COOH-terminal Extended Recombinant Amphiregulin with Bioactivity Comparable with Naturally Derived Growth Factor

Abstract

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 ␣ 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 ϳ32% 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.

Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line

“…Anti-EGFR antibodies and EGFR antisense RNA expression inhibited the transformed phenotype of NS2T2A1 cells (Ma et al, 1998), as was shown previously in other breast cancer cell lines or human tumors in vivo (review see Modjtahedi and Dean, 1994). AR is secreted as a monomer of either 81 or 87 amino acid residues after proteolytic cleavage of the 252 amino acid transmembrane precursor (Plowman et al, 1990;Shoyab et al, 1989;Thompson et al, 1996), with a molecular weight ranging from 9.5 ± 60 kD, depending on the degree of glycosylation and NH2-terminal processing (Brown et al, 1998;Martinez-Lacaci et al, 1995Johnson et al, 1993b;Shoyab et al, 1988). In our NS2T2A1 cells, only the 55 ± 60 kD form of the AR was detected by Western blot analysis in the total protein lysate with the AR 605 polyclonal antibody.…”

“…In most instances AR functions as a mitogen, but can inhibit the growth of human cancer cells, depending upon the concentration used, the presence of other growth factors, and the nature of the cell lines (Cook et al, 1991;Johnson et al, 1991Johnson et al, , 1992Johnson et al, , 1993aJohnson et al, , 1994Plowman et al, 1990;Shoyab et al, 1988;Thompson et al, 1996). In vitro, antisense oligonucleotides against AR were reported to partially inhibit AR expression and anchorage-dependent growth of immortalized-nontransformed human mammary epithelial 184A1N4 cells (Kenney et al, 1993) and human pancreatic cancer T3M4 cells (Funatomi et al, 1997).…”

Successful Piglet Production in a Chemically Defined System for In-vitro Production of Porcine Embryos: Dibutyryl Cyclic AMP and Epidermal Growth Factor-family Peptides Support In-vitro Maturation of Oocytes in the Absence of Gonadotropins

“…In the present study, EGF and betacellulin worked well at 10 ng/ml, whereas amphiregulin was not effective at 10 ng/ml, but was effective at 1000 ng/ml. This difference in the effective concentration may be due to the difference in binding affinity, since the affinity of amphiregulin is several orders of magnitude less than that of EGF [26,27].…”

“…In the present study, EGF and betacellulin worked well at 10 ng/ml, whereas amphiregulin was not effective at 10 ng/ml, but was effective at 1000 ng/ml. This difference in the effective concentration may be due to the difference in binding affinity, since the affinity of amphiregulin is several orders of magnitude less than that of EGF [26,27].Furthermore, it is very interesting that a combination of EGF with another EGF-family member (amphiregulin or betacellulin), as compared with EGF alone, improved the percentage of oocytes developing to the metaphase-II stage. A similar synergistic effect on oocyte maturation has been observed in buffalo COCs exposed to EGF plus IGF-I [28].…”

A Differential Requirement for the COOH-terminal Region of the Epidermal Growth Factor (EGF) Receptor in Amphiregulin and EGF Mitogenic Signaling

“…GRB2-glutathione S-transferase (GST) fusion protein were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The 87-amino acid residue form of recombinant human AR was generated as described previously (12). Recombinant human EGF was obtained from PeproTech, Inc. (Rocky Hill, NJ).…”