) in the EGF-induced cell rounding. Pretreatment of A-431 and their transfectan t cells with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 ng/ml), an activator of PKC, for 30 min inhibited both the EGF-induced cell roun ding and expression of high-affinity binding sites for EGF. However, b oth of these responses were recovered when cells were pretreated with TPA for 20 h, which treatment is known to result in depletion of PKC b y a process called ''down regulation''. A similar recovery was also ob served when cells were pretreated with forskolin (100 muM), an activat or of PKA, for 30 min. Both cell rounding and EGFR high-affinity bindi ng sites disappeared by activation of PKC, and reappeared by activatio n of PKA. These results suggest that the rounding of A-431 cells by EG F was induced via the high-affinity binding sites of EGFR.