The complete amino acid sequence of amphiregulin, a bifunctional cell growth modulator, was determined. The truncated form contains 78 amino acids, whereas a larger form of amphiregulin contains six additional amino acids at the amino-terminal end. The amino-terminal half of amphiregulin is extremely hydrophilic and contains unusually high numbers of lysine, arginine, and asparagine residues. The carboxyl-terminal half of amphiregulin (residues 46 to 84) exhibits striking homology to the epidermal growth factor (EGF) family of proteins. Amphiregulin binds to the EGF receptor but not as well as EGF does. Amphiregulin fully supplants the requirement for EGF or transforming growth factor-alpha in murine keratinocyte growth, but it is a much weaker growth stimulator in other cell systems.

A glycoprotein, termed amphiregulin (AR), inhibits growth of several human carcinoma cells in culture and stimulates proliferation of human fibroblasts and certain other tumor cells. It has been purified to apparent homogeneity from serum-free conditioned medium of MCF-7 human breast carcinoma cells that had been treated with phorbol 12-myristate 13-acetate. AR is, a single-chain extremely hydrophilic glycoprotein containing cysteines in disulfide linkage(s) that are essential for biological activity; it is stable between pH 2 and pH 12 and after heating for 30 min at 560C but unstable at 1OOC.The apparent molecular weights of AR and N-Glycanasetreated AR are 14,000 and 15,000, respectively, as assessed by gel chromatography, and 22,500 and %14,000, respectively, as determined by polyacrylamide gel electrophoresis. Treatment of AR with N-Glycanase, O-Glycanase, or neuraminidase does not affect its activity. The pI of AR is =7.8. The aminoterminal amino acid sequence of AR has been determined, and no significant sequence homology between AR and other proteins was found. The molecule thus appears to be a distinct growth regulatory protein.Cellular growth and differentiation appear to be initiated, promoted, maintained, and regulated by a multiplicity of stimulatory, inhibitory, and synergistic factors and hormones. The alteration and/or breakdown of the cellular homeostasis mechanism is a fundamental cause of growthrelated diseases including neoplasia (1-11). Physiological regulators of cell growth and differentiation, such as peptide growth factors (2, 4, 10, 11), hematopoietic regulatory proteins (5, 12-14), tumor necrosis factor types a and ,3 (8, 9), interferons (15) . In addition, PMA also alters the morphology of MCF-7 cells and PMA-treated cells exhibit the morphological characteristics of secretory cells (27,28 (vol/vol) heat-inactivated fetal bovine serum, L-glutamine, penicillin (60.6 Jug/ml), and streptomycin (100 ,ug/ml). Growth Modulatory Assay with ',I-Labeled DeoxyuridineIncorporation into DNA. The assays were performed in 96-well flat-bottomed plates (Falcon, catalog number 3072). Human epidermoid carcinoma of the vulva cells (A431) were used as test cells for growth inhibitory activity (GIA) and human forearm fibroblasts (SS) were used as indicator cells for growth stimulatory activity. A total of 3.5 x 104 cells in 50 tkl of DMEM, supplemented with 5% (vol/vol) heatinactivated fetal bovine serum, penicillin (60.6 pug/ml), streptomycin (100 tug/ml), and glutamine (test medium), was placed in all wells except peripheral wells. Three hours later, 50 jal of the test sample in test medium was added to each well; control wells received only 50 ,il of test medium. Three wells were used for each concentration of test sample. Plates were incubated at 370C for 2-3 days. After this, 100 t1d of solution of 125I-labeled deoxyuridine (Amersham) [4 Ci/mg; 0.5 Ci/ml (2 ,ul/ml in test medium); 1 Ci = 37 GBq] was added to each well and plates were incubated at 370C. After 4-6 hr, samples were processed as...