Coexpression of G-CSF with an unglycosylated G-CSF receptor mutant results in secretion of a stable complex.
Previously, we have shown that the entire extracellular domain of the granulocyte-colony stimulating factor receptor (sG-CSFr) produced in Chinese hamster ovary (CHO) cells forms a stable complex with its ligand G-CSF, at a stoichiometry of 2:2. A truncated receptor molecule consisting of the cytokine receptor homology domain and N-terminus Ig-like domain (Ig CRH) behaves quite similarly. Both of these forms of the receptor are highly glycosylated. To address the importance of glycosylation toward receptor activity and stability, and possibly obtain nonglycosylated receptor for crystallization, mutations were made to replace four Asn residues which are N-glycosylated in the truncated receptor. Virtually no receptor was recovered from conditioned media of CHO cells transfected with this mutant construct, although a high-level of mRNA coding for receptor was detected; this mRNA was translated as determined by Western blots of cell lysates. These results indicate that the translated product is apparently not secreted from these cells. Cells transfected with mutant receptor cDNA were cotransfected with a cDNA construct expressing G-CSF in which the single O-glycosylation site was eliminated by mutation. Upon fermentation of the cotransfectants, we observed a large amount of receptor-ligand complex in the conditioned media. The purified unglycosylated complex appeared to be of the same binding stoichiometry and approximate binding affinity as that of complex formed by addition of purified ligand and unmutated receptor. These results show that while glycosylation of sG-CSFr is not necessary for ligand binding, it appears to be crucial in folding and export from the cell.
Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320-1789, USA.
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TP
Horan
L
L
Simonet
R
R
Jacobsen
M
M
Mann
M
M
Haniu
J
J
Wen
T
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Arakawa
M
M
Kuwamoto
F
F
Martin
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017077:Culture Media, Conditioned; D015870:Gene Expression; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection
D017077:Culture Media, Conditioned; D046911:Macromolecular Substances; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D016179:Granulocyte Colony-Stimulating Factor