Specific involvement of tyrosine 764 of human granulocyte colony-stimulating factor receptor in signal transduction mediated by p145/Shc/GRB2 or p90/GRB2 complexes.
Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) occurs via multiple pathways, one of which involves activation of p21Ras and mitogen-activated protein kinase. The SH2 domain-containing proteins Shc and GRB2 have been implicated in this latter signaling route. We studied the role of these proteins in signal transduction from wild type (WT) G-CSF-R, C-terminal deletion mutants, and tyrosine-to-phenylalanine substitution mutants in transfectants of the mouse pro-B cell line, BAF3. G-CSF stimulation of BAF3 cells expressing WT G-CSF-R induced tyrosine phosphorylation of Shc. Anti-Shc antibodies co-immunoprecipitated tyrosine-phosphorylated 145-kD proteins (p145), whereas GRB2 immunoprecipitates contained phosphorylated Shc, Syp, and proteins of 145 and 90 kD (p90). Neither of these complexes were detected after activation of a C-terminal deletion mutant of G-CSF-R that lacked all four conserved cytoplasmic tyrosine residues. G-CSF induced formation of Syp/GRB2 complexes in all the tyrosine-substitution mutants, suggesting that this association did not depend on the presence of single specific tyrosine residues in G-CSF-R. In contrast, tyrosine 764 of G-CSF-R appeared to be exclusively required for tyrosine phosphorylation of Shc and its association with p145 and GRB2. In addition, tyrosine 764 also specifically mediated binding of GRB2 to p90 without the involvement of Shc. These findings indicate that tyrosine 764 of G-CSF-R has a prominent role in G-CSF signal transduction.
Institute of Hematology, Erasmus University, Rotterdam, The Netherlands.
J P
JP
de Koning
A M
AM
Schelen
F
F
Dong
C
C
van Buitenen
B M
BM
Burgering
J L
JL
Bos
B
B
Löwenberg
I P
IP
Touw
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001483:Base Sequence; D051380:GRB2 Adaptor Protein; D016179:Granulocyte Colony-Stimulating Factor / Q000502:physiology*; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008938:Mitosis / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000502:physiology*; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000502:physiology*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011506:Proteins; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016179:Granulocyte Colony-Stimulating Factor; D014443:Tyrosine