Chan S D, Antoniucci D M, Fok K S, Alajoki M L, Harkins R N, Thompson S A, Wada H G
Molecular Devices Corporation, Sunnyvale, California 94089, USA.
J Biol Chem. 1995 Sep 22;270(38):22608-13. doi: 10.1074/jbc.270.38.22608.
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., Akita, R.W., Lofgren, J.A., Fitzpatrick, V.D., Nuijens, A., Fendly, B.M., Cerione, R.A., Vandlen, R.L., and Carraway, K.L., III (1994) J. Biol. Chem. 269, 14661-14665; Plowman, G.D., Green, J.M., Culouscou, J.-M., Carlton, G.W., Rothwell, V.M., and Buckley, W. (1993) Nature 366, 473-475). Using the Cytosensor to measure the extracellular acidification rate, we have examined the effects of recombinant human heregulin-alpha on three mammary carcinoma cell lines expressing HER2 (MDA-MB-453, SK-BR-3, and MCF-7), an ovarian carcinoma cell line expressing HER2 (SK-OV-3), and CHO-K1 and 293-EBNA cells stably transfected with HER2. By reverse transcription polymerase chain reaction and Western blotting, we found that the breast cells also express HER3 and that the ovarian line co-expresses the HER4 message. A dramatic increase in the acidification rate was observed for the mammary carcinoma cells co-expressing high levels of HER2 and HER3. In contrast, the ovarian cells expressing high levels of HER2 and low levels of HER4 or CHO-K1 and 293-EBNA cells expressing HER2 alone were not responsive to heregulin. When these same transfected cells were exposed to monoclonal anti-HER2 antibody followed by anti-IgG to cause aggregation of the HER2 molecules, an increase in the acidification rate was observed, indicating coupling of transfected HER2 to the signal transduction pathway. Transfection of HER2 into MCF-7 cells, on the other hand, gave 4-fold enhanced acidification responses. These data, together with the previously reported high affinity heregulin binding and activation of tyrosine phosphorylation in HER2 and HER3 co-transfected cells support the role of HER2 and HER3 as components of the heregulin receptor in breast cells.
HER2是erbB-2/neu原癌基因产物，是一种与表皮生长因子受体相关的185 kDa跨膜糖蛋白。在包括乳腺癌和卵巢癌在内的几种人类腺癌中报道了HER2的过表达。一类糖蛋白，即神经调节蛋白/neu分化因子，已被鉴定并被认为是HER2的配体。最近，研究表明仅HER2不足以重建高亲和力的神经调节蛋白受体，HER3或HER4可能是乳腺癌细胞上神经调节蛋白受体的必需组成部分（斯利科夫斯基，M.X.，谢弗，G.，秋田，R.W.，洛夫格伦，J.A.，菲茨帕特里克，V.D.，努伊延斯，A.，芬德利，B.M.，塞里奥内，R.A.，万德伦，R.L.，以及卡拉韦，K.L.三世（1994年）《生物化学杂志》269卷，第14661 - 14665页；普洛曼，G.D.，格林，J.M.，库卢斯科，J.-M.，卡尔顿，G.W.，罗斯韦尔，V.M.，以及巴克利，W.（1993年）《自然》366卷，第473 - 475页）。我们使用细胞传感器测量细胞外酸化率，研究了重组人神经调节蛋白-α对三种表达HER2的乳腺癌细胞系（MDA-MB-453、SK-BR-3和MCF-7）、一种表达HER2的卵巢癌细胞系（SK-OV-3）以及稳定转染了HER2的CHO-K1和293-EBNA细胞的影响。通过逆转录聚合酶链反应和蛋白质免疫印迹法，我们发现乳腺细胞也表达HER3，卵巢细胞系共表达HER4信息。在共表达高水平HER2和HER3的乳腺癌细胞中观察到酸化率显著增加。相比之下，表达高水平HER2和低水平HER4的卵巢细胞或仅表达HER2的CHO-K1和293-EBNA细胞对神经调节蛋白无反应。当这些相同的转染细胞暴露于单克隆抗HER2抗体，然后再用抗IgG使HER2分子聚集时，观察到酸化率增加，表明转染的HER2与信号转导途径偶联。另一方面，将HER2转染到MCF-7细胞中，酸化反应增强了4倍。这些数据，连同先前报道的在HER2和HER3共转染细胞中神经调节蛋白的高亲和力结合和酪氨酸磷酸化的激活，支持了HER2和HER3作为乳腺细胞中神经调节蛋白受体组成部分的作用。
