Ligand-Mediated Autophosphorylation Activity of the Epidermal Growth Factor Receptor during Internalization

• Published in: The Journal of cell biology 1989-12, Vol.109 (6), p.2751-2760
• Main Authors: Lai, W. H., Cameron, P. H., J.-J. Doherty, II, Posner, B. I., Bergeron, J. J. M.
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; ANIMAL TISSUES; ANIMALS; AUTORADIOGRAPHY; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; BODY; Body weight; CELL CONSTITUENTS; CELL MEMBRANES; Cell physiology; CHEMICAL REACTIONS; DAYS LIVING RADIOISOTOPES; DIGESTIVE SYSTEM; Down regulation; ELECTRON CAPTURE RADIOISOTOPES; Endocytosis; Endosomes; Epidermal Growth Factor - metabolism; Epidermal Growth Factor - pharmacology; EPIDERMIS; EPITHELIUM; ErbB Receptors - drug effects; ErbB Receptors - metabolism; Fundamental and applied biological sciences. Psychology; GLANDS; GROWTH FACTORS; INTERMEDIATE MASS NUCLEI; Internalization; IODINE 125; IODINE ISOTOPES; Iodine Radioisotopes; ISOTOPES; KINETICS; Lectins; LIGANDS; LIVER; Liver - metabolism; Male; MAMMALS; MEMBRANE PROTEINS; MEMBRANES; MITOGENS; Molecular and cellular biology; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; ORGANS; PHAGOCYTOSIS; PHOSPHORYLATION; PROTEINS; RADIOISOTOPES; RATS; Rats, Inbred Strains; REACTION KINETICS; RECEPTORS; Reference Values; RODENTS; SKIN; TISSUES; VERTEBRATES
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.109.6.2751

The association of EGF with its receptor in endosomes isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 μg EGF/100 g body weight was 57%. This value increased to 77% for the dose of 10 μg EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10 μg125I- EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand-receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions. This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGF. The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of125I- EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity.