CLONING OF THE RAT ERBB3 CDNA AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN
CLONING OF THE RAT ERBB3 CDNA AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN
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DOI：
10.1016/0378-1119(95)00436-a
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发表时间：
1995-11-20
期刊：
影响因子：
3.5
通讯作者：
KOLAND, JG
中科院分区：
文献类型：
--
作者：
HELLYER, NJ;KIM, HH;KOLAND, JG
通过使用人 ERBB3 探针对大鼠肝脏 cDNA 文库进行低严格筛选，克隆了编码大鼠 ErbB3 蛋白除 N 末端以外的所有部分的 cDNA 片段。通过逆转录聚合酶链式反应方法生成cDNA的剩余5'端，并组装单个全长大鼠ErbB3 cDNA。对人和大鼠 ErbB3 的推导氨基酸 (aa) 序列进行了比较，并考虑了推定蛋白酪氨酸激酶结构域中某些 aa 取代的影响。随后，大鼠 ErbB3 cDNA 在培养的 NIH-3T3 小鼠成纤维细胞中表达，其中约高水平表达。生成了 180 kDa 重组 ErbB3 (re-ErbB3)。转染的成纤维细胞中产生的大鼠 re-ErbB3 对多肽、heregulin（一种已知的 ErbB3 配体）有反应。用调蛋白刺激转染的成纤维细胞刺激了大鼠 re-ErbB3 在 Tyr 残基上的磷酸化，并促进其与磷脂酰肌醇 3-激酶的 p85 亚基的结合。总之，这些结果表明功能齐全的大鼠ErbB3 cDNA已经被分离出来，并且表达该cDNA的成纤维细胞将适合研究ErbB3的信号转导机制。
Three cDNA fragments that encoded all but the extreme N terminus of the rat ErbB3 protein were cloned by low-stringency screening of a rat liver cDNA library with a human ERBB3 probe. The remaining 5'-end of the cDNA was generated by a reverse transcription-polymerase chain reaction method, and a single full-length rat ErbB3 cDNA was assembled. A comparison of the deduced amino acid (aa) sequences of human and rat ErbB3 was made, and the effects of certain aa substitutions in the putative protein tyrosine kinase domain were considered. The rat ErbB3 cDNA was subsequently expressed in cultured NIH-3T3 mouse fibroblasts, in which a high level of approx. 180-kDa recombinant ErbB3 (re-ErbB3) was generated. The rat re-ErbB3 produced in transfected fibroblasts was responsive to the polypeptide, heregulin, a known ligand for ErbB3. Challenge of transfected fibroblasts with heregulin stimulated the phosphorylation of rat re-ErbB3 on Tyr residues and promoted its association with the p85 subunit of phosphatidylinositol 3-kinase. Together, these results indicate that a fully functional rat ErbB3 cDNA has been isolated, and that fibroblast cells expressing this cDNA will be suitable for investigations of the signal transduction mechanism of ErbB3.