title: Unravelling the pivotal role of Alix in MVB sorting and silencing of the activated EGFR.
abstract: Endosomal sorting complex required for transport (ESCRT)-III-mediated membrane invagination and scission are a critical step in multivesicular body (MVB) sorting of ubiquitinated membrane receptors, and generally thought to be required for degradation of these receptors in lysosomes. The adaptor protein Alix is critically involved in multiple ESCRT-III-mediated, membrane-remodelling processes in mammalian cells. However, Alix knockdown does not inhibit degradation of the activated epidermal growth factor receptor (EGFR) in mammalian cell lines, leading to a widely held notion that Alix is not critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells. In the present study, we demonstrate that, despite its non-essential role in degradation of the activated EGFR, Alix plays a critical role in its MVB sorting and silencing Epidermal growth factor (EGF) stimulation of mammalian cell lines induces Alix's interaction with the ubiquitinated EGFR via the Alix V domain, and increases Alix's association with membrane-bound charged multivesicular body protein 4 (CHMP4) via the Alix Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of Alix's interaction with membrane-bound CHMP4, inhibition of Alix dimerization through the V domain or Alix knockdown dramatically inhibits MVB sorting of the activated EGFR and promotes sustained activation of extracellular-signal regulated kinase (ERK)1/2. Under the continuous EGF stimulation conditions, these cell treatments also retard degradation of the activated EGFR. These findings indicate that Alix is critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells.
authors.affiliation: *Department of Experimental Therapeutics, University of Texas, M.D. Anderson Cancer Center, Houston, Texas, U.S.A.
authors.forename: Sheng
authors.initials: S
authors.lastname: Sun
authors.affiliation: *Department of Experimental Therapeutics, University of Texas, M.D. Anderson Cancer Center, Houston, Texas, U.S.A.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhou
authors.affiliation: ‡The Key Laboratory for Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: †The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas, U.S.A.
authors.forename: Gary E
authors.initials: GE
authors.lastname: Gallick
authors.affiliation: *Department of Experimental Therapeutics, University of Texas, M.D. Anderson Cancer Center, Houston, Texas, U.S.A.
authors.forename: Jian
authors.initials: J
authors.lastname: Kuang
mesh_terms: D002135:Calcium-Binding Proteins / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism* / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000172:deficiency / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D057146:Multivesicular Bodies / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C117020:PDCD6IP protein, human; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors