Signal transduction mechanisms through granulocyte colony-stimulating factor receptor.

为了分析中性粒细胞分化的机制，克隆了编码转录因子的人MZF-1基因的小鼠同源基因(MZF-2)。参与中性粒细胞分化，被分离出来。含锌指结构域的MZF-2蛋白的C末端与MZF-1蛋白高度同源，但MZF-2蛋白的氨基末端与MZF-1蛋白不同，因为MZF-2蛋白含有亮氨酸富集区(LER区)和富含酸性氨基酸的区域。MZF-2的染色体基因由5个外显子组成，该基因的转录起始点交替产生MZF-1和MZF-2的mRNAs。对MZF-2氨基末端缺失衍生物的转录激活活性的分析表明，氨基末端区域与转录激活区域相互作用并抑制其自身的转录激活。另一方面，分离到依赖于G-CSF的磷酸化蛋白，可以与酪氨酸磷酸化的G-CSF受体结合。其中一个与SHIP的N端氨基酸序列完全相同，证明了其依赖于G-CSF的酪氨酸磷酸化和与G-CSF受体的相互作用。此外，我还检测了几个抗凋亡基因的G-CSF依赖的表达。其中BCL-2在G-CSF刺激后约3小时表达。由于该基因在携带第4胞浆酪氨酸残基突变的G-CSF受体的细胞中不表达，该受体的第4酪氨酸残基似乎参与了抗细胞凋亡的信号转导。此外，在G-CSF刺激15分钟后，α-D-甘露糖苷酶和巨噬细胞炎性蛋白1-α基因表达。

To analyze the mechanisms of neutrophil differentiation, murine homologue (MZF-2) of human MZF-1 cDNA, which encodes the transcription factor. involved in neutrophil differentiation, was isolated. C-terminus region of MZF-2 protein containing zinc finger domain is highly homologous to that of the MZF-1, However, the amino-terminus of MZF-2 protein was different from that of MZF-1 protein in that MZF-2 protein has leucine rich region (LeR domain) and a region rich in acidic amino acids. The chromosomal gene of MZF-2 consist of 5 exons and alternative transcription start sites of the gene produce MZF-1 and MZF-2 mRNAs. The analysis of transcription activation activities of amino-terminus deletion derivatives of MZF-2 revealed that amino-terminus region interacted to the transcription-activation domain and suppressed its own transcriptional activation.On the other hand, G-CSF dependent phospho-proteins, which can bind to the tyrosine-phosphorylated G-CSF receptor, were isolated. The N-terminal amino-acid sequence of one of those was identical to that of SHIP.Its G-CSF dependent tyrosine phosphorylation and interaction with G-CSF receptor were demonstrated.Furthermore, I examined G-CSF dependent expression of several anti-apoptotic genes. One of them, bcl-2, was found to be expressed about 3 hours after G-CSF stimulation. As the gene was not expressed in cells carrying G-CSF receptor with mutated 4th cytoplasmic tyrosine residue, the 4th tyrosine residue of the receptor seemed to be involved in the anti-apoptotic signal transduction.Moreover, alpha-D-mannnosidase and macrophage inflammatory protein 1-alpha genes were found to be expressed 15 minutes after G-CSF stimulation.