Biosynthetic analysis of the human interferon-gamma receptor. Identification of N-linked glycosylation intermediates.
Biosynthesis of the human IFN gamma receptor was studied using metabolic labeling techniques and immunoprecipitation with receptor-specific monoclonal antibodies. Colo-205 and HepG2 cells labeled with [35S]methionine gave rise to two components with molecular mass 75 and 90 kDa following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No bands were detected when immunoprecipitation was performed using irrelevant monoclonal IgG or in the presence of excess ligand, a condition known to block antibody-receptor interaction. When Colo-205 were labeled for increasing periods of time, the 75-kDa form was detected after 5 min, whereas the 90-kDa form appeared only after 60 min. Pulse-chase analysis established that the 75-kDa form was the precursor of the 90-kDa component. Only the 90-kDa form was detected on extrinsically radioiodinated Colo-205 cell surfaces. This observation was confirmed by Western blot analysis of isolated Colo-205 membranes. Digestion of labeled precipitates with peptide:N-glycosidase F caused a 22% reduction in the apparent molecular weight of the IFN gamma receptor. Receptor derived from tunicamycin-treated Colo-205 labeled for 5 min displayed a single molecular mass of 65 kDa and expressed ligand binding activity. Longer labeling periods in the presence of tunicamycin revealed the appearance of a second ligand-binding form of 70 kDa. Thus, Colo-205 IFN gamma receptors carry asparagine (N)-linked oligosaccharides and possibly some other form of post-translational modification.
Hershey
G K
GK
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Schreiber
R D
RD
D001216:Asparagine / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D011233:Precipitin Tests; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000378:metabolism; D017471:Receptors, Interferon
D009844:Oligosaccharides; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D001216:Asparagine; D007371:Interferon-gamma