Cellular 3/~m/8~, Vol. 3, No. 2, pp. 107-117, 1991. 0898-6568/91 $3.00+.00 Printed in Great Britain. © 1991 Pergamon Press plc DESENSITIZATION OF THE EGF RECEPTOR ALTERS ITS ABILITY TO UNDERGO EGF-INDUCED DIMERIZATION DHANDAPANI KUPPUSWAMY and LINDA J. PIKE* Howard Hughes Medical Institute, Washington University School of Medicine, Department of Biochemistry and Molecular Biophysics, 660 So. Euclid Avenue, St Louis, MO 63110, U.S.A. (Received 15 October 1990; and accepted 18 November 1990) Abstract--Treatment of A43 ! cells with EGF has been shown to induce the formation of EGF receptor dimers. Sucrose density gradient centrifugation as well as surface radio-iodination followed by crosslinking were used to study further the properties of EGF receptor monomers and dimers as well as the regulation of dimer formation. We have shown previously that treatment of A431 cells with high doses of EGF at 37°C leads to the desensitization of the EGF receptor without a significant loss of cell surface ~25I-EGFbinding [Kuppuswamy and Pike (1989) J. biol. Chem. 264, 3357-3363; Cunningham et aL (1989) J. biol. Chem. 264, 15351-15356]. Desensitization of the EGF receptor led to a decrease in the ability of receptor monomers to be induced to form dimers by EGF both in vivo and in vitro. These data suggest that the sensitivityof a cell to EGF may be modulated by altering the capacity of the EGF receptor to form oligomers. Key words: EGF receptor, desensitization, dimerization. INTRODUCTION EPIOERMAL growth factor (EGF) is a potent mitogen for a variety of cultured cells [1]. The receptor for EGF is an integral membrane pro- tein with a molecular weight of 170,000 that possesses intrinsic tyrosine protein kinase activity [2, 3]. The EGF receptor has three func- tional domains: an extracellular domain which recognizes and binds EGF, a hydrophobic region which anchors the receptor in the membrane through a single membrane- spanning domain and a cytoplasmic domain which is responsible for the tyrosine protein kinase activity of the receptor [4]. The EGF receptor tyrosine kinase catalyses an autophos- phorylation reaction as well as the phosphory- lation of exogenous substrates [5-7]. Studies of * Author to whomcorrespondenceshouldbe addressed. Abbreviations: BS 3, bis(sulphosuccinimidyl)suberate; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; HBSS, Hank's buffered saline solution; TPA, 12-O-tetradecanoylphorbol13-acetate. the structure and function of the EGF receptor using site-directed mutagenesis have demon- strated that this tyrosine kinase activity is abso- lutely required for transduction of the hormonal signal via the receptor [8-10]. While it is known that the binding of EGF to the extracellular domain stimulates the tyrosine kinase activity of the receptor, the exact mech- anism by which the receptor transduces the signal remains unknown. On the basis of bio- chemical data obtained from studies of purified EGF receptors, Yarden and Schlessinger [11] recently proposed an allosteric aggregation model for the activation of the kinase function of the receptor. In their model, EGF induces receptor dimerization and the ensuing receptor- receptor interactions lead to activation of the kinase. In support of this model, they provided physical evidence for the dimerization of puri- fied EGF receptors using non-denaturing gel electrophoresis [12]. Through chemical cross- linking, Schlessinger and co-workers subse- quently demonstrated that EGF receptors in living cells also dimerized [13]. 107