Studies on granulocyte colony-stimulating factor receptor

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Granulocyte colony-stimulating factor(G-CSF)is a growth and differentiation factor which works on cells restricted to the neutrophilic granulocyte lineage. To clarify the structure and function of G-CSF receptor(G-CSF-R), we isolated cDNAs for murine and human G-CSF-R. Sequence analysis revealed that G-CSF-R is an 812(mouse)or 813(human)amino acid polypeptide with a single transmembrane domain. The extracellulsr domain consists of an Ig-like domain, a cytokine-receptor-homologous(CRH)domain and three fibronectin type III(FNIII)domains. Mouse G-CSF-R expressed in COS cells was able to bind G-CSF with an affinity(Kd = 290 pM)similar to that of the receptor of NFS-60 cells, indicating that the single polypeptide is sufficient to form the high-affinity binding site for G-CSF. Molecular cloning and chromosomal mapping of the G-CSF-R gene indicated that the human G-CSF-R gene consists of 17 exons and is located on the p35-34.3 region of human chromosome 1. To explore signal transduction mechanism of the G-CSF-R, we expressed the G-CSF-R CDNA in various hematopoietic cell lines. Introduction of G-CSF-R CDNA into IL-3-dependent mouse myeloid cell line FDC-PL and pro-B cell line BAF-BO3 enabled them to proliferate in response to G-CSF. However, IL-2-dependent mouse CTLL-2 cells expressing G-CSF-R could not grow in the presence of G-CSF. Surface expression of some differentiation markers on the FDC-PL transformants was differentially regulated by G-CSF and IL-3. Mutational analysis of the G-CSF-R in FDC-Pl cells indicated that the N-terminal half of the CRH domain was essential for the recognition of G-CSF, but the Ig-like, FNIII and cytoplasmic domains were not. The CRH domain and a 76-amino acids portion of the cytoplasmic domain were indispensable for the transduction of the G-CSF-triggered growth signal.