title: Monoubiquitination of pro-amphiregulin regulates its endocytosis and ectodomain shedding.
abstract: All members of epidermal growth factor (EGF) family are expressed as transmembrane precursors on cell surfaces and then proteolytically converted to soluble ligands for EGF receptor (EGFR) by a disintegrin and metalloproteases (ADAMs). As enzyme-substrate complex formation is essential for this "ectodomain shedding", alteration of cell surface retention could affect their physical interaction with ADAMs and eventually contribute to shedding efficiency. Here, we showed that monoubiquitination of pro-amphiregulin (pro-AREG, an EGFR ligand) accelerated its half-life on cell surface. Monoubiquitination occurred at lysine 240 of pro-AREG as the primary acceptor site. Using a chimeric protein of pro-AREG and a monomeric ubiquitin mutant (pro-AREGmUb), immunocytochemical analysis and a cell surface biotinylation assay revealed that a significant portion of pro-AREGmUb was expressed on the cell surface, immediately endocytosed, and predominantly localized to early endosomes. Importantly, ectodomain shedding of pro-AREGmUb induced by tetradecanoyl phorbol acetate was significantly reduced in comparison to wild-type pro-AREG. These results suggested that pro-AREG monoubiquitination and the subsequent trafficking to intracellular organelles is a novel shedding regulatory mechanism that contributes to the secretion of EGFR ligands in growth factor signaling.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan. sfukuda@m.ehime-u.ac.jp
authors.forename: Shinji
authors.initials: S
authors.lastname: Fukuda
authors.forename: Hisayo
authors.initials: H
authors.lastname: Nishida-Fukuda
authors.forename: Hironao
authors.initials: H
authors.lastname: Nakayama
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Inoue
authors.forename: Shigeki
authors.initials: S
authors.lastname: Higashiyama
mesh_terms: D066258:Amphiregulin; D045744:Cell Line, Tumor; D066255:EGF Family of Proteins; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D054875:Ubiquitination
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D066246:ErbB Receptors; D008239:Lysine