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

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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