Epidermal growth factor stimulates phosphorylation in membrane preparations in vitro

Abstract

EPIDERMAL GROWTH FACTOR (EGF) forms a complex with plasma membrane receptors in intact cells that initiates a series of biochemical events resulting in increased cell growth in vivo and in vitro¹. The interaction of EGF with membrane receptors has been demonstrated in crude membrane preparations², but no biochemical alteration of the membrane resulting from hormone binding has been detected. To clarify the molecular mechanisms regulating cell proliferation, specific biochemical reactions initiated by mitogens such as EGF need to be investigated in cell-free systems. As the human epidermoid carcinoma cell line A-431 has an extraordinarily high concentration of EGF receptors^3,4 (2–3 x 10⁶ receptors per cell), we have used a crude membrane preparation from these cells to look for an EGF-dependent alteration of membrane structure and/or function. We report here that (1) membranes may be prepared from A-431 cells that retain the ability to bind ¹²⁵I-labelled EGF in a specific manner, and (2) the binding of EGF to these membranes in vitro results in a marked stimulation of the phosphorylation of endogenous proteins in the presence of [γ-³²P]ATP.