title: Postendocytic trafficking of epidermal growth factor-receptor complexes is mediated through saturable and specific endosomal interactions.
abstract: Intracellular trafficking of the epidermal growth factor receptor (EGF-R) is regulated by receptor occupancy. To investigate this, we developed an assay to study endosomal sorting under steady-state conditions. Using a cell line transfected with EGF-R variants, we found that the fraction of internalized EGF.EGF-R complexes sorted to lysosomes was a function of the number of intracellular complexes and required sequences in the cytoplasmic domain of the receptor. As the number of intracellular occupied wild-type receptors increased from 3 x 10(2) to 2 x 10(5)/cell, the fraction of internalized EGF that was degraded dropped from 70 to 20%. Transforming growth factor-alpha, which dissociates from the EGF-R at endosomal pH, was degraded to a uniform extent of approximately 50% at all intracellular ligand concentrations. EGF internalized by receptors lacking a cytoplasmic domain (c'647) was degraded to an extent of only 5-10% independent of the number of intracellular complexes. Mutant receptors truncated either at residues 1022 or 973 displayed sorting patterns intermediate between wild-type and c'647 receptors. Despite large differences in their internalization rates, the fractional sorting patterns of c'1022 and c'973 receptors were indistinguishable. Receptor tyrosine kinase activity appeared to have a small effect on sorting pattern, but only in the context of full-length receptors. Our results indicate that the default pathway of internalized receptors is rapidly recycling and that lysosomal targeting of occupied EGF-R is due to endosomal retention that is both specific and saturable. In addition, internalization and endosomal retention of EGF-R appear to be mediated by distinct structural elements.
authors.affiliation: Department of Chemical Engineering, University of Illinois, Urbana 61801.
authors.forename: A R
authors.initials: AR
authors.lastname: French
authors.forename: G P
authors.initials: GP
authors.lastname: Sudlow
authors.forename: H S
authors.initials: HS
authors.lastname: Wiley
authors.forename: D A
authors.initials: DA
authors.lastname: Lauffenburger
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D015388:Organelles / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000378:metabolism
chemical_list: D008024:Ligands; D011956:Receptors, Cell Surface; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases