title: The membrane-distal cytoplasmic region of human granulocyte colony-stimulating factor receptor is required for STAT3 but not STAT1 homodimer formation.
abstract: Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) involves the activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway. G-CSF induces tyrosine phosphorylation of Jak1, Jak2, STAT1, and STAT3. The membrane-proximal region of G-CSF-R is sufficient for activation of Jaks. It is still unclear how STAT proteins are activated by G-CSF-R. We investigated the possible involvement of the C-terminal region of G-CSF-R in the recruitment of STAT proteins using BAF3 cell transfectants expressing wild type (WT) G-CSF-R, C-terminal deletion mutants and tyrosine-to-phenylalanine substitution mutants. Electrophoretic mobility shift assays with STAT-binding oligonucleotides (m67) showed that activation of WT G-CSF-R induces three distinct STAT complexes, namely STAT3 homodimers, STAT1-STAT3 heterodimers, and STAT1 homodimers. However, STAT1 homodimers and STAT1-STAT3 heterodimers were predominantly formed after activation of a C-terminal deletion mutant d685, which lacks all four conserved cytoplasmic tyrosine residues, located at positions 704, 729, 744, and 764. Antiphosphotyrosine immunoblots of STAT3 immunoprecipitates showed that activation of WT G-CSF-R induced phosphorylation of STAT3. In contrast, no phosphorylation of STAT3 was observed after activation of deletion mutant d685. These findings establish that the C-terminal region of G-CSF-R plays a major role in the activation of STAT3. By using tyrosine-to-phenylalanine substitution mutants of G-CSF-R, we further show that tyrosine 704, present in a YXXQ consensus sequence shown to be essential for STAT3 binding to gp130, is not exclusively involved in the activation of STAT3 by G-CSF-R.
authors.affiliation: Institute of Hematology, Erasmus University, Rotterdam, The Netherlands.
authors.forename: J P
authors.initials: JP
authors.lastname: de Koning
authors.forename: F
authors.initials: F
authors.lastname: Dong
authors.forename: L
authors.initials: L
authors.lastname: Smith
authors.forename: A M
authors.initials: AM
authors.lastname: Schelen
authors.forename: R M
authors.initials: RM
authors.lastname: Barge
authors.forename: D C
authors.initials: DC
authors.lastname: van der Plas
authors.forename: L H
authors.initials: LH
authors.lastname: Hoefsloot
authors.forename: B
authors.initials: B
authors.lastname: Löwenberg
authors.forename: I P
authors.initials: IP
authors.lastname: Touw
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D016384:Consensus Sequence; D003593:Cytoplasm; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000502:physiology*; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D016188:Receptors, Granulocyte Colony-Stimulating Factor; 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; D019000:Phosphotyrosine