POINT MUTATIONS IN THE CONSERVED BOX-1 REGION INACTIVATE THE HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR-RECEPTOR FOR GROWTH SIGNAL-TRANSDUCTION AND TYROSINE PHOSPHORYLATION OF P75(C-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. (C) 1995 by The American Society of Hematology.