Cell surface ectodomain cleavage of human amphiregulin precursor is sensitive to a metalloprotease inhibitor. Release of a predominant N-glycosylated 43-kDa soluble form.

Brown CL, Meise KS, Plowman GD, Coffey RJ, Dempsey PJ
J Biol Chem. 1998 273 (27): 17258-68

PMID: 9642297 · DOI:10.1074/jbc.273.27.17258

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.

MeSH Terms (23)

Amino Acid Sequence Amphiregulin Animals Base Sequence Cell Line Cell Membrane Dogs EGF Family of Proteins Enzyme Inhibitors Glycoproteins Glycosaminoglycans Glycosylation Growth Substances Humans Hydrolysis Intercellular Signaling Peptides and Proteins Metalloendopeptidases Mutagenesis Oligodeoxyribonucleotides Protein Precursors Protein Processing, Post-Translational Solubility Tumor Cells, Cultured

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