Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain.
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.
Stoorvogel
Willem
W
Department of Cell Biology at the University Medical Center, Utrecht University, The Netherlands. W.Stoorvogel@vet.uu.nl
Kerstens
Sonja
S
Fritzsche
Ingo
I
den Hartigh
Jan C
JC
Oud
Ron
R
van der Heyden
Marcel A G
MA
Voortman
Jarno
J
van Bergen en Henegouwen
Paul M P
PM
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
D000199:Actins; D008024:Ligands; D066246:ErbB Receptors