A Mr = 190,000 glycoprotein phosphorylated on tyrosine residues in epidermal growth factor stimulated KB cells is the product of the c-erbB-2 gene

In human epidermoid carcinoma KB cells, a glycoprotein of Mr = 190,000 (gp190) has been shown to be phosphorylated on tyrosine residues upon EGF stimulation (Kadowaki et al., 1987, J. Biol. Chem. in press). Using a specific antibody to the c-terminal portion of the human c-erbB-2 gene product, we have found that gp190 is the human c-erbB-2 gene product which is structurally closely related to the epidermal growth factor (EGF) receptor. Since monoclonal antibody specific for the EGF receptor abolished both EGF binding to its receptor and tyrosine phosphorylation of the c-erbB-2 gene product, we have concluded that activation of EGF receptor tyrosine kinase activity upon EGF binding leads to the phosphorylation of the c-erbB-2 gene product on its tyrosine residues.     

Structural basis for epidermal growth factor receptor function

The receptor for epidermal growth factor (EGF) has been the subject of intense study primarily as a consequence of the pioneering studies of Cohen on growth factors and also because of its homology to the transforming protein encoded by the avian oncogene v-erbB, which is a truncated receptor, and its consequent role in cancer. Although similar structural mutation of the EGF receptor has not yet been found in human tumours, aberrant overexpression of both EGF receptors and c-erbB2, a closely related putative receptor [1], have been found to occur in squamous cell carcinomas and glial tumours, and mammary carcinomas respectively [2–4]. In addition to EGF, the related polypeptides transforming growth factor α (TGFα) and vaccinia virus growth factor [5] are also ligands for the EGF receptor. Expression of TGFα occurs during embryonal development and in specific adult tissues; it may also play a role in cellular transformation (reviewed in Ref. 6). These important properties, as well as the potential roles of both TGFα and EGF in wound repair, have emphasized the need to understand EGF receptor structure, function and regulation. This review discusses the structural properties of the EGF receptor and how these can be related to receptor function and regulation.     

Identification of c-erbB-3 binding sites for phosphatidylinositol 3''-kinase and SHC using an EGF receptor/c-erbB-3 chimera.

c-erbB-3 is a member of the type I (EGF receptor-related) family of growth factor receptors for which no ligand has been identified. To facilitate ligand stimulation we have constructed a chimeric receptor which possesses an activatable kinase and promotes the growth of NIH 3T3 fibroblasts. In this study we have shown that SHC and phosphatidylinositol 3'-kinase bind to the activated EGF receptor/c-erbB-3 chimera. Whereas p85 is not phosphorylated to a significant extent, SHC appears to be a major substrate for phosphorylation on tyrosine. In contrast to EGF receptor and c-erbB-2, we were unable to detect binding of activated c-erbB-3 to GRB2. Using synthetic peptides corresponding to each of 13 potential phosphorylation sites on c-erbB-3, we have shown that tyrosine 1309 is responsible for SHC binding. Peptides containing the motif YXXM inhibit p85 association. By comparison with recently reported SHC binding sites on Middle T antigen and Trk we have identified a SHC binding motif, NPXY.     

HER2 cytoplasmic domain generates normal mitogenic and transforming signals in a chimeric receptor.

We have investigated the biological function of an unidentified human growth factor, the ligand of the putative HER2 receptor, by characterizing the signalling properties of its receptor. HER2 (or c-erbB-2), the human homolog of the rat neu proto-oncogene, encodes a transmembrane glycoprotein of the tyrosine kinase family that appears to play an important role in human breast carcinoma. Since a potential ligand for HER2 has not yet been identified, it has been difficult to analyze the biochemical properties and biological function of this cell surface protein. For this reason, we replaced the HER2 extracellular domain with the closely related ligand binding domain sequences of the epidermal growth factor (EGF) receptor, and examined the ligand-induced biological signalling potential of this chimeric HER1-2 protein. This HER1-2 receptor is targetted to the cell surface of transfected NIH 3T3 cells, forms high and low affinity binding sites, and generates normal mitogenic and cell transforming signals upon interaction with EGF or TGF alpha. The constitutive activation of wild-type HER2 in transfected NIH 3T3 cells suggests the possibility that these cells synthesize the as yet unidentified HER2 ligand and activate HER2 by an autocrine mechanism.     

Mitogenic activity of neu differentiation factor/heregulin mimics that of epidermal growth factor and insulin-like growth factor-I in human mammary epithelial cells

Recently, a family of growth factors has been described that activates erbB-2 receptors. These factors, known as the neu differentiation factors (NDF) or heregulins (HRG), induce tyrosine phosphorylation of erbB-2 receptors as a result of their direct interaction with either erbB-3 or erbB-4 receptors. Although it is known that expression of erbB-2 receptors has relevance in human breast cancer progression, how erbB-2, -3 and -4 receptors regulate mammary epithelial cell proliferation is not known. Therefore, experiments were carried out to study the mitogenic activity of NDF/HRG on the human mammary epithelial cell line MCF-10A which can be cultured continuously under serum-free conditions. MCF-10A cells, like primary cultures of normal human mammary epithelial cells, express an absolute requirement for exogenous epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) for growth. The results of these experiments indicate that NDF/HRG can induce tyrosine phosphorylation of p185erbB-2 in MCF-10A cells and is mitogenic for these cells. This is consistent with the coexpression of erbB-2 and erbB-3 mRNA that we have observed in MCF-10A cells. In addition, we found that NDF/HRG can substitute for either EGF or IGF-I to stimulate proliferation of these cells. The ability to substitute for both EGF and IGF-I is a unique property of NDF/HRG and is not shared by other members of the EGF or IGF family of growth factors, nor by other factors that we have studied. A striking isoform specificity was also observed which indicated that the β-isoforms of NDF/HRG were greater than ten times more mitogenic than the α-isoforms. We also examined the mitogenic activity of NDF/HRG on MCF-10A cells that overexpress the erbB-2 receptor as a result of infection with a retroviral vector containing the human c-erbB-2 gene (MCF-10AerbB-2 cells). These studies indicated that MCF-10AerbB-2 cells have increased sensitivity to the mitogenic effects of NDF/HRG and that these cells are responsive to the α-isoforms of NDF/HRG at physiological concentrations. Thus, NDF/HRG is a dual specificity growth factor for human mammary epithelial cells, and the responsiveness of the cells to NDF/HRG is influenced by the level of expression of erbB-2 receptors. © 1995 Wiley-Liss, Inc.     

Purification of Her-2 extracellular domain and identification of its cleavage site

The EGF family of receptors belongs to the tyrosine kinase receptor (TKR) family and plays an important role during embryonic and postnatal development and also in the progression of tumors. Her-2/neu/c-erbB-2, a member of the epidermal growth factor receptor family, can be cleaved into a soluble extra cellular domain (ECD) and a membrane-bound stub fragment. Her-2 ECD from a breast cancer cell line SKBR3 was immunopurified and analyzed with matrix-assisted laser desorption ionization (MALDI) and carboxyl terminal amino acid sequencing. A sequence within the juxtamembrane region (only 11 amino acid residues) PAEQR ASP was identified most likely as a primary site of cleavage, PA EQRASP as a minor site, that generate the ECD. The sites of cleavage are within the signature motif P/GX(5-7)P/G highly conserved in the EGF receptor family.     

Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.     

Heregulin-beta is especially potent in activating phosphatidylinositol 3-kinase in nontransformed human mammary epithelial cells

The neu differentiation factors/heregulins (HRGs) comprise a family of polypeptide growth factors that activate p185(erbB-2) through direct binding to either erbB-3 or erbB-4 receptor tyrosine kinases. We have previously shown that HRG-beta is mitogenic for various human mammary epithelial cell lines that coexpress c-erbB-2 and c-erbB-3. Phosphatidylinositol 3-kinase (PI3K) is activated by p185(erbB-2) /erbB-3 heterodimers in cells stimulated by HRG, and PI3K is constitutively activated by p185(erbB-2) /erbB-3 in breast carcinoma cells that overexpress c-erbB-2. To better understand the relative abilities of HRGs, epidermal growth factor (EGF), or insulin to activate PI3K under normal physiological conditions, we compared the levels of recruitment of the 85-kDa regulatory subunit of PI3K when activated by the type I (erbB) or type II [insulin-like growth factor (IGF)] receptor tyrosine kinases in two different nontransformed human mammary epithelial cell lines. The nontransformed H16N-2 cells isolated from normal tissue express EGFR, p185(erbB-2), and erbB-3, and are highly responsive to the mitogenic effects of HRG-beta as well as to the combination of EGF and insulin in serum-free culture. We measured the stoichiometry of p85 recruited by tyrosine-phosphorylated proteins induced in H16N-2 cells by either the alpha or the beta isoform of HRG. HRG-beta was greater than 10-fold more potent in inducing p85 recruitment than was the less biologically active HRG-alpha isoform. HRG-beta was also a more potent inducer of p85 recruited by tyrosine-phosphorylated proteins than was either EGF, insulin, or EGF and insulin combined. Furthermore, erbB-3 principally mediated the direct recruitment of p85 in cells stimulated by HRG or EGF, indicating that, in addition to the high-level activation of PI3K by p185(erbB-2) / erbB-3, EGFR/erbB-3 heterodimer interaction is essential for the weak but significant level of PI3K activated by EGF in cells that express normal EGFR levels. Studies using the PI3K inhibitor wortmannin also indicated that PI3K activation was required for the proliferation of H16N-2 cells induced by either HRG-beta or EGF and insulin in serum-free culture. Finally, HRG-beta was also an especially potent inducer of PI3K in the nontransformed MCF-10A cells, which were derived spontaneously from normal reduction mammoplasty tissue. These data show, for the first time, a side-by-side quantitative comparison of the relative degree of PI3K activated by different growth factors in nontransformed growth factor-dependent cells under precisely defined conditions in culture.     

Hormonal regulation of c-erbB-2 oncogene expression in breast cancer cells.

Expression of the c-erbB-2 (neu, HER-2) oncogene is found to be subjected to hormonal and developmental regulation in normal as well as neoplastic mammary cells. We have previously reported that estrogens inhibit c-erbB-2 expression at both the mRNA and protein level in estrogen receptor (ER)-positive, but not in ER-negative, breast cancer cell lines. Reversion of c-erbB-2 inhibition is seen with tamoxifen. The effect on c-erbB-2 expression of several other hormones and factors, which influence mammary cell growth and differentiation, has been studied. Our observations indicate that, in normal and neoplastic mammary cells, c-erbB-2 expression is inversely related to cell proliferation. While estrogens, anti-estrogens and cAMP clearly regulate c-erbB-2 mRNA levels, epidermal growth factor dramatically decreases the c-erbB-2 protein without affecting the level of c-erbB-2 mRNA. Therefore, different signals converging in terms of cell proliferation regulate c-erbB-2 expression by different molecular mechanisms.     

Tumor promoter and epidermal growth factor stimulate phosphorylation of the c-erbB-2 gene product in MKN-7 human adenocarcinoma cells.

Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.     

The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling.     

Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line.

The c-erbB-2 gene is a v-erbB-related proto-oncogene which is distinct from the gene encoding the epidermal growth factor receptor. By using two independent methods, hybridization of both sorted chromosomes and metaphase spreads with cloned c-erbB-2 DNA, we mapped the c-erbB-2 locus on human chromosome 17 at q21, a specific breakpoint observed in a translocation associated with acute promyelocytic leukemia. Furthermore, we observed amplification and elevated expression of the c-erbB-2 gene in the MKN-7 gastric cancer cell line. These data suggest possible involvement of the c-erbB-2 gene in human cancer.     

Significance of the Grb2 and son of sevenless (Sos) proteins in human bladder cancer cell lines

The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine-nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP. However, the significance of Grb2 and Sos proteins, which is related to EGF-triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU-7, UMUC-2, UMUC-6) and two kinds of cultured normal urothelial cells (HMKU-1, HMKU-2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c-H-ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H-ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer.     

Cloning and biological activity of epigen, a novel member of the epidermal growth factor superfamily

High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putative N-linked glycosylation sites. Epigen shows 24-37% identity to members of the EGF superfamily including EGF, transforming growth factor alpha, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor alpha in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy.     

An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors

Recent crystallographic studies have provided significant new insight into how receptor tyrosine kinases from the EGF receptor or ErbB family are regulated by their growth factor ligands. EGF receptor dimerization is mediated by a unique dimerization arm, which becomes exposed only after a dramatic domain rearrangement is promoted by growth factor binding. ErbB2, a family member that has no ligand, has its dimerization arm constitutively exposed, and this explains several of its unique properties. We outline a mechanistic view of ErbB receptor homo- and heterodimerization, which suggests new approaches for interfering with these processes when they are implicated in human cancers.     

Light and electron microscopical demonstration of c-erbB-2 gene product-like immunoreactivity in human malignant tumors

C-erbB-2 is a human protooncogene homologous with the well-known c-erbB. Genes and gene products of the EGF receptor and c-erbB are known to be closely related and to be closely homologous in their intracellular domain. Inspection of the deduced amino acid sequence suggested that the c-erbB-2 gene encodes a receptor for a yet unidentified growth factor. An immunohistological study was performed by introducing an antibody raised in the rabbit by immunization with a synthetic peptide corresponding to a part of the intracytoplasmic domain of predicted gene product. Specimens from 13 normal human organs, fresh frozen tissue from 41 surgically excised human malignant tumors and eight cell lines maintained in nude mice were studied. Positive staining was found in 4 of the 41 (9.8%) malignant tumors. All of the positive tumors were adenocarcinomas and two adenocarcinoma cell lines were also positive. Amongst the normal human tissues, epithelial cells in stomach, small and large intestine were faintly stained. When the positively stained cell lines were studied by immunoelectronmicroscopy, the reaction was most prominent in the membrane of microvilli, but part of the nuclear membrane, the endoplasmic reticulum and the outer cell membrane were also stained. DNA and mRNA blot assays, as well as our immunoprecipitation test, revealed that immunohistologically positive cell lines bore amplified c-erbB-2 DNA, c-erbB-2 mRNA and 185 kD protein which is supposed to be the gene product, while negative cell lines did not.