scholarly journals Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3298-3306 ◽  
Author(s):  
M Weiss ◽  
C Yokoyama ◽  
Y Shikama ◽  
C Naugle ◽  
B Druker ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Beta Subunits ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.

scholarly journals Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3298-3306 ◽  
Author(s):  
M Weiss ◽  
C Yokoyama ◽  
Y Shikama ◽  
C Naugle ◽  
B Druker ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Beta Subunits ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.

Reconstituted human granulocyte-macrophage colony-stimulating factor receptor transduces growth-promoting signals in mouse NIH 3T3 cells: comparison with signalling in BA/F3 pro-B cells

1993 ◽  
Vol 13 (3) ◽  
pp. 1440-1448
Author(s):  
S Watanabe ◽  
A L Mui ◽  
A Muto ◽  
J X Chen ◽  
K Hayashida ◽  
...  
Keyword(s):  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Growth Promoting ◽  
Macrophage Colony ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.

scholarly journals Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation

2008 ◽  
Vol 295 (1) ◽  
pp. L114-L122 ◽  
Author(s):  
Megan N. Ballinger ◽  
Leah L. N. Hubbard ◽  
Tracy R. McMillan ◽  
Galen B. Toews ◽  
Marc Peters-Golden ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Host Defense ◽  
Receptor Expression ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Bacterial Killing ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Impaired host defense post-bone marrow transplant (BMT) is related to overproduction of prostaglandin E2(PGE2) by alveolar macrophages (AMs). We show AMs post-BMT overproduce granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas GM-CSF in lung homogenates is impaired both at baseline and in response to infection post-BMT. Homeostatic regulation of GM-CSF may occur by hematopoietic/structural cell cross talk. To determine whether AM overproduction of GM-CSF influenced immunosuppression post-BMT, we compared mice that received BMT from wild-type donors (control BMT) or mice that received BMT from GM-CSF−/− donors (GM-CSF−/− BMT) with untransplanted mice. GM-CSF−/− BMT mice were less susceptible to pneumonia with Pseudomonas aeruginosa compared with control BMT mice and showed antibacterial responses equal to or better than untransplanted mice. GM-CSF−/− BMT AMs displayed normal phagocytosis and a trend toward enhanced bacterial killing. Surprisingly, AMs from GM-CSF−/− BMT mice overproduced PGE2, but expression of the inhibitory EP2receptor was diminished. As a consequence of decreased EP2receptor expression, we found diminished accumulation of cAMP in response to PGE2stimulation in GM-CSF−/− BMT AMs compared with control BMT AMs. In addition, GM-CSF−/− BMT AMs retained cysteinyl leukotriene production and normal TNF-α response compared with AMs from control BMT mice. GM-CSF−/− BMT neutrophils also showed improved bacterial killing. Although genetic ablation of GM-CSF in hematopoietic cells post-BMT improved host defense, transplantation of wild-type bone marrow into GM-CSF−/− recipients demonstrated that parenchymal cell-derived GM-CSF is necessary for effective innate immune responses post-BMT. These results highlight the complex regulation of GM-CSF and innate immunity post-BMT.

scholarly journals Differential activation of leukotriene biosynthesis by granulocyte- macrophage colony-stimulating factor and interleukin-5 in an eosinophilic substrain of HL-60 cells

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3507-3516 ◽  
Author(s):  
KA Scoggan ◽  
AW Ford-Hutchinson ◽  
DW Nicholson
Keyword(s):  
Binding Sites ◽  
Affinity Binding ◽  
Colony Stimulating Factor ◽  
High Affinity Binding ◽  
Interleukin 5 ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Cytokines can stimulate eosinophils to produce cysteinyl leukotrienes (LTs) in the lung that provoke tissue destruction associated with asthma. Priming of an eosinophilic substrain of HL-60 cells (HL-60#7) with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) before ionophore challenge was found to produce an apparent 45% increase in total LT production in a dose-dependent manner (ED50 = 150 pmol/L) that could be accounted for by a decrease in the time required for maximal formation of LTs. GM-CSF had no effect on the kinetic parameters of LTC4 synthase and therefore probably acts upstream of this catalytic event. Incubation with interleukin-5 (IL-5), however, had no effect on LT biosynthesis. This differential priming ability was not a consequence of different receptor populations or differences in the affinity or stability of the ligand-receptor complexes of GM-CSF and IL-5. GM-CSF and IL-5 each displayed similar populations of high-affinity binding sites and neither GM-CSF nor IL-5 were able to cross-compete for the other's receptor binding sites. Analysis of phosphotyrosine patterns suggest that IL-5 is incapable of transducing a signal in eosinophilic HL-60#7 cells even though IL-5 and GM-CSF receptors mediate signal transduction via a common beta-chain component that is also necessary for high-affinity binding. Overall, this unique system may permit the dissection of distinct events responsible for specific intracellular signals transduced separately by GM-CSF or IL-5.

Simultaneous Antagonism of Interleukin-5, Granulocyte-Macrophage Colony-Stimulating Factor, and Interleukin-3 Stimulation of Human Eosinophils by Targetting the Common Cytokine Binding Site of Their Receptors

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1943-1951 ◽  
Author(s):  
Q. Sun ◽  
K. Jones ◽  
B. McClure ◽  
B. Cambareri ◽  
B. Zacharakis ◽  
...  
Keyword(s):  
Affinity Binding ◽  
Colony Stimulating Factor ◽  
High Affinity Binding ◽  
Receptor Dimerization ◽  
Interleukin 5 ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific  chain and a shared subunit (βc). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor  chains is the first step in receptor activation, it is the recruitment of βc that allows high-affinity binding and signal transduction to proceed. Thus, βc is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of βc. BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of125I–IL-5, 125I–GM-CSF, and125I–IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of βc. Interestingly, epitope analysis using several βc mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of βc, suggesting that ligand contact with βc is a prerequisite for recruitment of βc, receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.

scholarly journals STAT5A-Deficient Mice Demonstrate a Defect in Granulocyte-Macrophage Colony-Stimulating Factor–Induced Proliferation and Gene Expression

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1768-1776 ◽  
Author(s):  
Gerald M. Feldman ◽  
Louis A. Rosenthal ◽  
Xiuwen Liu ◽  
Mark P. Hayes ◽  
Anthony Wynshaw-Boris ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding Proteins ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Stat Proteins ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
In Cells

Abstract Responses of cells to cytokines typically involve the activation of a family of latent DNA binding proteins, referred to as signal transducers and activators of transcription (STAT) proteins, which are critical for the expression of early response genes. Of the seven known STAT proteins, STAT5 (originally called mammary gland factor) has been shown to be activated by several cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5, which are known to play important roles in growth and differentiation of hematopoietic precursors. In this report we have used mice that are deficient in STAT5A (one of two homologues of STAT5) to study the role of STAT5A in GM-CSF stimulation of cells. When bone marrow–derived macrophages were generated by differentiation with macrophage-CSF (M-CSF), exposure of cells from wild-type mice to GM-CSF resulted in a typical pattern of assembly of DNA binding proteins specific for the gamma activation sequence (GAS) element within the β-casein promoter. However, in cells from the STAT5A null mouse one of the shifted bands was absent. Immunoblotting analysis in the null mice showed that lack of STAT5A protein resulted in no alteration in activation of STAT5B by tyrosine phosphorylation. Proliferation experiments revealed that, when exposed to increasing concentrations of GM-CSF, cells derived from the null mice grew considerably more slowly than cells derived from the wild-type mice. Moreover, expression of GM-CSF–dependent genes, CIS and A1, was markedly inhibited in cells derived from null mice as compared with those of wild-type mice. The decreased expression observed with A1, a bcl-2 like gene, may account in part for the suppression of growth in cells from the null mice. These data suggest that the presence of STAT5A during the GM-CSF–induced assembly of STAT5 dimers is critical for the formation of competent transcription factors that are required for both gene expression and cell proliferation.

Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2539-2553 ◽  
Author(s):  
PD Doshi ◽  
JF DiPersio
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Surface Expression ◽  
Colony Stimulating Factor ◽  
Cos Cells ◽  
Gm Csf ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.

Erythropoietin receptor haploinsufficiency and in vivo interplay with granulocyte-macrophage colony-stimulating factor and interleukin 3

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2603-2605 ◽  
Author(s):  
Armin G. Jegalian ◽  
Adriana Acurio ◽  
Glenn Dranoff ◽  
Hong Wu
Keyword(s):  
Erythropoietin Receptor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Definitive Erythropoiesis ◽  
Stimulating Factor

Erythropoietin (EPO) and its receptor (EPOR) are critical for definitive erythropoiesis, as mice lacking either gene product die during embryogenesis with severe anemia. Here we demonstrate that mice expressing just one functional allele of the EpoR have lower hematocrits and die more frequently than do wild-type littermates on anemia induction. Furthermore, EpoR+/−erythroid colony-forming unit (CFU-E) progenitors are reduced both in frequency and in responsiveness to EPO stimulation. To evaluate the interaction between EPO and granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3),GM-CSF−/− orIL-3−/− mice were interbred withEpoR+/− mice. Deletion of either GM-CSF or IL-3 also leads to reduction in CFU-E numbers and hematocrits but does not significantly alter steady-state erythroid burst-forming unit numbers. These results suggest EpoR haploinsufficiency and promotion of in vivo erythropoiesis by GM-CSF and IL-3.

Simultaneous Antagonism of Interleukin-5, Granulocyte-Macrophage Colony-Stimulating Factor, and Interleukin-3 Stimulation of Human Eosinophils by Targetting the Common Cytokine Binding Site of Their Receptors

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1943-1951 ◽  
Author(s):  
Q. Sun ◽  
K. Jones ◽  
B. McClure ◽  
B. Cambareri ◽  
B. Zacharakis ◽  
...  
Keyword(s):  
Affinity Binding ◽  
Colony Stimulating Factor ◽  
High Affinity Binding ◽  
Receptor Dimerization ◽  
Interleukin 5 ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific  chain and a shared subunit (βc). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor  chains is the first step in receptor activation, it is the recruitment of βc that allows high-affinity binding and signal transduction to proceed. Thus, βc is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of βc. BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of125I–IL-5, 125I–GM-CSF, and125I–IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of βc. Interestingly, epitope analysis using several βc mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of βc, suggesting that ligand contact with βc is a prerequisite for recruitment of βc, receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.

scholarly journals Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2539-2553 ◽  
Author(s):  
PD Doshi ◽  
JF DiPersio
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Surface Expression ◽  
Colony Stimulating Factor ◽  
Cos Cells ◽  
Gm Csf ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.

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