title: Cell surface ectodomain cleavage of human amphiregulin precursor is sensitive to a metalloprotease inhibitor. Release of a predominant N-glycosylated 43-kDa soluble form.
abstract: Biosynthesis and processing of amphiregulin (AR) have been investigated in human colorectal (HCA-7, Caco-2) and mammary (MCF-7) cancer cell lines, as well as in Madin-Darby canine kidney cells stably expressing various human AR precursor (pro-AR) forms. Both cells expressing endogenous and transfected AR produce multiple cellular and soluble forms of AR with an N-glycosylated 50-kDa pro-AR form being predominant. Our results demonstrate that sequential proteolytic cleavage within the ectodomain of the 50-kDa pro-AR form leads to release of a predominant N-glycosylated 43-kDa soluble AR, as well as the appearance of other cellular and soluble AR forms. Cell surface biotinylation studies using a C-terminal epitope-tagged pro-AR indicate that all cell surface forms are membrane-anchored and support that AR is released by ectodomain cleavage of pro-AR at the plasma membrane. We also show that pro-AR ectodomain cleavage is a regulated process, which can be stimulated by phorbol 12-myristate 13-acetate and inhibited by the metalloprotease inhibitor, batimastat. In addition, we provide evidence that high molecular mass AR forms may retain the full-length N-terminal pro-region, which may influence the biological activities of these forms.
authors.affiliation: Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2279, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Brown
authors.forename: K S
authors.initials: KS
authors.lastname: Meise
authors.forename: G D
authors.initials: GD
authors.lastname: Plowman
authors.forename: R J
authors.initials: RJ
authors.lastname: Coffey
authors.forename: P J
authors.initials: PJ
authors.lastname: Dempsey
mesh_terms: D000595:Amino Acid Sequence; D066258:Amphiregulin; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D066255:EGF Family of Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006031:Glycosylation; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012995:Solubility; D014407:Tumor Cells, Cultured
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D066255:EGF Family of Proteins; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D006025:Glycosaminoglycans; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D008666:Metalloendopeptidases