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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
 6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A synthetic segment (110-127) of the carboxyl terminus of recombinant human granulocyte-macrophage colony-stimulating factor (rh-GM-CSF) was used to generate a rabbit polyclonal antibody (345-6), which recognized both peptide and full-length Escherichia coli-derived rh-GM-CSF in a direct enzyme-linked immunosorbent assay. Antibody 345-6 was shown to antagonize the binding of 125I-labeled rh-GM-CSF to its receptor on the KG-1 cell line and to inhibit human GM-CSF-dependent proliferation of the AML-193 cell line. The purified IgG fraction of neutralizing antibody 345-6 was used as immunogen to obtain sheep anti-serum 1418. Antibody 1418 recognized antibody 345-6 on direct enzyme-linked immunosorbent assay but did not recognize rh-GM-CSF or the peptide 110-127 to which antibody 345-6 was raised. Antiserum 1418, as well as a purified IgG fraction of this serum, inhibited both rh-GM-CSF-stimulated cell proliferation and 125I-labeled rh-GM-CSF receptor binding but not 125I-labeled recombinant human interleukin-4 receptor binding. The anti-idiotypic antibody response derived from the anti-(110-127) antibody strongly suggests that the carboxyl-terminal region of rh-GM-CSF may be directly involved in the receptor-ligand interaction of this protein. The high affinity receptor consists of two different components (GM-R alpha beta) a cytokine-specific alpha-subunit and a beta-subunit that is shared by human GM-CSF, interleukin-3, and interleukin-5. In an effort to localize the epitope of antibody 1418 to either GMR alpha or GMR beta, several cell lines containing high, low, or both high and low affinity receptors were examined. Each was specifically and completely inhibited by antibody 1418. Interleukin-3-dependent cell proliferation of the AML-193 cell line was found to be unaffected by the antibody 1418. Thus, the carboxyl-terminal region of rh-GM-CSF is likely to be involved in the interaction of the ligand with the alpha-subunit of the high affinity receptor.
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PMID:A role for the carboxyl terminus of human granulocyte-macrophage colony-stimulating factor in the binding of ligand to the alpha-subunit of the high affinity receptor. 811 89

The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of at least two subunits, alpha and beta. In addition to the conserved cysteine residues and a "WSxWS" motif, the extracellular segments of both subunits have domains that are structurally related to a fibronectin type III domain. This structure is conserved in all members of the cytokine receptor superfamily. We isolated and characterized genomic DNA clones containing the entire coding sequences of the alpha subunit of the human GM-CSF receptor (hGMR alpha). The gene spans approximately 44 kilobases and has 13 exons. The major transcription initiation site was determined to be 195 base pairs upstream of the translation initiation site. The putative promoter region lacks a typical TATA motif and an Sp1 binding site, but contains a purine-rich stretch about 30 base pairs upstream of the transcription initiation site. This stretch is also found in the human interleukin 2 receptor gamma subunit and granulocyte colony-stimulating factor receptor genes. We compared the exon-intron organization of the hGMR alpha gene with other members of the cytokine receptor superfamily and found the genomic organizations to be remarkably well conserved. On the basis of these observations, we propose a model for evolution of this gene family.
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PMID:Structure of the gene encoding the alpha subunit of the human granulocyte-macrophage colony stimulating factor receptor. Implications for the evolution of the cytokine receptor superfamily. 814 76

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.
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PMID:The alpha subunit of the human granulocyte-macrophage colony-stimulating factor receptor signals for glucose transport via a phosphorylation-independent pathway. 814 50

Residues within the first and fourth helices of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) were analyzed for their role in biologic activity and interaction with the alpha- and beta-chains of the hGM-CSF receptor. Within the first helix substitution of the surface residues Glu14, Asn17, Gln20, Arg23, Arg24, and Asn27 or the buried residues Ala18, Leu25, and Leu28 did not significantly impair bioactivity or receptor binding. Substitutions at the buried residues Ala22 and Leu26 had intermediate bioactivity. However, substitutions of the surface residue Glu21 or the buried residue Ile19 reduced the relative bioactivity of the analogues to as little as 0.45% and 0.3%, respectively. Substitution of the charged surface residues of the fourth helix showed that substitution at Glu104, Lys107, and Lys111 had no significant effect on bioactivity, but substitution at Glu108 and Asp112 reduced the potency of the analogues to 34% and 7%, respectively. Receptor binding studies showed that, whereas Glu21 is the critical residue for binding to the hGM-CSF-receptor beta-chain, Asp112 is likely to be involved in binding to the GM-CSF-receptor alpha-chain. These results establish the relative contribution of residues in the first and fourth helices for GM-CSF bioactivity and receptor binding, and support a model where the fourth helix of GM-CSF interacts with the alpha-chain, and the first helix with the beta-chain of the GM-CSF receptor.
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PMID:Identification of residues in the first and fourth helices of human granulocyte-macrophage colony-stimulating factor involved in biologic activity and in binding to the alpha- and beta-chains of its receptor. 820 77

High-affinity receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3, and interleukin 5 consist of ligand-specific alpha chains (low-affinity subunits) and a common beta chain (beta c) that converts each complex to a high-affinity form. Although beta c alone has no detectable cytokine-binding activity, amino acid substitutions for Glu-21 of human GM-CSF significantly reduce high-affinity but not low-affinity binding, implying that beta c interacts directly with GM-CSF during formation of the high-affinity receptor but only in the presence of the alpha chain. A potential GM-CSF-binding determinant was identified in the second hemopoietin domain of beta c, and the role of individual residues within this region was investigated by determining the ability of mutated beta c chains to confer high-affinity binding when coexpressed with the alpha subunit of the GM-CSF receptor in COS cells. Substitutions involving Met-363, Arg-364, Tyr-365, and Glu-366 did not affect high-affinity binding. However, substitution of His-367 by lysine or glutamine abolished high-affinity binding, suggesting that this residue may form an important part of the high-affinity GM-CSF-binding determinant. Consistent with the loss of high-affinity binding, higher concentrations of human GM-CSF were required to stimulate proliferation of CTLL-2 cell lines transfected with cDNAs for GM-CSF receptor alpha chain and His-367 beta c mutant than those expressing GM-CSF receptor alpha subunit and beta c wild type.
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PMID:Histidine-367 of the human common beta chain of the receptor is critical for high-affinity binding of human granulocyte-macrophage colony-stimulating factor. 827 75

Human fetal liver (FL) and neonatal cord blood (CB) granulocyte-monocyte colony-forming progenitor cells (GM-CFC) are unique in their physiological environment and in certain proliferative and differentiative capacities. Tumor necrosis factor (TNF) and interferon (IFN) may inhibit or stimulate the growth of human bone marrow GM-CFC in vitro. The effects of recombinant human (rh) TNF-alpha, rhIFN-alpha, and rhIFN-tau on recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF)-stimulated clonogenic cultures of day 7 GM-CFC from FL and umbilical CB were compared with rhGM-CSF-stimulated GM-CFC from normal human bone marrow (BM). We demonstrate that, in comparison to BM progenitor cells, GM-CFC from both FL and CB were highly resistant to growth inhibition by all three cytokines. Furthermore, clonogenic growth of progenitors from FL and CB was markedly potentiated by IFN-tau in GM-CSF-stimulated cultures and was stimulated by IFN-tau in the absence of GM-CSF. Depletion of potential accessory cells resulted in a marked stimulatory response of CB cells to TNF-alpha, in the presence of GM-CSF, while it did not alter the responses to IFN. The stimulatory effects of IFN-tau and TNF-alpha may be indirectly mediated, at least in part, through induction of increased GM-CSF production and increased GM-CSF receptor expression by fetal cells. Divergent responses of myelopoietic cells, derived from various hematopoietic compartments, to regulatory actions of cytokines may provide a basis for further understanding the role of the environment in maturation and differentiation of granulocytes and monocytes.
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PMID:Differential responses of fetal, neonatal, and adult myelopoietic progenitors to interferon and tumor necrosis factor. 829 33

Studies in recent years have suggested that human tumor cell lines are capable of responding in vitro to hematopoietic growth factors. In the present study, we investigate the transcription of the alpha and beta subunits of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the alpha and beta subunits of interleukin 3 (IL-3) receptor, and the single subunit of interleukin 6 (IL-6) receptor and its associated gp130 transduction protein by PCR amplification of reverse-transcribed cellular mRNA in 34 malignant cell lines derived from a variety of histological cell types. mRNA for only a single subunit polypeptide was found in a significant minority of cell lines (23%), while in 20% both the alpha and beta subunits of either the GM-CSF receptor or the IL-3 receptor were detected among a number of different histological cell types. Transcription of the gene encoding the IL-6 receptor was found in 38% of cell lines, and all lines transcribed the gp130 transduction protein, consistent with previous observations on the ubiquity of that polypeptide. In order to test the in vitro effect of exogenously added growth factors on those malignant cell lines transcribing complete cytokine receptor, either GM-CSF, IL-3, or IL-6 was added in therapeutic concentrations (20-500 ng/ml) and cellular proliferation was measured by incorporation of [3H]thymidine. No stimulation was seen at either 3 and 6 days of culture. Production of cytokine by these cell lines was investigated at the level of transcription and by assay of peptide product. None transcribed mRNA for either GM-CSF or IL-3, while 5 of 6 (STD, DOZ, ADE, Hep-2, and Detroit) expressed IL-6 mRNA. Of these latter, 2 cell lines (ADE and Hep-2) produced IL-6 as determined by bioassay, while none produced GM-CSF or IL-3 by enzyme-linked immunosorbent assay. This suggests that in the case of GM-CSF and IL-3, failure to proliferate on addition of cytokine is not due to the prior presence of endogenous production. In contrast, at least a subset of malignant cell lines may involve a closed IL-6 autocrine loop saturating cell surface sites. These findings suggest that the ability to transcribe the genes encoding cytokine receptor is by itself insufficient to render cells cytokine responsive and that malignant cells may lack the cellular machinery for cytokine-induced proliferation. This in turn suggests that therapeutic administration of either GM-CSF, IL-3, or IL-6 may involve no additional risk of tumor regrowth in vivo.
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PMID:Transcription of genes encoding granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 6 receptors and lack of proliferative response to exogenous cytokines in nonhematopoietic human malignant cell lines. 831 22

When granulocyte-macrophage colony-stimulating factor (GM-CSF) is chemically conjugated to the ribosome-inactivating protein saporin, the resulting protein conjugate is highly toxic for cells expressing the GM-CSF receptor. Structural and Western blot analyses of the purified conjugate establish that it contains equimolar amounts of the starting materials and is free of any contamination by the non-conjugated components. The resulting bifunctional reagent is specifically cytotoxic to cells expressing the GM-CSF receptor, but is ineffective to cells that do not express the receptor. The cytotoxic activity is inhibited in a dose-dependent manner by GM-CSF, but not by any one of five other peptide growth factors. This is the first report of a mitotoxin for cells that express the GM-CSF receptor and which promises to be a valuable tool to study the expression of the GM-CSF receptor in normal and pathological states.
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PMID:Characterization of a saporin mitotoxin specifically cytotoxic to cells bearing the granulocyte-macrophage colony-stimulating factor receptor. 834 50

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 proB 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 NIH3T3 cells, but its ability to transduce signals in fibroblasts remained unanswered. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR both in NIH3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH3T3 fibroblasts and BA/F3 cells in response to human GM-CSF to activate transcription of c-fos, c-jun and c-myc protooncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. The ability of hGM-CSFR to transduce signals was affected by inhibitors of tyrosine kinase. These results indicated that the hGM-CSFR is functional in fibroblasts, that signal transduction via the hGM-CSFR in fibroblasts involves tyrosine kinase(s) and that association of hGM-CSFR with factor(s) specific to hematopoietic cell lineage is not essential to transduce growth promoting signals.
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PMID:Reconstitution of functional human GM-CSF receptor in mouse NIH3T3 fibroblasts and BA/F3 proB cells. 836 Dec 10

A panel of monoclonal antibodies was raised against the low-affinity human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor alpha-chain expressed as recombinant protein on murine FDC-P1 cells. All the selected antibodies were of the IgG2A isotype and bound to protein A. They each recognized both native and recombinant receptors by indirect surface immunofluorescence and by immunoprecipitation. Several of the antibodies also recognized presumably denatured receptors as detected by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three different epitopes on the extracellular domain of the GM-CSF receptor alpha-chain were defined by these antibodies, and two of the epitopes did not appear to be involved in binding hGM-CSF or in interactions with the beta-chain of the GM-CSF receptor that are required for high-affinity binding of GM-CSF. On the other hand, the epitope recognized by antibody 2B7-17-A appeared to be critically involved in the binding of GM-CSF because this antibody completely abrogated both high- and low-affinity binding of GM-CSF to native and recombinant receptors. Antibody 2B7-17-A had a relatively high affinity for the GM-CSF receptor alpha-chain (kd = 3 nmol/L) and slow dissociation kinetics (kd = 0.002 min-1). These properties made the 2B7-17-A antibody a potent inhibitor of hGM-CSF biologic action in several different bioassays, with a half-maximal inhibitory dose of about 6 nmol/L (1 microgram/mL). This antibody could prove useful in alleviating any pathologic states mediated by excess GM-CSF levels and in defining the domains of the GM-CSF receptor required for ligand binding.
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PMID:Neutralizing and nonneutralizing monoclonal antibodies to the human granulocyte-macrophage colony-stimulating factor receptor alpha-chain. 840 Feb 29


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