Totzke F, Marmé D, Hug H
University of Freiburg, Institute of Molecular Cell Biology, Federal Republic of Germany.
Eur J Biochem. 1992 Feb 1;203(3):633-9. doi: 10.1111/j.1432-1033.1992.tb16593.x.
Growth factors such as platelet-derived growth factor (PDGF) have been shown to activate phospholipase C-gamma 1 (PLC-gamma 1). We have overexpressed the human PLC-gamma 2 (hPLC-gamma 2) cDNA in murine NIH 3T3 fibroblasts using the interferon-type-I inducible murine Mx promoter. Northern blot analysis revealed an induction of hPLC-gamma 2 mRNA by interferon (IFN) alpha of about 25-fold as compared to the uninduced transcript level. Western blot analysis with anti(bovine PLC-gamma 2) antiserum showed increased hPLC-gamma 2 protein levels in hPLC-gamma 2 transfected cells. Induction with IFN alpha resulted only in a slight further increase. After labelling the cells with [35S]methionine an increase of radioactive label in a protein migrating at 148 kDa could be detected in IFN-alpha-stimulated, hPLC-gamma 2 overexpressing cells. PLC activity in homogenates from hPLC-gamma 2 overexpressing cells was increased as compared to control cells transfected with the vector lacking the hPLC-gamma 2 cDNA insert. There was no difference between in vitro PLC activity in homogenates from PDGF B-chain homodimer (BB) treated and untreated cells. PLC activity was mainly present in the soluble fraction. After incubation of hPLC-gamma 2 overexpressing cells with IFN alpha, the in vitro activity of PLC increased significantly in the soluble fraction. Stimulation with PDGF BB increased inositol phosphate production about 3.5-fold in control cells and about 10-fold in hPLC-gamma 2 overexpressing cells. PDGF A-chain homodimer (AA) showed slightly smaller effects. These results demonstrate that human PLC-gamma 2 can be expressed functionally in murine NIH 3T3 fibroblasts and can be activated by both murine PDGF receptors, alpha and beta type.
诸如血小板衍生生长因子（PDGF）等生长因子已被证明可激活磷脂酶C-γ1（PLC-γ1）。我们使用I型干扰素诱导的小鼠Mx启动子，在鼠源NIH 3T3成纤维细胞中过表达了人PLC-γ2（hPLC-γ2）cDNA。Northern印迹分析显示，与未诱导的转录水平相比，干扰素（IFN）α可使hPLC-γ2 mRNA诱导增加约25倍。用抗（牛PLC-γ2）抗血清进行的Western印迹分析表明，在hPLC-γ2转染细胞中hPLC-γ2蛋白水平增加。用IFNα诱导仅导致轻微的进一步增加。在用[35S]甲硫氨酸标记细胞后，在IFN-α刺激的、hPLC-γ2过表达细胞中，可检测到在148 kDa处迁移的蛋白质中放射性标记增加。与用缺乏hPLC-γ2 cDNA插入片段的载体转染的对照细胞相比，hPLC-γ2过表达细胞匀浆中的PLC活性增加。PDGF B链同二聚体（BB）处理和未处理细胞的匀浆中的体外PLC活性没有差异。PLC活性主要存在于可溶性部分。用IFNα孵育hPLC-γ2过表达细胞后，可溶性部分的PLC体外活性显著增加。用PDGF BB刺激可使对照细胞中的肌醇磷酸生成增加约3.5倍，在hPLC-γ2过表达细胞中增加约10倍。PDGF A链同二聚体（AA）的作用略小。这些结果表明，人PLC-γ2可在鼠源NIH 3T3成纤维细胞中功能性表达，并可被鼠源PDGF受体α型和β型激活。
