title: Endocytosis of Ubiquitylation-Deficient EGFR Mutants via Clathrin-Coated Pits is Mediated by Ubiquitylation.
abstract: Signaling by epidermal growth factor receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here, we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP-2-binding sites (21KRΔAP2) was internalized at relatively high rates via the clathrin-dependent pathway in human duodenal adenocarcinoma HuTu-80 cells. RNA interference analysis revealed that this residual internalization is strongly inhibited by depletion of Grb2 and the E2 ubiquitin-conjugating enzyme UbcH5b/c, and partially affected by depletion of the E3 ubiquitin ligase Cbl and ubiquitin-binding adaptors, indicating that an ubiquitylation process is involved. Several new ubiquitin conjugation sites were identified by mass spectrometry in the 21KRΔAP2 mutant, suggesting that cryptic ubiquitylation may mediate endocytosis of this mutant. Total internal reflection fluorescence microscopy imaging of HuTu-80 cells transfected with labeled ubiquitin adaptor epsin1 demonstrated that the ubiquitylation-deficient EGFR mutant was endocytosed through a limited population of epsin-enriched clathrin-coated pits (CCPs), although with a prolonged CCP lifetime. Native EGFR was recruited with the same efficiency into CCPs containing either AP-2 or epsin1 that were tagged with fluorescent proteins by genome editing of MDA-MD-231 cells. We propose that two redundant mechanisms, ubiquitylation and interaction with AP-2, contribute to EGFR endocytosis via CCPs in a stochastic fashion.
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA,, USA.
authors.forename: Arola
authors.initials: A
authors.lastname: Fortian
authors.affiliation: Department of Pharmacology, University of Colorado Anschutz Medical Center, Aurora, CO,, USA.
authors.forename: Lai K
authors.initials: LK
authors.lastname: Dionne
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA,, USA.
authors.forename: Sun H
authors.initials: SH
authors.lastname: Hong
authors.affiliation: Department of Cell Biology, University of Harvard School of Medicine, Boston, MA,, USA.
authors.forename: Woong
authors.initials: W
authors.lastname: Kim
authors.affiliation: Department of Cell Biology, University of Harvard School of Medicine, Boston, MA,, USA.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA,, USA.
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA,, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D011499:Protein Processing, Post-Translational / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D002966:Clathrin; D025801:Ubiquitin; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: EGF; clathrin; endocytosis; ubiquitin