title: Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase.
abstract: EGF-receptor (EGF-R) tyrosine kinase is required for the down-regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of kinase-deficient receptors occurs inefficiently and at the same basal rate of endocytosis of unoccupied receptors or receptors lacking any cytoplasmic domain. Sequestration of deletion mutants of the EGF-R that lack autophosphorylation sites also requires an active tyrosine kinase. This suggests that a tyrosine kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Addition of a soluble EGF-R tyrosine kinase fully and specifically restores the recruitment of kinase-deficient EGF-R into coated pits providing a powerful functional assay for identification of these substrate(s).
authors.affiliation: Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
authors.forename: C
authors.initials: C
authors.lastname: Lamaze
authors.forename: S L
authors.initials: SL
authors.lastname: Schmid
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D015536:Down-Regulation; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007739:L Cells; D008239:Lysine; D008715:Methionine; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D008715:Methionine; D066246:ErbB Receptors; D008239:Lysine