Signal transduction mechanisms of neutrophil differentiation through G-CSF receptor.

G-CSF刺激导致髓前体细胞增殖数天，然后停止增殖，随后分化为成熟的中性粒细胞。G-CSF受体远端膜区酪氨酸残基和STAT3的激活参与了中性粒细胞分化过程中的生长停滞。然而，受体的其他残基和其他信号分子在生长阻滞中的作用仍有待确定。我们在受体的细胞质区域引入突变，并将其转染到不表达内源性G-CSF受体的粒细胞前体细胞中。分离出一株在G-CSF存在下持续增殖的细胞系，即在含有G-CSF的培养基中不表现出生长停滞。从获得的细胞系染色体DNA中分离出突变的G-CSF受体基因。将每个突变基因重新导入粒细胞前体细胞，观察g - csf反应。在含有G-CSF的培养基中连续增殖的细胞系，在其c端缺失了59个氨基酸的G-CSF受体，表明该c端结构域负责转导生长停滞信号。构建了一系列被截断的受体，并建立了表达这些被截断受体的细胞系。含有这些截断受体的细胞系的G-CSF反应表明，G-CSF受体c端25个氨基酸残基是转导生长停滞信号所必需的。表达截断受体的细胞在g - csf依赖性诱导嗜中性粒细胞形态变化的过程中也表现出缺陷。在表达截断受体的细胞中，G-CSF依赖性的STATS激活延长。因此，c端25个氨基酸残基负责STAT5的持续激活，导致G-CSF依赖性生长停滞和形态改变的缺陷。少

G-CSF stimulation leads to the myeloid precursor cells to proliferate for a few days, then they stop proliferating, followed by differentiation to the mature neutrophils. The tyrosine residues in membrane-distal region of the G-CSF receptor as well as the activation of STAT3 was involved in the growth arrest during the neutrophil differentiation. However, involvement of other residues of the receptor and other signaling molecules in the growth arrest remains to be determine. We introduced mutations in the cytoplasmic region of the receptor and transfected them into granulocyte precursor cells which did not express endogenous G-CSF receptor. A cell line was isolated that proliferated continuously in the presence of G-CSF, that is, that did not show growth-arrest in the medium containing G-CSF. The mutated G-CSF receptor genes were isolated by PCR from the chromosomal DNA of the obtained cell line. Each mutant gene was re-introduced into the granulocyte precursor cells and G-CSF-response … More s of the obtained stable transformants were examined. A cell line which proliferated continuously in the medium with G-CSF, carved G-CSF receptor with its 59 amino acid deletion in its C-terminus, suggesting this C-terminus domain is responsible for transducing the growth arrest signals. A series of truncated receptors were constructed and cell lines expressing these truncated receptors were established. G-CSF-responses of the cell lines harboring these truncated receptors revealed that C-terminal 25 amino acid residues of the G-CSF receptor was necessary for the transducing growth arrest signals. Cells expressing the truncated receptors also showed the defects in the G-CSF-dependent induction of neutrophilic morphological changes with lobulated nucleus. The G-CSF dependent activation of STATS was prolonged in the cells expressing the truncated receptor. Therefore, the C-terminal 25 amino acid residues were responsible for the sustained activation of STAT5, resulting in the defects in the G-CSF dependent growth arrest and morphological changes. Less