Perinuclear Location and Recycling of Epidermal Growth Factor Receptor Kinase: Immunofluorescent Visualization Using Antibodies Directed to Kinase and ExtraceUular Domains

This paper describes studies on the migratory behavior of epidermal growth factor (EGF) receptor kinase using antibodies that are specific for either the kinase domain or the extracellular domain of the receptor. Antiserum was raised to a 42,000-D subfragment of EGF receptor, which was shown earlier to carry the kinase catalytic site but not the EGF-binding site. Another antiserum was raised to the pure intact 170,000-D EGF receptor. The specificities of these antibodies were established by immunoprecipitation and immunoblotting experiments. The domain specificity was examined by indirect immunofluorescent staining of fixed cells. The anti-42-kD peptide antibody could bind specifically to EGF receptors of both human and murine origin and was found to be directed to the cytoplasmic part of the molecule. It did not bind to EGF receptor-negative cells, which contained other types of tyrosine kinases. The antibodies raised against the intact receptor recognized only EGF receptorspecific epitopes and were directed to the extracellular part of the molecule. The anti-receptor antibodies described above were used to visualize the cyclic locomotory behavior of EGF receptor kinase under various conditions of EGF stimulation and withdrawal. The receptor was examined in fixed and permeabilized cells by indirect immunofluorescent staining. The results demonstrate the following: (a) the receptor kinase domain migrates to the perinuclear region upon challenge with EGF; (b) both extracellular and cytoplasmic domains of the receptor are involved in migration as a unit; (c) withdrawal of EGF results in rapid recycling of the perinuclear receptors to the plasma membrane; (d) this return to the cell surface is inhibited by methylamine, chloroquine, and monensin; and (e) neither the internal migration nor the recycling process is blocked by inhibitors of protein biosynthesis. T HE epidermal growth factor (EGF) 1 receptor is a plasma membrane protein that has been implicated in the regulation of cell proliferation and cancer causation (17). It is a single chain polypeptide of 170,000--180,000 D that has an externally facing EGF-binding domain and a cytoplasmically facing tyrosine-specific protein kinase site (10, 13, 41). Although no significant biological function has yet been assigned to the tyrosine kinase site, the importance of tyrosine kinases in biology is apparent in the stringent conservation of these molecules through prolonged evolutionary times, and in the remarkable homologies seen between different tyrosine kinases in their catalytic domains (25). Some of the questions that can be asked regarding receptor tyrosine kinases are as follows: (a) What is the molecular basis for growth factor-induced activation of the receptor kinase site? (b) What is the subcellular site of action of the receptor kinase? Is it at the plasma membrane or elsewhere? If the site of action is other than plasma membrane, what 1. Abbreviations used in this paper: DME, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; EBSS, Earle's balanced salt solution. structural features control the migration to the inside of the cell? We have answered the first question to some extent. Our in vitro studies provide experimental evidence for the supposition that the EGF receptor is an allosteric enzyme that can interconvert between active (monomeric) and inactive (dimeric) forms, and that EGF, the ligand, activates the kinase by shifting the equilibrium towards the catalytically active monomeric form (6). These observations on the activation/inactivation cycle of the kinase site led us to the studies described here. Receptors for many ligands, such as asialoglycoproteins, transferrin, and low density lipoprotein, are known to display a characteristic migratory behavior (3, 9, 22). After binding to their ligands, these receptors cluster or segregate in coated pits and then internalize. Within internal organelles (endosomes), the receptor dissociates from its ligand, and the free receptor recycles back to the cell surface. This round-trip itinerary serves to accumulate the ligand (often a nutrient) inside the cell. EGF bound to cell-surface receptor is also known to un© The Rockefeller University Press, 0021-9525/86/08/333/10 $1.00 The Journal of Cell Biology, Volume 103, August 1986 333-342 333 on Jauary 8, 2018 jcb.rress.org D ow nladed fom dergo a rapid endocytic fate (14, 23, 29, 30). The receptor is co-internalized and is subjected to slow proteolysis (t,~ > 1 h) (4, 14, 16, 19, 38). Since EGF is not a simple nutrient, what could be the biological significance of this internal migration of the receptor? It is thought that this process serves a regulatory function by mediating rapid lysosomal destruction of excess EGF and receptor. However it is likely that this migratory process may have yet another biological role that is more closely related to the mitogenic function of EGF receptor. This is suggested by the fact that the EGF receptor is an enzyme. If the target substrate for the receptor kinase is located within the interior of the cell, it will be necessary for the receptor and its substrate to come into close contact; one way to achieve this contact would be through receptor internalization. To visualize the movement of receptor kinase domain during EGF stimulation and withdrawal, we needed to develop an antibody directed to that domain. Recently we isolated, by proteolysis, a 42,000-D receptor domain that retains the kinase catalytic activity of the intact 170-kD EGF receptor but loses EGF-binding and kinase-regulatory functions (2). Here we describe the development of an immunological probe for this human EGF receptor-derived 42-kD tyrosine kinase. The antibody was found to recognize only the cytoplasmic part of EGF receptor and showed no reaction with other receptor kinases. The specificity of this antibody and other anti-EGF-receptor antibodies used here was established by immunoprecipitation and immunoblot analyses. We used these specific antibodies in indirect immunofluorescent staining studies to visualize the subcellular location of EGF receptor kinase under various conditions of stimulation and withdrawal. The results demonstrate that the EGF receptor kinase displays a cyclic locomotion that is controlled by its state of activation. The cycle of locomotion can be disturbed by certain drugs that inhibit receptor mitogenicity but not by inhibitors of protein biosynthesis. Some implications of these findings are discussed. Materials and Methods