title: Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain.
abstract: Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the second transport step requires a domain of the EGFR that encompasses residues 985-996 and was previously found to interact with actin. Deletion of domain 989-994 (Delta989-994 EGFR) did not interfere with EGFR uptake but completely abrogated its degradation. In contrast, both uptake and degradation were affected for K721A EGFR, a kinase-deficient EGFR mutant. To measure intracellular EGFR sorting, we developed a novel cell fractionation assay toward which cells were co-transfected for chicken hepatic lectin, a receptor for agialoglycoproteins. These cells were incubated with agialofetuin-coupled colloidal gold, which was targeted to lysosomes after receptor-mediated endocytosis. Compartments within the lysosomal pathway gained buoyant density because of the presence of colloidal gold and could be isolated from cell homogenates by ultracentrifugation through a high-density sucrose cushion. In contrast to endocytosed wild type EGFR, both Delta989-994 EGFR and K721A EGFR were largely not retrieved in gold-containing endocytic compartments. These results are supported with morphological data. We conclude that sorting of endocytosed EGFR into the degradation pathway requires both its kinase activity and actin-binding domain.
authors.affiliation: Department of Cell Biology at the University Medical Center, Utrecht University, The Netherlands. W.Stoorvogel@vet.uu.nl
authors.forename: Willem
authors.initials: W
authors.lastname: Stoorvogel
authors.forename: Sonja
authors.initials: S
authors.lastname: Kerstens
authors.forename: Ingo
authors.initials: I
authors.lastname: Fritzsche
authors.forename: Jan C
authors.initials: JC
authors.lastname: den Hartigh
authors.forename: Ron
authors.initials: R
authors.lastname: Oud
authors.forename: Marcel A G
authors.initials: MA
authors.lastname: van der Heyden
authors.forename: Jarno
authors.initials: J
authors.lastname: Voortman
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen en Henegouwen
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002451:Cell Compartmentation; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D021381:Protein Transport
chemical_list: D000199:Actins; D008024:Ligands; D066246:ErbB Receptors