ERBB-3 PROTEIN REVEALS STRUCTURAL FEATURES OF A TRANSMEMBRANE TYROSINE KINASE

我们之前已经通过克隆第三个cdna来鉴定和表征 人类erbB/表皮生长中的功能基因，命名为erbB-3 酪氨酸激酶因子(EGF)亚家族。它的预测编码 生长因子受体的结构特征 酪氨酸激酶与两者的总体相似性要大得多 表皮生长因子受体(EGFR)及其产物比任何其他 酪氨酸激酶。用于编码基因的体外高水平表达 ErbB-3基因产物。其完整的编码序列已在 基于ltr的真核表达载体及其存在下的转染 将一个可选择的标记基因导入NIH/3T3成纤维细胞。利用 抗小鼠免疫球蛋白表位特异性多肽的多克隆抗血清 预测的erbB-3编码序列，成熟的erbB-3产物 在标记筛选的转染体中鉴定为180 kDa蛋白 含有重组表达载体，对照中不存在 NIH/3T3细胞。ErbB-3蛋白的生化特性表明 它的生物合成是145 kDa的前体多肽，成为 翻译后被N-连接的糖基化修饰为180 kDa 成熟的糖蛋白。表位特异性免疫组织化学分析 抗血清鉴定了成熟erbB-3的跨膜拓扑结构 蛋白。它在酪氨酸残基上的自磷酸化能力 显示出一种内在的酪氨酸激酶活性，与 Gpl80erbB-3蛋白的细胞质部分。初步约束和 触发研究没有揭示EGF或 转化生长因子α(TGFα)。两个原型配体 对于密切相关的EGF受体，与成熟的erbB-3蛋白一起用于 信号转导。这表明在erbB/EGFR家族中。这个 ErbB-3蛋白可能是一种独特的生长因子受体 目前还没有确定的配基。

We have previously identified and characterized by cDNA cloning a third functional gene, designated erbB-3, in the human erbB/epidermal growth factor (EGF) subfamily of tyrosine kinases. Its predicted coding structure revealed features characteristic of a growth factor receptor tyrosine kinase with significantly greater overall similarities to both epidermal growth factor receptor (EGFR) and products than to any other tyrosine kinase. For high level in vitro expression of the encoded erbB-3 gene product. its complete coding sequence was introduced in LTR-based eukaryotic expression vectors and transfected in the presence of a selectable marker gene into NIH/3T3 fibroblasts. Utilizing polyclonal antisera raised against epitope-specific peptides in the predicted erbB-3 coding sequence, the mature erbB-3 product was identified as a 180-kDa protein in marker-selected transfectants containing the recombinant expression vector and was absent from control NIH/3T3 cells. Biochemical characterization of the erbB-3 protein showed its biosynthesis as a 145-kDa precursor polypeptide which becomes posttranslationally modified by N-linked glycosylation into a 180-kDa mature glycoprotein. Immunohistochemical analysis with epitope-specific antisera identified a transmembrane topology of the mature erbB-3 protein. Its ability to autophosphorylate on tyrosine residues demonstrated an intrinsic tyrosine kinase activity associated with the cytoplasmic portion of the gpl80erbB-3 protein. Preliminary binding and triggering studies did not reveal direct interaction of EGF or transforming growth factor alpha (TGFalpha). the two prototype ligands for the closely related EGF receptor, with the mature erbB-3 protein for signal transduction. This suggests that within the erbB/EGFR family. the erbB-3 protein may function as a distinct growth factor receptor for an as yet unidentified ligand.