G-csf receptor tyrosines are involved in signal transduction in normal haemopoietic cells

The four tyrosine residues (Y1-4) in the cytoplasmic region of the granulocytic colony-stimulating factor (G-CSF) receptor are crucial for signal transduction in cultured cell lines. However, their role in vivo is controversial. This project aimed to determine the role of Y residues in proliferation/differentiation signal transduction in normal murine bone marrow cells. To bypass signaling by the endogenous G-CSF receptor, a chimeric receptor containing the extracellular domain of the epidermal growth factor and the cytoplasmic domain of the G-CSF receptor was constructed (EGR WT). To prevent phosphorylation on Y, constructs with phenyalanine (F) substitution for Y were created, for each Y1-4 individually and all four Y1-4 together. Expression of these six constructs in bone marrow cells from 5-FU treated G-CSF-deficient mice was achieved using the pMSCVpac retroviral expression vector. Bone marrow cells expressing EGR (Y1-4 null) mutation showed reduced sensitivity to EGF compared to those expressing the EGR WT in an agar colony formation assay. Moreover, colony formation was reduced in the bone marrow cells expressing EGR (Y4→F) mutation compared to those expressing EGR WT. Preliminary results suggest that the bone marrow cells stimulated via EGR (Y2→F) produce fewer granulocytic colonies. However, those remaining express myeloperoxidase. In summary, Y residues are required for full response in agar at lower concentrations of EGF. Y4 supports cell proliferation and Y2 may have a role in neutrophil differentiation.