COOH-terminal extended recombinant amphiregulin with bioactivity comparable with naturally derived growth factor.
The mature secreted form of the epidermal growth factor (EGF) receptor ligand amphiregulin (AR) is reported to be an 84-amino acid residue polypeptide, which is generated by proteolytic processing of a 252-amino acid precursor. This form of recombinant AR (rAR84) and two forms with COOH-terminal extensions corresponding to sequences from the AR precursor (rAR87 and rAR92) were expressed at high levels in Escherichia coli, oxidized to the correct disulfide arrangement, and purified to homogeneity. rAR84 competed poorly for binding of radiolabeled EGF to the EGF receptor and had little ability to stimulate growth of Balb/c/3T3 cells. In striking contrast, rAR87 and rAR92 possessed 42- and 20-fold greater receptor binding activity and 55- and 14-fold greater bioactivity, respectively. Furthermore, addition of the COOH-terminal four amino acids from transforming growth factor alpha to the COOH terminus of rAR84 improved the activity of rAR84 by 100- and 1000-fold, respectively, in these assays. rAR87 was found to have approximately 32% of the specific activity of natural AR from MCF-7 cells when compared in two different bioassays. These findings strongly suggest that the 84-amino acid sequence is not the correct structure of the naturally occurring secreted form of AR and that natural AR contains additional amino acid residues at the COOH-terminal end.
Thompson
S A
SA
Department of Protein Chemistry and Biophysics, Berlex Biosciences, Richmond, California 94804, USA.
Harris
A
A
Hoang
D
D
Ferrer
M
M
Johnson
G R
GR
D016475:3T3 Cells; D000595:Amino Acid Sequence; D066258:Amphiregulin; D000818:Animals; D001681:Biological Assay; D004305:Dose-Response Relationship, Drug; D066255:EGF Family of Proteins; D066246:ErbB Receptors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics* / Q000494:pharmacology*; D006133:Growth Substances / Q000235:genetics* / Q000494:pharmacology*; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D051379:Mice; D008934:Mitogens / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship
D066258:Amphiregulin; C587849:Areg protein, mouse; D066255:EGF Family of Proteins; D006023:Glycoproteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D008934:Mitogens; D011994:Recombinant Proteins; D066246:ErbB Receptors