Experimental Cell Research 166 (1986) 312-326 Subcellular Distribution of the External and Internal Domains of the EGF Receptor in A-431 Cells JEAN-LOUIS CARPENTIER,‘, * ANTHONY R. REES,2 MARY GREGORIOU,2 RICHARD KRIS3 JOSEPH SCHLESSINGER,3 and LELIO ORCI’ ‘Institute of Histology and Embryology, School of Medicine, University of Geneva, Geneva, Switzerland, ‘Laboratory of Molecular Biophysics, University of Oxford, Oxford, UK, and ‘Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85 % of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulo- vesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly. @ 19% Academic Press, Inc. The epidermal growth factor (EGF) receptor is an integral membrane protein of 1186 residues that spans the entire membrane and exhibits three functional domains: a glycoprotein EGF-binding domain located on the external surface, a transmembrane segment and a cytoplasmic domain containing a tyrosine-specific protein kinase activity [IA]. The receptor is mobile in the plane of the plasma membrane and shows both lateral and rotational movements 15-81. Following EGF binding, the EGF receptor is phosphorylated, internalized by an adsorptive endocytotic process and the major proportion of the population proceeds to lysosomes where it is apparently degraded, while a minor proportion may be recycled [P-15]. At present, however, it is not clear whether the two domains of the EGF receptor are processed similarly inside the cell or whether fragments are cleaved in a prelysosomal or lysosomal step and further routed towards other intracytoplasmic organelles [ll, 161. This question is of special interest, since it was shown that the v-erb-B protein of avian erythroblastosis virus (AEV) [17, 181 is a truncated EGF receptor which has lost most of the’extracellular EGF-binding domain but retains the cytoplasmic region with intrinsic tyrosine-kinase activity [191. The differential routing of deregulated kinase may therefore play a role in the subverted mitogenic signal induced by the truncated receptor [ 161. * To whom offprint requests should be sent. Address: Institute of Histology and Embryology, CMU, 1, rue Michel Servet, 1211 Geneva 4, Switzerland.