This report describes the isolation and recombmiant expression of a cDNA clone encoding HER4, the fourth member of the human epidermal growth factor receptor (EGFR) family. The HER4/erbB4 gene encodes a 180-kDa transmembrane tyrosine kinase (HER4/p18WbB) whose extraceilular domain is most similar to the orphan receptor HER3/pl6OerbD3, whereas its cytoplasmic kinase domain exhibits 79% and 77% identity with EGFR and HER2/pj85erbB2, respectively. HER4 is most predominaudy expressed in several breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and cerebellum. In addition, we describe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found to specifically stimulate the intrinsic tyrosine kinase activity of HER4/plW'rbB4 while having no direct effect on the phosphorylation of EGFR, HER2, or HER3. Furthermore, this heparin-binding protein induces phenotypic differentiation, and tyrosine phosphorylation, of a human mammary tumor cell line that overexpresses both HER4 and HER2. These findings suggest that this ligand-receptor interaction may play a role in the growth and differentiation of some normal and transformed cells.

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.

We have isolated the gene for a novel growth regulator, amphiregulin (AR), that is evolutionarily related to epidermal growth factor (EGF) and transforming growth factor a (TGF-a). AR is a bifunctional growth modulator: it interacts with the EGF/TGF-a receptor to promote the growth of normal epithelial cells and inhibits the growth of certain aggressive carcinoma cell lines. The 84-amino-acid mature protein is embedded within a 252-amino-acid transmembrane precursor, an organization similar to that of the TGF-a precursor. Human placenta and ovaries were found to express significant amounts of the 1.4-kilobase AR transcript, implicating AR in the regulation of normal cell growth. In addition, the AR gene was localized to chromosomal region 4q13-4q21, a common breakpoint for acute lymphoblastic leukemia.Cell growth and differentiation are regulated in part by the specific interaction of secreted growth factors and their membrane-bound receptors. Receptor-ligand interaction results in activation of intracellular signals leading to specific cellular responses. Epidermal growth factor (EGF), plateletderived growth factor, insulin, insulinlike growth factor 1, colony-stimulating factor 1, and fibroblast growth factor all transmit their growth-modulating signals by binding to and activating receptors with intrinsic tyrosine kinase activity (reviewed in reference 30). Characterization of the physiologic and chemical effects that result from the binding of EGF and transforming growth factor a (TGF-a) to the EGF receptor has served as a useful model for understanding receptor-ligand interactions, signal transduction, and the regulation of cell growth and oncogenesis (reviewed in reference 76).We have recently reported the purification and sequence analysis of a glycoprotein isolated from the conditioned media of 12-0-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells (65,66). The protein was termed amphiregulin (AR) to reflect its bifunctional activities: it inhibits the growth of many human tumor cells, and it stimulates the proliferation of normal fibroblasts and keratinocytes. The secreted protein exists as a monomer of either 78 or 84 amino acids (aa), with the shorter form lacking the six N-terminal residues of the larger molecule. Sequence analysis reveals that AR has a region with striking homology to EGF (38%) and TGF-a (32%), yet it also has an N-terminal extension of 43 aa composed primarily of very basic, hydrophilic residues (Lys, Arg, and Asn). In addition, AR has functional homology with this class of growth factors; it partially competes for binding of EGF to the EGF receptor and can supplant the need for EGF or TGF-a to maintain keratinocytes in culture. AR differs from EGF and TGF-a in that it fails to promote anchorage-independent growth of normal rat kidney (NRK) fibroblasts in the presence of TGF-P and inhibits the growth of certain tumor cells that proliferate in response to EGF or TGF-a.In this report, we describe the isolation and characterization of cDNA and genomic clones for human AR, t...

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...

TPA (12-O-tetradecanoyl-phorbol-13-acetate) reversibly inhibits the binding of (125)I-labelled epidermal growth factor (EGF) to treated mouse and human cells, but does not affect the binding of various other ligands to their membrane receptors. It alters the affinity of the receptors for EGF without changing the total number of available receptors per cell. Those phorbol esters which stimulate cell growth in culture and have tumour-promoting activity in vivo alter the EGF-receptor affinity, while the biologically inactive derivatives fail to change the affinity of EGF for its receptors.