title: Characterization of high and low molecular weight forms of amphiregulin that differ in glycosylation and peptide core length. Evidence that the NH2-terminal region is not critical for bioactivity.
abstract: Human amphiregulin (AR) is a polypeptide growth regulator which acts by binding to and activating the epidermal growth factor (EGF) receptor tyrosine kinase. AR consists of an EGF-like domain and an NH2-terminal extension which contains potential glycosylation sites and nuclear localization signals. Two high molecular weight species which had molecular masses of approximately 16.5 kDa (HMW-AR1 and HMW-AR2) and a approximately 9.5-kDa low molecular weight form (LMW-AR) were isolated from the conditioned medium of phorbol 12-myristate 13-acetate-treated MCF-7 human breast carcinoma cells by sequential heparin affinity, immunoaffinity, and reverse phase-high performance liquid chromatography. HMW-AR1 and HMW-AR2 were found to possess complex or hybrid type N-linked oligosaccharide structures that contained sialic acid. Additionally, HMW-AR1 and HMW-AR2 contained the disaccharide, Gal beta(1-->3)GalNAc, linked to Ser/Thr residues. No carbohydrate moieties were detected in LMW-AR. Mapping of the peptide cores of these molecules using antipeptide antibodies revealed that HMW-AR1 and HMW-AR2 were intact molecules, whereas LMW-AR contained the EGF-like domain, but possessed a truncated NH2-terminal extension. LMW-AR, HMW-AR1, and HMW-AR2 were all found to be potent stimulators of DNA synthesis in MCF-10A human mammary epithelial cells. These results suggest that the NH2-terminal region of the AR molecule is not critical to the ability of AR to activate the EGF receptor tyrosine kinase.
authors.affiliation: Division of Cytokine Biology, Food and Drug Administration, Bethesda, Maryland 20892.
authors.forename: G R
authors.initials: GR
authors.lastname: Johnson
authors.forename: S A
authors.initials: SA
authors.lastname: Prigent
authors.forename: W J
authors.initials: WJ
authors.lastname: Gullick
authors.forename: K
authors.initials: K
authors.lastname: Stromberg
mesh_terms: D000595:Amino Acid Sequence; D066258:Amphiregulin; D001943:Breast Neoplasms / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D004187:Disaccharides / Q000032:analysis; D066255:EGF Family of Proteins; D004815:Epidermal Growth Factor / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006133:Growth Substances / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping; D012794:Sialic Acids / Q000032:analysis; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D017077:Culture Media, Conditioned; D004187:Disaccharides; D066255:EGF Family of Proteins; D006023:Glycoproteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009844:Oligosaccharides; D012794:Sialic Acids; D004815:Epidermal Growth Factor; D006493:Heparin; D019158:N-Acetylneuraminic Acid; D013755:Tetradecanoylphorbol Acetate