CDB15:0000376 CSF2 — CSF2RA

ABSTRACT: Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, we obtained a homologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells--i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. We therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the alpha and beta subunits of the GM-CSF receptor, respectively.

ABSTRACT: Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine derived from activated T cells, endothelial cells, fibroblasts, and macrophages. It stimulates myeloid and erythroid progenitors to form colonies in semisolid medium in vitro, as well as enhancing multiple differentiated functions of mature neutrophils, macrophages, and eosinophils. We have examined the binding of human GM-CSF to a variety of responsive human cells and cell lines. The most mature myelomonocytic cells, specifically human neutrophils, macrophages, and eosinophils, express the highest numbers of a single class of high affinity receptors (Kd approximately 37 pM, 293-1000 sites/cell). HL-60 and KG-1 cells exhibit an increase in specific binding at high concentrations of GM-CSF; computer analysis of the data is nonetheless consistent with a single class of high affinity binding sites with a Kd approximately 43 pM and 20-450 sites/cell. Dimethyl sulfoxide induces a 3-10-fold increase in high affinity receptors expressed in HL-60 cells, coincident with terminal neutrophilic differentiation. Finally, binding of 125I-GM-CSF to fresh peripheral blood cells from six patients with chronic myelogenous leukemia was analyzed. In three of six cases, binding was similar to the nonsaturable binding observed with HL-60 and KG-1 cells. GM-CSF binding was low, or in some cases, undetectable on myeloblasts obtained from eight patients with acute myelogenous leukemia. The observed affinities of the receptor for GM-CSF are consistent with all known biological activities. Affinity labeling of both normal neutrophils and dimethyl sulfoxide-induced HL-60 cells with unglycosylated 125I-GM-CSF yielded a band of 98 kDa, implying a molecular weight of approximately 84,000 for the human GM-CSF receptor.

ABSTRACT: The receptor for the hemopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of two chains, both of which belong to the superfamily of cytokine receptors. The alpha chain confers low affinity binding only, whereas the beta chain (betac) confers high affinity binding when associated with alpha. Ectopic expression of both chains of the receptor in murine NIH-3T3 fibroblasts results in signal transduction, mitogenesis, and morphologic transformation. The cytoplasmic domain of the GM-CSF receptor alpha subunit (GMR-alpha) comprises 54 amino acids that have been shown to be important for signal transduction through the beta chain. The present study was designed to address the possibility of receptor oligomerization and its functional implication. Cross-linking studies with 125I-GM-CSF on NIH-3T3 transfectants is consistent with the presence of alpha and betac dimers and of receptor oligomers. We have, therefore, generated an inert alpha chain through polymerase chain reaction-mediated truncation of 47 amino acids of the COOH-terminal domain of alpha (alphat), and coexpressed alphat, alpha, and betac in NIH-3T3. In cells in which alphat and alpha are present in stoichiometric proportion within the GM-CSF-binding complex, we provide evidence that alphat is dominant negative over wild type alpha on the basis of two different functional assays: cell proliferation and foci formation. Hence, our results suggest the requirement for at least two functional alpha chains for signal transduction. Together with the cross-linking studies, our data indicate that the functional GMR is an oligomer that contains at least two alpha chains.

ABSTRACT: Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage progenitor cells. The biologic actions of GM-CSF are mediated by binding to a specific receptor consisting of two chains designated as alpha and beta subunits. We have demonstrated that the murine FDC-P1-derived cell line WT-19 transfected with the human GM-CSF receptor alpha and beta subunits (GM-CSFRalpha and beta) can be induced to differentiate by the addition of human GM-CSF (hGM-CSF). By expressing a series of GM-CSFRalpha mutants in WT19 cells, we have determined the amino acid domains of the GM-CSFRalpha cytoplasmic domain that regulate cell differentiation, proliferation, and survival. We found that the membrane proximal proline-rich domain and adjacent 16 residues are essential for both hGM-CSF-dependent cell proliferation and differentiation. In contrast, the C-terminal region of the GM-CSFRalpha cytoplasmic domain was not necessary for cell differentiation mediated by hGM-CSF, but the removal of this region severely impaired the ability of hGM-CSF to support cell survival. While the activation of JAK2, Shc, Erk, and STAT5 proteins correlated with hGM-CSF-mediated cell growth, cellular differentiation occurred in the absence of activation of these signal transduction pathways.