The ␣ subunit of the receptor for human granulocytemacrophage colony-stimulating factor (GM-CSF) is a glycoprotein containing 11 potential N-glycosylation sites in the extracellular domain. We examined the role of N-glycosylation on ␣ subunit membrane localization and function. Tunicamycin, an N-glycosylation inhibitor, markedly inhibited GM-CSF binding, GM-CSF-induced deoxyglucose uptake, and protein tyrosine phosphorylation in HL-60(eos) cells but did not affect cell surface expression of the ␣ subunit as detected by an anti-␣ subunit monoclonal antibody. In COS cells expressing the ␣ subunit and treated with tunicamycin, N-unglycosylated ␣ subunit was expressed and transported to the cell surface but was not capable of binding GM-CSF. High affinity binding in COS cells expressing both ␣ and ␤ subunits was also blocked by tunicamycin treatment. These studies indicate that N-linked oligosaccharides are essential for ␣ subunit ligand binding and signaling by the human GM-CSF receptor.Granulocyte-macrophage colony-stimulating factor (GM-CSF) 1 is a hematopoietic growth factor that promotes the proliferation and maturation of myeloid progenitor cells and enhances the function of mature granulocytes and mononuclear phagocytes (1). GM-CSF exerts its effect via its cognate receptor on the cell surface. The human GM-CSF receptor is composed of an ␣ subunit that binds GM-CSF with low affinity (K d 1-10 nM) (2-4) and a ␤ subunit that has no intrinsic GM-CSF binding capacity but associates with the ␣ subunit to form a high affinity receptor with a K d of 10 -50 pM (5, 6). The high affinity receptor signals for proliferation and functional activation via protein phosphorylation pathways (7-11). We recently found that the isolated ␣ subunit signals for glucose uptake through a protein phosphorylation-independent pathway (12).The ␣ subunit of the human GM-CSF receptor is an 84-kDa glycoprotein that readily binds lectins and has 11 potential N-glycosylation sites in the cDNA-deduced amino acid sequence, all located in the extracellular domain (13). The calculated molecular mass of the ␣ subunit based on amino acid sequence is 40 kDa. The difference between the apparent and calculated molecular mass (44 kDa) is in part due to N-glycosylation. It is not known what role, if any, the N-linked carbohydrates present in the extracellular domain play in the function of the ␣ subunit. We used the N-glycosylation inhibitor tunicamycin to probe the role of N-glycosylation in the function and surface expression of the GM-CSF receptor in the HL-60(eos) cell line, which expresses a high affinity GM-CSF receptor, and in COS cells transfected with ␣ subunit cDNA alone or both ␣ and ␤ subunit cDNA. Our results indicate that Nglycosylation of the ␣ subunit is essential for ligand binding and signaling by the human GM-CSF receptor. EXPERIMENTAL PROCEDURESCell Culture-A previously described eosinophilic subline of HL-60, HL-60(eos) (14, 15), was cultured in Iscove's modified Dulbecco's medium (IMDM) (pH 7.6) supplemented with 10% heat-inactiv...

The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of an a and B3 subunit, which together form the high-affinity receptor. The a subunit by itself binds ligand at low affinity, whereas the isolated (8 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 a subunit (GMRa) does not transduce a signal. Xenopus laevis oocytes injected with RNA encoding human GMRa expressed up to 1010 low-affinity sites for GM-CSF (Kd = 6 nM). GM-CSF binding to the a 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 GMRa. Engagement of the isolated a subunit in oocytes did not lead to protein phosphorylation or tyrosine phosphorylation of mitogen-activated protein kinase (MAP kinase). Staurosporin and genistein inhibited GM-CSFinduced tyrosine phosphorylation of MAP kinase in human neutrophils and HL-60 cells without affecting GM-CSFstimulated uptake of 2-deoxyglucose. These results provide direct evidence that the isolated a 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.The receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of an a and ( subunit, and the a subunit (GMRa) by itselfbinds ligand at low affinity (Kd = 3-7 nM) (1-6). The isolated 8 subunit (GMR(3) does not bind GM-CSF; however, it associates with GMRa to form a high-affinity receptor (Kd = 20-40 pM) (3-6). GM-CSF receptors are present on myeloid progenitors and mature neutrophils, eosinophils, and mononuclear phagocytes (7)(8)(9)(10)(11)(12)(13). GMRa is also present in some nonhematopoietic cells, such as placenta and melanoma cells (1,14,15), while the GMR(8 is mainly restricted to hematopoietic cells (3-5, 12).It is generally believed that the biological functions exerted by GM-CSF are mediated by the high-affinity receptor and that the isolated GMRa does not transduce a signal (3-6, 16, 17). GMRa, however, binds GM-CSF and in association with GMR,8 transduces signals involving protein phosphorylation (2,(18)(19)(20). To examine the functional capability of the isolated a subunit, we expressed it in Xenopus laevis oocytes and discovered that it could signal for glucose transport without involving the phosphorylation of mitogen-activated protein kinase (MAP kinase). MATERIALS AND METHODSComplementary DNAs for the human GMRa (21) and the human glucose transporter 4 (GLUT4) (22) cloned in pBluescript (Stratagene) were linearized by digestion with restriction enzymes, and the sense or antisense RNA was transcribed in vitro (23). Stage V and VI oocy...