Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells.

Abstract

Abstract. We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR 1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4"C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37"C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20"C the ligand-receptor complex accumulated in the pericentriolar compartment.Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step. EPIDERMAL growth factor (EGF) 1 is a potent mitogenic polypeptide, which, upon binding to specific receptors on the plasma membrane of its target cell, initiates a chain ofintracellular events that have been studied extensively (1, 4-6, 12, 26, 28, 35, 38). By the use of labeled derivatives of EGF it has been shown both biochemically and morphologically that ceil-bound EGF is rapidly internalized by its target cells into an endosomal compartment and is subsequently degraded in lysosomes (5,16,17,30, 34). In most cell types continuous incubation at 37"C in the presence of EGF results not only in the degradation of EGF but also in the down-regulation of the EGF receptor. This led to the idea, for which Krupp et at. (29) morphological evidence that down-regulation of the EGF receptor in KB cells is due to receptor internalization into an endosomal compartment followed by degradation in lysosomes.Much of the work on internalization and processing of the EGF receptor and its ligand has been carried out in epidermoid carcinoma (A431) cells, mainly because of the large number of receptors for EGF that these cells express ( 1-3 × 106/cell). However, although EGF receptors have recently been identified by immunocytochemistry in these cells (3), the morphological pathway of internalization of the EGF receptor has so far not been documented. Two monoclonal antibodies, TL5 (35) and EGFR1 (39), have been described that recognize, respectively, the carbohydrate do...

E3-13.7 Integral Membrane Proteins Encoded by Human Adenoviruses Alter Epidermal Growth Factor Receptor Trafficking by Interacting Directly with Receptors in Early Endosomes

“…Hence, EGF receptor down-regulation likely depends on several discrete sorting events activated during MVE maturation. Because ligandoccupied EGF receptors also accumulate in the internal vesicles of MVEs en route to lysosomes (Miller et al, 1986), we hypothesize that E3-13.7 has usurped a mechanism normally used during ligand-induced receptor down-regulation. Our model predicts that although some EGF receptor lysosomal sorting signals, including the leucine signal usurped by E3-13.7, may be activated by ligand-induced oligomerization, others acting distally may function in the context of monomers.…”

“…The transferrin receptor, for example, is found on the limiting membrane of relatively immature MVEs (Hopkins, 1983), the CI-M6PR is enriched in MVEs with an intermediate degree of maturation (Hirst et al, 1998), and Lamp I is found predominantly in mature MVEs (van Deurs et al, 1993). In addition, selected cargo, including ligand-occupied EGF receptors (Miller et al, 1986), is sequestered in internal vesicles. Hence, EGF receptor down-regulation likely depends on several discrete sorting events activated during MVE maturation.…”

Endocytosis and intracellular trafficking of ErbBs

“…2). EGF and EGFR begin to accumulate in the intralumenal membranes of MVB that are located in the perinuclear area of the cell after 15-20 min of EGF-induced endocytosis [80,81,88,89]. Serial sectioning electron microscopy demonstrated that internal membranes of MVB represent vesicles that are not connected to the limiting membrane [90].…”

“…Clathrin-coated vesicles containing EGF-receptor complexes rapidly release their coat and fuse with early endosomes, compartments of a heterogeneous morphology consisting of vesicular and tubular membranes and located at the periphery of the cell [78][79][80][81] (Fig. 2).…”

Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells.

“…Anti-plOO and synthetic EGF (with the addition of four extra lysine residues on the carboxyl terminus) were conjugated to horseradish peroxidase using SPDP (Sigma Chemical) as described previously (Hopkins and Trowbridge, 1983;Miller et al, 1986). a-2 M/HRP was prepared as described previously by Hopkins et al (1994).…”