Kinetics of binding, endocytosis, and recycling of EGF receptor mutants.
This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF. We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (less than 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.
Felder
S
S
Department of Molecular Biology, Rhone-Poulenc Rorer, Inc., King of Prussia, Pennsylvania 19406.
LaVin
J
J
Ullrich
A
A
Schlessinger
J
J
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine; D014162:Transfection; D015854:Up-Regulation
D000911:Antibodies, Monoclonal; D013912:Threonine; D004815:Epidermal Growth Factor; D004247:DNA; D066246:ErbB Receptors; D013755:Tetradecanoylphorbol Acetate