Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor.
Endocytosis of the epidermal growth factor receptor (EGFR) is important for the regulation of EGFR signaling. However, EGFR endocytosis mechanisms are poorly understood, which precludes development of approaches to specifically inhibit EGFR endocytosis and analyze its impact on signaling. Using a combination of receptor mutagenesis and RNA interference, we demonstrate that clathrin-dependent internalization of activated EGFR is regulated by four mechanisms, which function in a redundant and cooperative fashion. These mechanisms involve ubiquitination of the receptor kinase domain, the clathrin adaptor complex AP-2, the Grb2 adaptor protein, and three C-terminal lysine residues (K1155, K1158, and K1164), which are acetylated, a novel posttranslational modification for the EGFR. Based on these findings, the first internalization-defective EGFR mutant with functional kinase and normal tyrosine phosphorylation was generated. Analysis of the signaling kinetics of this mutant revealed that EGFR internalization is required for the sustained activation of protein kinase B/AKT but not for the activation of mitogen-activated protein kinase.
Department of Pharmacology, School of Medicine, University of Colorado Denver, Aurora, CO 80045, USA.
Lai Kuan
LK
Goh
Fangtian
F
Huang
Woong
W
Kim
Steven
S
Gygi
Alexander
A
Sorkin
D000107:Acetylation; D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D013552:Swine; D014162:Transfection; D054875:Ubiquitination / Q000502:physiology
D033962:Adaptor Protein Complex 2; D002966:Clathrin; D051380:GRB2 Adaptor Protein; D034741:RNA, Small Interfering; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D008239:Lysine