Point mutations in the conserved box 1 region inactivate the human granulocyte colony-stimulating factor receptor for growth signal transduction and tyrosine phosphorylation of p75c-rel.
The human granulocyte colony-stimulating factor receptor (hG-CSFR) belongs to the cytokine receptor superfamily. As with other members of this family, the cytoplasmic domain of hG-CSFR lacks intrinsic tyrosine kinase activity. To identify critical regions mediating growth signal transduction by hG-CSFR, deletions or site-directed amino acid substitutions were introduced into the cytoplasmic domain of hG-CSFR, and the mutant cDNAs were transfected into the murine interleukin-3 (IL-3)-dependent Ba/F3 and FDCP cell lines. Truncation of the carboxy-terminal end of the receptor to the membrane-proximal 53 amino acids of the cytoplasmic domain, which retained the conserved Box 1 and Box 2 sequence motifs, decreased the ability of hG-CSFR to transduce G-CSF-mediated growth signals without an associated loss in receptor binding affinity. Substitution of proline by alanine at amino acid positions 639 and 641 within Box 1 completely abolished the G-CSF-mediated growth signal. Rapid induction of tyrosine phosphorylation of several cellular proteins, including a 75-kD protein (p75) identified as c-rel, was an early event associated with transduction of proliferative signals by hG-CSFR in Ba/F3 transfectants. Mutant receptors containing Pro-to-Ala substitutions that inactivated the receptor for mitogenic activity also inactivated the receptor for tyrosine-specific phosphorylation of p75. These results show that the conserved Box 1 sequence motif (amino acids 634 to 641) is critical for mitogenesis and activation of cellular tyrosine kinases by hG-CSFR.
Avalos
B R
BR
Arthur G. James Cancer Hospital and Research Institute, Ohio State University, Columbus 43210, USA.
Hunter
M G
MG
Parker
J M
JM
Ceselski
S K
SK
Druker
B J
BJ
Corey
S J
SJ
Mehta
V B
VB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D017354:Point Mutation; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000502:physiology; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D014162:Transfection
D018076:DNA, Complementary; D007377:Interleukin-3; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D020794:Receptor Protein-Tyrosine Kinases