Ligand-Mediated Internalization, Recycling, and Downregulation of the Epidermal Growth Factor Receptor in vivo

• Published in: The Journal of cell biology 1989-12, Vol.109 (6), p.2741-2749
• Main Authors: Lai, W. H., Cameron, P. H., Wada, I., J.-J. Doherty, II, Kay, D. G., Posner, B. I., Bergeron, J. J. M.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Body weight; Cell Fractionation; Cell physiology; Dose response relationship; Down regulation; Endocytosis; Epidermal Growth Factor - pharmacology; ErbB Receptors - drug effects; ErbB Receptors - genetics; ErbB Receptors - metabolism; Fundamental and applied biological sciences. Psychology; Hepatocytes; Internalization; Kinetics; Ligands; Liver; Liver - metabolism; Liver cells; Male; Molecular and cellular biology; Organelles - metabolism; Organelles - ultrastructure; Rats; Rats, Inbred Strains; Receptors; Recycling; Subcellular Fractions - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.109.6.2741

EGF receptor internalization, recycling, and downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to ≤50% surface receptor occupancy (i.e., ≤1 μg/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF >50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 μg/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.