High-affinity Granulocyte-macrophage Colony-stimulating Factor Research Articles

Malignant Progenitors From Patients With Acute Myelogenous Leukemia Are Sensitive to a Diphtheria Toxin–Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractWe have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 × 10−12 mol/L (range, 5 to >4,000 × 10−12 mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing ≥35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 × 10−9 mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.

Granulocyte-Macrophage Colony-stimulating Factor Signals for Increased Glucose Transport via Phosphatidylinositol 3-Kinase- and Hydrogen Peroxide-dependent Mechanisms

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates cellular glucose uptake by decreasing the apparent K(m) for substrate transport through facilitative glucose transporters on the plasma membrane. Little is known about this signal transduction pathway and the role of the alpha subunit of the GM-CSF receptor (alpha GMR) in modulating transporter activity. We examined the function of phosphatidylinositol 3-kinase (PI 3-kinase) in GM-CSF-stimulated glucose uptake and found that PI 3-kinase inhibitors, wortmannin and LY294002, completely blocked the GM-CSF-dependent increase of glucose uptake in Xenopus oocytes expressing the low affinity alpha GMR and in human cells expressing the high affinity alpha beta GMR complex. We identified a Src homology 3 domain-binding motif in alpha GMR at residues 358-361 as a potential interaction site for the PI 3-kinase regulatory subunit, p85. Physical evidence for p85 binding to alpha GMR was obtained by co-immunoprecipitation with antibodies to alpha GMR and p85, and an alpha GMR mutant with alteration of the Src homology 3 binding domain lost the ability to bind p85. Experiments with a construct eliminating most of the intracellular portion of alpha GMR showed a 50% reduction in GM-CSF-stimulated glucose uptake with residual activity blocked by wortmannin. Searching for a proximally generated diffusible factor capable of activating PI 3-kinase, we identified hydrogen peroxide (H(2)O(2)), generated by ligand or antibody binding to alpha GMR, as the initiating factor. Catalase treatment abrogated GM-CSF- or anti-alpha GMR antibody-stimulated glucose uptake in alpha GMR-expressing oocytes, and H(2)O(2) activated PI 3-kinase and led to some stimulation of glucose uptake in uninjected oocytes. Human myeloid cell lines and primary explant human lymphocytes expressing high affinity GM-CSF receptors responded to alpha GMR antibody with increased glucose uptake. These results identify the early events in the stimulation of glucose uptake by GM-CSF as involving local H(2)O(2) generation and requiring PI 3-kinase activation. Our findings also provide a mechanistic explanation for signaling through the isolated alpha subunit of the GM-CSF receptor.

High-affinity binding to the GM-CSF receptor requires intact N-glycosylation sites in the extracellular domain of the β subunit

AbstractThe human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor consists of 2 glycoprotein subunits, GMRα and GMRβ. GMRα in isolation binds to GM-CSF with low affinity. GMRβ does not bind GM-CSF by itself, but forms a high-affinity receptor in association with GMRα. Previously, it was found that N-glycosylation of GMRα is essential for ligand binding. The present study investigated the role of N-glycosylation of the β subunit on GM-CSF receptor function. GMRβ has 3 potential N-glycosylation sites in the extracellular domain at Asn58, Asn191, and Asn346. Single mutants and triple mutants were constructed, converting asparagine in the target sites to aspartic acid or alanine. A single mutation at any of the 3 consensus N-glycosylation sites abolished high-affinity GM-CSF binding in transfected COS cells. Immunofluorescence and subcellular fractionation studies demonstrated that all of the GMRβ mutants were faithfully expressed on the cell surface. Reduction of apparent molecular weight of the triple mutant proteins was consistent with loss of N-glycosylation. Intact N-glycosylation sites of GMRβ in the extracellular domain are not required for cell surface targeting but are essential for high-affinity GM-CSF binding.

High-affinity binding to the GM-CSF receptor requires intact N-glycosylation sites in the extracellular domain of the β subunit

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor consists of 2 glycoprotein subunits, GMR and GMRβ. GMR in isolation binds to GM-CSF with low affinity. GMRβ does not bind GM-CSF by itself, but forms a high-affinity receptor in association with GMR. Previously, it was found that N-glycosylation of GMR is essential for ligand binding. The present study investigated the role of N-glycosylation of the β subunit on GM-CSF receptor function. GMRβ has 3 potential N-glycosylation sites in the extracellular domain at Asn58, Asn191, and Asn346. Single mutants and triple mutants were constructed, converting asparagine in the target sites to aspartic acid or alanine. A single mutation at any of the 3 consensus N-glycosylation sites abolished high-affinity GM-CSF binding in transfected COS cells. Immunofluorescence and subcellular fractionation studies demonstrated that all of the GMRβ mutants were faithfully expressed on the cell surface. Reduction of apparent molecular weight of the triple mutant proteins was consistent with loss of N-glycosylation. Intact N-glycosylation sites of GMRβ in the extracellular domain are not required for cell surface targeting but are essential for high-affinity GM-CSF binding.

Kinetic Resolution of Two Mechanisms for High-Affinity Granulocyte-Macrophage Colony-Stimulating Factor Binding to Its Receptor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic cytokine that exerts its effects by interaction with the GM-CSF receptor (GMR) on the surface of responsive cells. The GM-CSF receptor consists of two subunits: GMRalpha, which binds GM-CSF with low affinity, and GMRbeta, which lacks intrinsic ligand-binding capability but complexes with GMRalpha to form a high-affinity receptor (GMRalpha/beta). We conducted dynamic kinetic analyses of GM-CSF receptors to define the role of GMRbeta in the interaction of ligand and receptor. Our data show that GMRalpha/beta exhibits a higher k(on) than GMRalpha, indicating that GMRbeta facilitates ligand acquisition to the binding pocket. Heterogeneity with regard to GM-CSF dissociation from GMRalpha/beta points to the presence of loose and tight ligand-receptor complexes in high-affinity binding. Although the loose complex has a k(off) similar to GMRalpha, the lower k(off) indicates that GMRbeta inhibits GM-CSF release from the tight receptor complex. The two rates of ligand dissociation may provide for discrete mechanisms of interaction between GM-CSF and its high-affinity receptor. These results show that the beta subunit functions to stabilize ligand binding as well as to facilitate ligand acquisition.

The soluble granulocyte-macrophage colony-stimulating factor receptor's carboxyl-terminal domain mediates retention of the soluble receptor on the cell surface through interaction with the granulocyte-macrophage colony-stimulating factor receptor beta-subunit.

The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates its activity through binding to cell-surface receptors. The high-affinity GM-CSF receptor (GMR) consists of two transmembrane-anchored subunits: a ligand-specific, low-affinity subunit (GMRalpha); and a signal-transducing beta-subunit (GMRbeta). The human GMRalpha subunit also exists in a soluble isoform (SOLalpha) which antagonizes GM-CSF activity in vitro. Previous studies by us have shown that coexpression of SOLalpha and a mutated GMRbeta in BHK cells results in retention of SOLalpha on the cell surface and the formation of an intermediate affinity binding complex (Kd approximately 300 pM). This paper investigates the mechanism of the retention of SOLalpha on the cell surface. The data demonstrate that SOLalpha is anchored by a direct, ligand-independent interaction with GMRbeta which also occurs when SOLalpha is coexpressed with wild-type GMRbeta. However, SOLalpha and wild-type GMRbeta form a complex which binds GM-CSF with high affinity (Kd = 39 pM), indistinguishable from the binding characteristics of the TMalpha/GMRbeta complex. The experiments further reveal that the interaction between SOLalpha and GMRbeta is abrogated by removal of the unique 16 amino acid carboxyl-terminal domain of SOLalpha. Specific mutation of cysteine 323 in this carboxyl-domain to alanine also eliminates the cell-surface retention of SOLalpha identifying this residue as being necessary for the formation of the SOLalpha/GMRbeta complex.

Malignant progenitors from patients with acute myelogenous leukemia are sensitive to a diphtheria toxin-granulocyte-macrophage colony-stimulating factor fusion protein.

We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 x 10(-12) mol/L (range, 5 to >4,000 x 10(-12) mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing > or = 35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 x 10(-9) mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.

Expression of Granulocyte-Macrophage Colony-Stimulating Factor Receptors in Human Prostate Cancer

We studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, alpha and beta. While the isolated alpha subunit binds GM-CSF at low-affinity, the isolated beta subunit does not bind GM-CSF by itself; but complexes with the alpha subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the alpha and beta subunits of the GM-CSF receptor in LNCaP cells, and the presence of the alpha and beta proteins was confirmed by immunolocalization with anti-alpha and anti-beta antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF alpha and beta subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-alpha subunit antibodies were also reactive with anti-beta subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.

Expression of Granulocyte-Macrophage Colony-Stimulating Factor Receptors in Human Prostate Cancer

AbstractWe studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, α and β. While the isolated α subunit binds GM-CSF at low-affinity, the isolated β subunit does not bind GM-CSF by itself; but complexes with the α subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the α and β subunits of the GM-CSF receptor in LNCaP cells, and the presence of the α and β proteins was confirmed by immunolocalization with anti-α and anti-β antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF α and β subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-α subunit antibodies were also reactive with anti-β subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.

Modulation of the Apoptotic Response of Human Myeloid Leukemia Cells to a Diphtheria Toxin Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractIt has previously been shown that human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be fused to a truncated diphtheria toxin (DT) to produce a recombinant fusion toxin that kills GM-CSF receptor–bearing cells. We now report that DT388–GM-CSF induces apoptosis and inhibition of colony formation in semisolid medium in receptor positive cells, and that the induction of apoptosis correlates with GM-CSF–receptor occupancy at low ligand concentrations. Also, the induction of apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic proteins (Bcl2 and Bc1XL ). Nine myeloid leukemia cells lines and four nonmyeloid leukemia cell lines were incubated with 0.7 nmol/L of 125I–GM-CSF in the presence or absence of excess cold GM-CSF and bound label measured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombinant fusion toxin for 48 hours and incorporation of 3H-leucine (protein synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. DT388–GM-CSF at 4 × 10−9 mol/L inhibited colony formation 1.5 to 3.0 logs for receptor positive cell lines. Protein synthesis and apoptosis IC50s varied among cell lines from greater than 4 × 10−9 mol/L to 3 × 10−13 mol/L. GM-CSF–receptor occupancy at 0.7 nmol/L GM-CSF–ligand concentration correlated with the protein synthesis IC50 . Similarly, the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclXL , in which 25- to 150-fold inhibition of apoptosis was observed. We conclude that DT388–GM-CSF can kill acute myeloid leukemia blasts but that apoptotic sensitivities will depend on the presence of at least 100 high affinity GM-CSF receptors/cell and the absence of overexpressed antiapoptotic proteins.

Granulocyte-Macrophage colony-Stimulating factor receptor-Targeted therapy of chemotherapy- and radiation-Resistant human myeloid leukemias

Contemporary therapies for acute myeloid leukemia (AML) commonly fail to cure patients because of the emergence of drug resistance. Drug resistance in AML is multifactorial but can be associated with the overexpression of transmembrane transporter molecules, including P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP), or associated with inactivation of the p53 tumor suppressor gene, as well as overexpression of the anti-apoptotic protein bcl-2. We are investigating if novel recombinant biotherapeutics can circumvent these resistance mechanisms to effectively treat refractory AML. To target the lethal action of diphtheria toxin (DT) to high affinity granulocyte-macrophage colony-stimulating factor (GMCSF) receptors on AML blasts, we have produced a recombinant chimeric fusion toxin, DTctGMCSF. Since DTctGMCSF enters and kills its target cells by unique mechanisms (GMCSF-receptor binding and protein synthesis inhibition) and is not similar in structure to Pgp or MRP substrates, we postulated that it would be an active agent against therapy-resistant AML. DTctGMCSF was selectively cytotoxic (IC50 1–10ng/ml) to GMCSF-receptor positive AML cells expressing the Pgp- or MRP-associated multi-drug resistant phenotypes, despite high level resistance to conventional chemotherapeutic agents. DTctGMCSF also efficiently killed AML cells deficient in p53 expression, as well as radiation-resistant AML cells and mixed lineage leukemia cells expressing high levels of bcl-2. In addition, DTctGMCSF killed>99% of primary leukemic progenitor cells from therapy-refractory AML patients under conditions that we have previously found to not adversely affect the proliferative capacity or differentiation of pluripotent normal hematopoietic progenitor cells. DTctGMCSF may prove useful in treating myeloid leukemias that are otherwise resistant to a wide range of conventional therapies.

A Recombinant fusion toxin targeted to the granulocyte-Macrophage colony-Stimulating factor receptor

Human granulocyte-macrophage colony stimulating factor (GMCSF) and its high affinity receptor function to regulate the proliferation and differentiation of myeloid lineage hematopoietic cells, and may participate in the pathogenesis of many malignant myeloid diseases. We have used genetic engineering based on the elucidated molecular structures of human granulocyte-macrophage colony-stimulating factor and diphtheria toxin (DT) to pro-duce a recombinant fusion toxin, DTctGMCSF, that targets diphtheria toxin to high affinity GMCSF receptors expressed on the surface of blast cells from a large fraction of patients with acute myeloid leukemia (AML). DTctGMCSF was specifically immunoreactive with anti-diphtheria toxin and anti-GMCSF antiseras, and exhibited the characteristic catalytic activity of diphtheria toxin, catalyzing the in vitro ADP-ribosylation of purified elongation factor 2. The cytotoxic effects of DTctGMCSF were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium (MTT) bromide assay of cell viability and in vivo assays of protein synthesis inhibition. DTctGMCSF were specifically cytotoxic to human leukemia cell lines bearing high affinity receptors for human GMCSF with IC50 of 10−9 to 10−11M. It was not toxic to mammalian hematopoietic cell lines lacking human GMCSF (hGMCSF) receptors. In receptor positive cells, cytotoxicity can be specifically blocked by a large excess of hGMCSF, confirming that its cytotoxicity is mediated through the hGMCSF receptor. Though DTctGMCSF inhibited granulocyte-macrophage colony formation by committed myeloid progenitor cells (CFU-GM), it did not significantly affect erythroid burst formation by committed erythroid progenitor cells (BFU-E), or mixed granulocyte-erythroid-macrophage-megakaryocyte colony formation by pluripotent multilineage progenitor cells (CFU-GEMM). DTctGMCSF holds promise for the treatment of myeloid lineage malignancies, and is a useful reagent to study hematopoiesis.

Chimeric Granulocyte/Macrophage Colony-stimulating Factor/Transforming Growth Factor-β (TGF-β) Receptors Define a Model System for Investigating the Role of Homomeric and Heteromeric Receptors in TGF-β Signaling

Transforming growth factor-beta (TGF-beta) belongs to a family of ligands that regulate cell growth and differentiation. The most commonly observed receptors are referred to as the type I, type II, and type III (betaglycan) TGF-beta receptors. Two receptor models have been presented to account for the various cellular responses to TGF-beta. The first proposes that all TGF-beta signaling results from the formation of a heteromeric type I/type II complex, while the second suggests that distinct type I or type II TGF-beta receptor combinations mediate aspects of TGF-beta signaling. We have addressed this general question relating to TGF-beta signaling by constructing chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor (GM-CSF) alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF-beta receptor. Since high affinity GM-CSF binding requires dimerization of the alpha and beta ligand binding subunits, the response elicited by defined type I and/or type II TGF-beta receptor cytoplasmic domain homomers or heteromers can be examined. We show in mesenchymal AKR-2B cells that while TGF-beta-dependent transient luciferase activity, endogenous gene activity, and long-term biological responses are similarly induced by activating the chimeric heteromeric receptors with GM-CSF as the endogenous TGF-beta receptor, chimeric homomeric type I/type I or type II/type II receptors are signaling-incompetent.

Ligand-independent Cell Surface Expression of the Human Soluble Granulocyte-Macrophage Colony-stimulating Factor Receptor α Subunit Depends on Co-expression of the Membrane-associated Receptor β Subunit

The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates its activity through binding to cell surface receptors. The receptor for GM-CSF belongs to a superfamily of cytokine receptors characterized by a conserved extracellular motif. The high affinity GM-CSF receptor (GMR) consists of two transmembrane anchored subunits; a ligand binding alpha subunit (transmembrane GMRalpha) and a signal transducing beta subunit (GMRbeta), both of which belong to the cytokine receptor superfamily. The human GM-CSF receptor alpha subunit also exists in a soluble form (solGMRalpha), which antagonizes GM-CSF activity in vitro. We directly tested the potential for solGMRalpha to interact with GMRbeta in vitro. Our experiments demonstrated that exogenous solGMRalpha, even in the presence of GM-CSF, does not interact with GMRbeta on the cell surface. However, when solGMRalpha and GMRbeta are co-expressed in baby hamster kidney cells, solGMRalpha is retained on the cell surface and forms a functional intermediate affinity GM-CSF binding complex (Kd = 331 pM). In addition, the cell surface expression of solGMRalpha is independent of the presence of GM-CSF as demonstrated using flow cytometry. Cells expressing only solGMRalpha do not show cell surface retention or form functional GM-CSF cell surface binding complexes. Sequencing of our GMRbeta clone revealed a nucleotide substitution (A --> C) resulting in the substitution of Ala for Glu at position 9 from the amino terminus of the mature GMRbeta peptide. Because the GMRbeta (A --> C) clone is capable of forming functional high affinity receptors with transmembrane GMRalpha (Kd = 64 pM), we feel that the cell surface retention of solGMRalpha is independent of the GMRbeta mutation. We suggest that the co-expression and interaction of solGMRalpha and GMRbeta represents a previously unrecognized GM-CSF receptor complex and a novel, ligand-independent mechanism of cytokine receptor assembly.

Apoptosis of hemopoietic cells by the human granulocyte-macrophage colony-stimulating factor mutant E21R.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) binds to a high-affinity heterodimeric receptor composed of a specific alpha chain and a common beta chain (beta(c)), which is shared with the receptors for interleukins 3 and 5. Hemopoietic cell survival requires GM-CSF binding this high-affinity receptor. We have recently developed the GM-CSF mutant E21R, which selectively binds to the alpha chain and behaves as a competitive GM-CSF antagonist. We have now examined the role of E21R on the survival of hemopoietic cells and found that E21R causes apoptosis (programmed cell death) of normal and malignant cells directly in the absence of GM-CSF. The direct apoptotic effect of E21R occurred in a dose- and time-dependent manner. Apoptosis by E21R was dependent on cells expressing the high-affinity GM-CSF receptor and could be blocked by GM-CSF. Significantly, apoptosis of the cells occurred even in the presence of the survival factors granulocyte CSF and stem cell factor but was prevented by engagement of beta(c) with interleukin 3. The initiation of apoptosis required phosphorylation, transcriptional activity, and protein synthesis. These findings support a model whereby binding of E21R to the alpha chain leads to apoptosis, while beta(c) plays an important role in cell survival. This model may be applicable to other multimeric cytokine receptors and offers a novel approach for the treatment of human leukemia.

The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway.

The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of an alpha and beta subunit, which together form the high-affinity receptor. The alpha subunit by itself binds ligand at low affinity, whereas the isolated beta subunit does not bind GM-CSF. It is generally believed that the high-affinity receptor is responsible for the multiple functions of GM-CSF and that the isolated alpha subunit (GMR alpha) does not transduce a signal. Xenopus laevis oocytes injected with RNA encoding human GMR alpha expressed up to 10(10) low-affinity sites for GM-CSF (Kd = 6 nM). GM-CSF binding to the alpha subunit expressed in Xenopus oocytes caused activation of 2-deoxyglucose transport through endogenous glucose transporters. 2-Deoxyglucose transport was stimulated by similar low concentrations of GM-CSF in HL-60 leukemia cells as well as normal human neutrophils and Xenopus oocytes expressing GMR alpha. Engagement of the isolated alpha subunit in oocytes did not lead to protein phosphorylation or tyrosine phosphorylation of mitogen-activated protein kinase (MAP kinase). Staurosporin and genistein inhibited GM-CSF-induced tyrosine phosphorylation of MAP kinase in human neutrophils and HL-60 cells without affecting GM-CSF-stimulated uptake of 2-deoxyglucose. These results provide direct evidence that the isolated alpha subunit signals for hexose transport and can do so without engagement of the kinase cascade. Our data also indicate that signaling for hexose uptake may occur in a phosphorylation-independent manner in cells expressing the high-affinity GM-CSF receptor.

Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling.

The high-affinity receptors for granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and IL-5 consist of two subunits, alpha and beta. The alpha subunits are specific to each cytokine and the same beta subunit (beta c) is shared by these three receptors. Although none of these receptor subunits has intrinsic kinase activity, these cytokines induce protein tyrosine phosphorylation, activation of Ras, Raf-1 and MAP kinase, and transcriptional activation of nuclear proto-oncogenes such as c-myc, c-fos and c-jun. In this paper, we describe a detailed analysis of the signaling potential of the beta c subunit by using a series of cytoplasmic deletion mutants. The human beta c consists of 881 amino acid residues. A C-terminal deletion mutant of beta c at amino acid 763 (beta 763) induced phosphorylation of Shc and activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, whereas a deletion at amino acid 626 (beta 626) induced none of these effects. The beta 763 mutant, as well as the full-length beta c, induced transcription of c-myc, c-fos and c-jun. Deletions at amino acid 517 (beta 517) and 626 (beta 626) induced c-myc and pim-1, but no induction of c-fos and c-jun was observed. GM-CSF increased phosphatidylinositol 3 kinase (PI3-K) activity in anti-phosphotyrosine immunoprecipitates from cells expressing beta 763 as well as beta c, whereas it was only marginally increased from cells expressing beta 517 or beta 626. Thus, there are at least two distinct regions within the cytoplasmic domain of beta c that are responsible for different signals, i.e. a membrane proximal region of approximately 60 amino acid residues upstream of Glu517 is essential for induction of c-myc and pim-1, and a distal region of approximately 140 amino acid residues (between Leu626 and Ser763) is required for activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, as well as induction of c-fos and c-jun.

Differential regulation of early response genes and cell proliferation through the human granulocyte macrophage colony-stimulating factor receptor: selective activation of the c-fos promoter by genistein.

Granulocyte macrophage colony-stimulating factor (GM-CSF) binds to the high-affinity GM-CSF receptor (GMR) consisting of alpha and beta subunits and induces rapid tyrosine phosphorylation, activation of early response genes, and proliferation of hematopoietic cells. The alpha subunit is the primary cytokine binding component and the beta subunit is required for high-affinity binding as well as for signal transduction. Using tyrosine kinase inhibitors and cytoplasmic deletion mutants of the beta subunit, we obtained evidence that there are at least two distinct pathways downstream of the GMR in BA/F3 cell, one which is essential for proliferation, leads to the c-myc gene activation, and is sensitive to herbimycin and genistein. Activation of this pathway depends on the cytoplasmic region between amino acid positions 455 and 517 of the beta subunit. The second pathway, which leads to activation of c-fos and c-jun genes, is only partially sensitive to herbimycin, is resistant to genistein and depends on the region between amino acid positions 626 and 763 of the beta subunit. Unexpectedly, the c-fos mRNA induction was augmented by genistein. The enhanced expression of c-fos mRNA by genistein also occurred with stimulation with cAMP, PMA, or EGF in NIH3T3 cells. It thus seems likely that genistein affects a common pathway downstream of these signals.

Granulocyte-macrophage colony-stimulating factor receptors alter their binding characteristics during myeloid maturation through up-regulation of the affinity converting beta subunit (KH97)

Acute myeloid leukemia blasts express dual affinity (high and low) granulocyte-macrophage colony-stimulating factor (GM-CSF) binding, and the high affinity GM-CSF binding is counteracted by excess interleukin-3 (IL-3). Neutrophils express a single class of GM-CSF-R with intermediate affinity that lack IL-3 cross-reactivity. Here we demonstrate the differentiation associated changes of GM-CSF binding characteristics in three models representative of different stages of myeloid maturation. We find that high affinity GM-CSF binding is converted into intermediate affinity binding, which still cross-reacts with IL-3, beyond the stage of promyelocytes. During terminal maturation towards neutrophils, IL-3 cross-reactivity is gradually lost. We sought to determine the mechanism underlying the affinity conversion of the GM-CSF-R. Northern and reverse transcriptase-polymerase chain reaction analysis of GM-CSF-R alpha and -beta c (KH97) transcripts did not provide indications for the involvement of GM-CSF-R splice variants in the formation of the intermediate affinity GM-CSFR complex. In COS-cell transfectants with increasing amounts of beta c in the presence of a fixed number of GM-CSF-R alpha chains, the high affinity GM-CSF binding converted into intermediate affinity GM-CSF binding. These results are discussed in view of the concept that increasing expression of beta c subunits may cause alternative oligomerization of the GM-CSF-R alpha and -beta c subunits resulting in the formation of intermediate rather than high affinity GM-CSFR alpha.beta c complexes.

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.

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.