title: Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G-CSF-mediated activation of signaling complexes of the p21ras route.
abstract: Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G-CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G-CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.
authors.affiliation: Institute of Hematology, Erasmus University, Rotterdam, The Netherlands.
authors.forename: R M
authors.initials: RM
authors.lastname: Barge
authors.forename: J P
authors.initials: JP
authors.lastname: de Koning
authors.forename: K
authors.initials: K
authors.lastname: Pouwels
authors.forename: F
authors.initials: F
authors.lastname: Dong
authors.forename: B
authors.initials: B
authors.lastname: Löwenberg
authors.forename: I P
authors.initials: IP
authors.lastname: Touw
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D016179:Granulocyte Colony-Stimulating Factor / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D053614:Janus Kinase 2; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D014364:Tryptophan / Q000502:physiology*; D018909:src Homology Domains
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494080:Stat1 protein, mouse; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D016179:Granulocyte Colony-Stimulating Factor; D014364:Tryptophan; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)