Heregulin Activation of Extracellular Acidification in Mammary Carcinoma Cells Is Associated with Expression of HER2 and HER3

Abstract

HER2, the erbB-2/neu proto-oncogene product, is a 185-kDa transmembrane glycoprotein related to the epidermal growth factor receptor. Overexpression of HER2 was reported in several human adenocarcinomas, including mammary and ovarian carcinomas. A family of glycoproteins, the heregulin/neu differentiation factors, was characterized and implicated as the ligands for HER2. Recently, it has been shown that HER2 alone is not sufficient to reconstitute high affinity heregulin receptors and that HER3 or HER4 may be the required components of the heregulin receptors on mammary carcinoma cells (Sliwkowski, M. X., Schaefer, G. A family of EGF 1 receptor-related proteins has recently been described that includes the proto-oncogene products of erbB-2/ neu (HER2), erbB-3 (HER3), and erbB-4 (HER4) (1, 2), which have a high degree of sequence homology (40 -50%) and similar molecular structure, containing a glycosylated extracellular domain, a transmembrane domain, and a conserved tyrosine kinase domain. The HER2 receptor was the first of these discovered by two groups who screened human genomic libraries at low stringency using EGF receptor-based probes (3, 4). Subsequent to this, the HER3 (5) and HER4 (6) receptors were identified using similar techniques. Despite the structural similarity between these receptors, they have no binding affinity for any of the EGF ligands, which leaves open the possibility that ligands exist for each of these receptors.The identity of the ligands for these receptors has been the object of much investigation resulting in the identification of several candidate ligands for HER2. A 30-kDa protein (7) and heregulin (8) were purified from the conditioned medium of MDA-MB-231 mammary carcinoma cells. Neu differentiating factor was purified from the conditioned medium of ras-transformed fibroblasts (9), and Neu activating factor was purified from the conditioned medium of human ATL-2 T cells (10). Each of these putative ligands was shown to activate phosphorylation of the HER2 protein, p185. Subsequent to the cloning of Neu differentiating factor (11) and heregulin (8), peptide ligands with a high degree of sequence homology with heregulin/Neu differentiating factor, the glial growth factors-I, -II, and -III (12) and a protein that stimulates muscle acetylcholine receptor synthesis (ARIA) (13) were identified. These findings indicate that a family of heregulin-like ligands exist that are important in the development and regeneration of the nervous system and are products of the same gene, produced by alternatively spliced mRNA (12,13).Whether the heregulin/Neu differentiating factor is truly a ligand for HER2 was recently disputed by the finding that heregulin did not activate phosphorylation of p185 in ovarian cell lines expressing HER2 and in ovarian cells transfected with HER2 (14). These results suggest that another component in addition to HER2 is required for the heregulin receptor. More recently, both HER4 (16) and HER3 (17, 18) have been separately reported to be the additional component requ...

c-erbB3 and c-erbB4 expression is a feature of the endocrine responsive phenotype in clinical breast cancer

“…The developed RT ± PCR procedure for the detection of c-erbB3 and c-erbB4 mRNA was tested further using a small sub-group of breast tumour samples and was found to be highly reproducible, with a mean densitometry variance of only *4% (not illustrated). Furthermore, analysis of c-erbB3 mRNA expression by RT ± PCR for a series of breast cancer cell lines was in concurrence with the literature, both for those reported as positive (ZR75.1, MCF-7, MDA 453; Lemoine et al, 1992;Kraus et al, 1993;Chan et al, 1995) and negative (MDA 231, A431;Rajkumar and Gullick, 1994) respectively for c-erbB3 mRNA and protein (not illustrated).…”

“…Con®dence in the optimised RT ± PCR procedure for the detection of c-erbB3 mRNA was initially tested on a series of cell monolayers reported in the literature as positive (ZR75.1, MCF-7, MDA 453; Lemoine et al, 1992;Chan et al, 1995;Rajkumar et al, 1995) or negative (MDA 231, A431; Rajkumar and Gullick, 1994) respectively for c-erbB3 protein or mRNA. Furthermore, assay conditions were carefully optimized by performing simultaneous RT ± PCR of each speci®c primer in combination with aactin on 10 of the breast cancer samples using a range of cycle numbers (20 ± 32) and using conditions identical to those described previously (Knowlden et al, 1997).…”

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

“…In addition to the lack of EGFR expression, HER4 expression was minimally detected. It has been reported that HER2 expression is not detectable by Northern blotting in CHO cells (Chan et al, 1995), but we were able to detect low levels of HER2 expression by both RT-PCR and flow cytometry. HER3 expression in CHO cells was minimally detected by RT-PCR and flow cytometry.…”

Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase

“…Our Northern blot analysis (Figure 2) demonstrated that in the adult, two of these ErbB-4-positive tissues, pancreas and muscle, express three molecular weight species of NRG-4. Likewise, multiple mRNA species of NRG-1 and NRG-2 were reported (Chan et al, 1995;Wen et al, 1992). Whether or not the multiplicity of NRG-4 mRNAs is related to the existence of many isoforms of NRG-1 and NRG-2 (Bus®eld et al, 1997;Carraway et al, 1997;Chang et al, 1997;Marchionni et al, 1993;Wen et al, 1994) is currently unknown.…”