title: Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells.
abstract: Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10(4)-fold increase of colony-forming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.
authors.affiliation: Institute of Hematology, Erasmus University of Rotterdam, The Netherlands.
authors.forename: Mirjam H A
authors.initials: MH
authors.lastname: Hermans
authors.forename: Gert-Jan
authors.initials: GJ
authors.lastname: van de Geijn
authors.forename: Claudia
authors.initials: C
authors.lastname: Antonissen
authors.forename: Judith
authors.initials: J
authors.lastname: Gits
authors.forename: Daphne
authors.initials: D
authors.lastname: van Leeuwen
authors.forename: Alister C
authors.initials: AC
authors.lastname: Ward
authors.forename: Ivo P
authors.initials: IP
authors.lastname: Touw
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D003114:Colony-Forming Units Assay; D005260:Female; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D009154:Mutation; D023461:Myeloid Progenitor Cells / Q000166:cytology* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism / Q000502:physiology; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics* / Q000502:physiology; D012097:Repressor Proteins; D015398:Signal Transduction; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D014161:Transduction, Genetic; D014443:Tyrosine / Q000502:physiology*
chemical_list: D011506:Proteins; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D012097:Repressor Proteins; C491411:Socs3 protein, mouse; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D016179:Granulocyte Colony-Stimulating Factor; D014443:Tyrosine; D020928:Mitogen-Activated Protein Kinases