Constitutive mutants of the GM-CSF receptor reveal multiple pathways leading to myeloid cell survival, proliferation, and granulocyte-macrophage differentiation

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 507-516 ◽  
Author(s):  
Anna L. Brown ◽  
Michelle Peters ◽  
Richard J. D'Andrea ◽  
Thomas J. Gonda
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Myeloid Cell ◽  
Proximal Region ◽  
Macrophage Differentiation ◽  
Gm Csf ◽  
Myeloid Cell Line ◽  
Constitutively Active Mutants ◽  
Macrophage Colony ◽  
Membrane Proximal Region

Abstract Activation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) family of receptors promotes the survival, proliferation, and differentiation of cells of the myeloid compartment. Several signaling pathways are activated downstream of the receptor, however it is not clear how these induce specific biologic outcomes. We have previously identified 2 classes of constitutively active mutants of the shared signaling subunit, human (h) βc, of the human GM-CSF/interleukin-3 (IL-3)/IL-5 receptors that exhibit different modes of signaling. In a factor-dependent bipotential myeloid cell line, FDB1, an activated mutant containing a substitution in the transmembrane domain (V449E) induces factor-independent proliferation and survival, while mutants in the extracellular domain induce factor-independent granulocyte-macrophage differentiation. Here we have used further mutational analysis to demonstrate that there are nonredundant functions for several regions of the cytoplasmic domain with regard to mediating proliferation, viability, and differentiation, which have not been revealed by previous studies with the wild-type GM-CSF receptor. This unique lack of redundancy has revealed an association of a conserved membrane-proximal region with viability signaling and a critical but distinct role for tyrosine 577 in the activities of each class of mutant.

Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor β subunit

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 120-127 ◽  
Author(s):  
Matthew P. McCormack ◽  
Thomas J. Gonda
Keyword(s):  
Cell Lines ◽  
Transmembrane Domain ◽  
Myeloid Cell ◽  
Cell Types ◽  
Β Subunit ◽  
Gm Csf ◽  
Constitutively Active Mutants ◽  
Macrophage Colony ◽  
Constitutively Active

Several activating mutations have recently been described in the common β subunit for the human interleukin(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (hβc). Two of these, FIΔ and I374N, result, respectively, in a 37–amino acid duplication and an isoleucine-to-asparagine substitution in the extracellular domain. A third, V449E, leads to valine-to–glutamic acid substitution in the transmembrane domain. Previous studies have shown that when expressed in murine hemopoietic cells in vitro, the extracellular mutants can confer factor independence on only the granulocyte-macrophage lineage while the transmembrane mutant can do so to all cell types of the myeloid and erythroid compartments. To further study the signaling properties of the constitutively active hβc mutants, we have used novel murine hemopoietic cell lines, which we describe in this report. These lines, FDB1 and FDB2, proliferate in murine IL-3 and undergo granulocyte-macrophage differentiation in response to murine GM-CSF. We find that while the transmembrane mutant, V449E, confers factor-independent proliferation on these cell lines, the extracellular hβc mutants promote differentiation. Hence, in addition to their ability to confer factor independence on distinct cell types, transmembrane and extracellular activated hβc mutants deliver distinct signals to the same cell type. Thus, the FDB cell lines, in combination with activated hβc mutants, constitute a powerful new system to distinguish between signals that determine hemopoietic proliferation or differentiation. (Blood. 2000;95:120-127)

Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor β subunit

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 120-127 ◽  
Author(s):  
Matthew P. McCormack ◽  
Thomas J. Gonda
Keyword(s):  
Cell Lines ◽  
Transmembrane Domain ◽  
Myeloid Cell ◽  
Cell Types ◽  
Β Subunit ◽  
Gm Csf ◽  
Constitutively Active Mutants ◽  
Macrophage Colony ◽  
Constitutively Active

Abstract Several activating mutations have recently been described in the common β subunit for the human interleukin(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (hβc). Two of these, FIΔ and I374N, result, respectively, in a 37–amino acid duplication and an isoleucine-to-asparagine substitution in the extracellular domain. A third, V449E, leads to valine-to–glutamic acid substitution in the transmembrane domain. Previous studies have shown that when expressed in murine hemopoietic cells in vitro, the extracellular mutants can confer factor independence on only the granulocyte-macrophage lineage while the transmembrane mutant can do so to all cell types of the myeloid and erythroid compartments. To further study the signaling properties of the constitutively active hβc mutants, we have used novel murine hemopoietic cell lines, which we describe in this report. These lines, FDB1 and FDB2, proliferate in murine IL-3 and undergo granulocyte-macrophage differentiation in response to murine GM-CSF. We find that while the transmembrane mutant, V449E, confers factor-independent proliferation on these cell lines, the extracellular hβc mutants promote differentiation. Hence, in addition to their ability to confer factor independence on distinct cell types, transmembrane and extracellular activated hβc mutants deliver distinct signals to the same cell type. Thus, the FDB cell lines, in combination with activated hβc mutants, constitute a powerful new system to distinguish between signals that determine hemopoietic proliferation or differentiation. (Blood. 2000;95:120-127)

scholarly journals Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Y Kanakura ◽  
B Druker ◽  
KW Wood ◽  
HJ Mamon ◽  
K Okuda ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Line ◽  
Human Factor ◽  
Myeloid Cell ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Myeloid Cell Line ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor- dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

scholarly journals JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

1994 ◽  
Vol 14 (7) ◽  
pp. 4335-4341 ◽  
Author(s):  
F W Quelle ◽  
N Sato ◽  
B A Witthuhn ◽  
R C Inhorn ◽  
M Eder ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Proximal Region ◽  
Mitogen Activated Protein Kinase ◽  
Colony Stimulating Factor ◽  
Alpha Chain ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Membrane Proximal Region ◽  
Stimulating Factor

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.

JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region

1994 ◽  
Vol 14 (7) ◽  
pp. 4335-4341
Author(s):  
F W Quelle ◽  
N Sato ◽  
B A Witthuhn ◽  
R C Inhorn ◽  
M Eder ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Proximal Region ◽  
Mitogen Activated Protein Kinase ◽  
Colony Stimulating Factor ◽  
Alpha Chain ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Membrane Proximal Region ◽  
Stimulating Factor

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.

scholarly journals Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Y Kanakura ◽  
B Druker ◽  
KW Wood ◽  
HJ Mamon ◽  
K Okuda ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Line ◽  
Human Factor ◽  
Myeloid Cell ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Myeloid Cell Line ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor- dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

scholarly journals The Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Exists as a Preformed Receptor Complex That Can Be Activated by GM-CSF, Interleukin-3, or Interleukin-5

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3005-3017 ◽  
Author(s):  
Joanna M. Woodcock ◽  
Barbara J. McClure ◽  
Frank C. Stomski ◽  
Michael J. Elliott ◽  
Christopher J. Bagley ◽  
...  
Keyword(s):  
Myeloid Cell ◽  
Cytokine Receptor ◽  
Activation Mechanism ◽  
Receptor Complex ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The granulocyte-macrophage colony-stimulating factor (GM-CSF ) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor α chain (GMRα) and to the common β chain of the GM-CSF, IL-3, and IL-5 receptors (βc ) immunoprecipitated both GMRα and βc from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMRα and βc , as shown by the ability of soluble βc to associate with membrane-anchored GMRα or soluble GMRα. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMRα-associated βc . These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.

Sign in / Sign up

Export Citation Format

Share Document