Interferon-gamma induces receptor dimerization in solution and on cells.
The extracellular domain (ECD) of the human interferon-gamma (IFN gamma) receptor was stably expressed in Chinese hamster ovary cells and purified to homogeneity. Scatchard analysis of 125I-IFN gamma binding to ECD preparations revealed the formation of a ligand-receptor complex which displayed a Ka of 6.4 +/- 0.9 x 10(8) M-1. Two types of complexes were identified by sucrose density gradient ultracentrifugation. The stoichiometry of the major ECD-ligand complex was determined by high performance liquid chromatography gel filtration. When IFN gamma was incubated with a 2-fold molar excess of ECD, a 190-kDa complex was isolated that contained 2 mol of ECD per 1 mol of IFN gamma. IFN gamma also induced dimerization of IFN gamma receptors expressed at the cell surface as detected by chemically cross-linking receptor bound ligand and analyzing cell lysates by SDS-polyacrylamide gel electrophoresis and immunoblotting. Finally, labeled forms of ECD bound to cells preincubated at 4 degrees C with excess amounts of IFN gamma indicating that the ligand could associate with more than one receptor molecule in the absence of chemical cross-linking agents. These results demonstrate that IFN gamma effects dimerization of its receptor under physiologic conditions and suggest that IFN gamma receptor dimerization may be an important event in inducing IFN gamma-dependent biologic responses.
Greenlund
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AC
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Schreiber
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RD
Goeddel
D V
DV
Pennica
D
D
D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism* / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D017471:Receptors, Interferon / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D000096743:Interferon gamma Receptor
D000911:Antibodies, Monoclonal; D007371:Interferon-gamma; D046911:Macromolecular Substances; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D000096743:Interferon gamma Receptor