Kumar S, de Vellis J
Department of Neurobiology, UCLA School of Medicine, USA.
J Neurosci Res. 1996 Jun 1;44(5):490-8. doi: 10.1002/(SICI)1097-4547(19960601)44:5<490::AID-JNR9>3.0.CO;2-A.
The role of the NT-3 has been implicated in the survival of progenitor oligodendrocytes in culture. The object of this study was to investigate the expression of the TrkC receptor and its responsiveness in glial cells. We report the expression of two TrkC receptor isoforms in rat primary oligodendrocyte cultures, a glial progenitor cell line, CG-4, and in C6 glioma cells. The reverse transcription-polymerase chain reaction-aided amplification of glial trkC with specific primers from the kinase domain, followed by its cloning and sequencing, shows the presence of two trkC transcripts. The sequence of one of the transcripts is homologous to a previously identified trkC isoform which encodes a functional receptor. The other transcript contains a 42-bp insert in the kinase domain. A Western blot of CG-4 and C6 probed with antibody to a TrkC revealed the presence of gp145-kDa protein band. The investigations revealed a rapid autophosphorylation of gp145TrkC in CG-4 and C6 cells in the presence of its specific ligand, NT-3. Furthermore, K252a, a neurotrophin-specific inhibitor, abolishes the NT-3-mediated receptor autophosphorylation. We also examined other NT-3-dependent phosphorylation of cellular substrates in oligodendroglial cells. Interestingly, we observed phosphorylation of phospholipase C gamma-1 in CG-4 and C6 cells, and phosphorylation of phosphatidylinositol 3-kinase in C6 cells in the presence of NT-3. Both the NT-mediated phosphorylation of phospholipase C gamma-1 and phosphorylation of phosphatidylinositol 3-kinase are blocked in the presence of K252a. The detection of the NT-3-mediated early signal transduction events demonstrates that TrkC receptor exhibits NT-3-mediated intracellular response in oligodendroglial cells.
已发现神经营养因子-3（NT-3）在培养的少突胶质前体细胞存活中发挥作用。本研究的目的是调查TrkC受体在神经胶质细胞中的表达及其反应性。我们报告了大鼠原代少突胶质细胞培养物、神经胶质前体细胞系CG-4以及C6胶质瘤细胞中两种TrkC受体亚型的表达情况。用来自激酶结构域的特异性引物通过逆转录-聚合酶链反应辅助扩增神经胶质trkC，随后进行克隆和测序，结果显示存在两种trkC转录本。其中一种转录本的序列与先前鉴定的编码功能性受体的trkC亚型同源。另一种转录本在激酶结构域中有一个42个碱基对的插入片段。用抗TrkC抗体对CG-4和C6进行蛋白质印迹分析，结果显示存在145-kDa的糖蛋白条带。研究发现，在其特异性配体NT-3存在的情况下，CG-4和C6细胞中的gp145TrkC会快速发生自身磷酸化。此外，神经营养因子特异性抑制剂K252a可消除NT-3介导的受体自身磷酸化。我们还检测了少突胶质细胞中其他NT-3依赖的细胞底物磷酸化情况。有趣的是，我们观察到在NT-3存在的情况下，CG-4和C6细胞中磷脂酶Cγ-1发生磷酸化，C6细胞中磷脂酰肌醇3激酶发生磷酸化。在K252a存在的情况下，NT介导的磷脂酶Cγ-1磷酸化和磷脂酰肌醇3激酶磷酸化均被阻断。NT-3介导的早期信号转导事件的检测表明，TrkC受体在少突胶质细胞中表现出NT-3介导的细胞内反应。
