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

Belinda R. Avalos, Melissa G. Hunter, Jennifer M. Parker, Sarah K. Ceselski, Brian Druker, Seth J. Corey, Veela B. Mehta

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)3117-3126
Number of pages10
JournalBlood
Volume85
Issue number11
StatePublished - Jun 1 1995
Externally publishedYes

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Granulocyte Colony-Stimulating Factor Receptors
Signal transduction
Phosphorylation
Point Mutation
Tyrosine
Signal Transduction
Growth
Amino Acids
Substitution reactions
Granulocyte Colony-Stimulating Factor
Protein-Tyrosine Kinases
Amino Acid Motifs
Cytokine Receptors
Aptitude
Interleukin-3
Amino Acid Substitution
Proline
Alanine
Amino Acid Sequence
Proteins

ASJC Scopus subject areas

  • Hematology

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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. / Avalos, Belinda R.; Hunter, Melissa G.; Parker, Jennifer M.; Ceselski, Sarah K.; Druker, Brian; Corey, Seth J.; Mehta, Veela B.

In: Blood, Vol. 85, No. 11, 01.06.1995, p. 3117-3126.

Research output: Contribution to journalArticle

Avalos, Belinda R. ; Hunter, Melissa G. ; Parker, Jennifer M. ; Ceselski, Sarah K. ; Druker, Brian ; Corey, Seth J. ; Mehta, Veela B. / 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. In: Blood. 1995 ; Vol. 85, No. 11. pp. 3117-3126.
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abstract = "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.",
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