Immune interferon receptor: chemical and enzymatic sensitivity.

Human immune interferon-gamma (HuIFN-gamma) labeled with 32P was used to study the structure of IFN-gamma receptor. When [32P]HuIFN-gamma was bound and crosslinked to IFN-gamma the receptor of human cells with a bifunctional crosslinker disuccinimidyl suberate (DSS), a single diffused 32P-labeled band corresponding to the IFN-gamma.receptor complex was visualized by SDS-polyacrylamide gel electrophoresis and autoradiography. The size of the [32P]-HuIFN-gamma.receptor complex was about 100-120 kD. Separation of crosslinked complex in reducing and nonreducing gels showed no size differences, suggesting the absence of interchain disulfide linkage. However, binding and formation of the crosslinked IFN-gamma. receptor complex on cells was diminished in the presence of the disulfide reducing agent dithiothreitol (DTT). The reduction was DTT-dose-dependent, suggesting that intramolecular disulfides of the receptor are important for binding. Also, [32P]HuIFN-gamma did not bind if cells were pretreated with and then washed free of DTT, suggesting an irreversible reduction of intrachain disulfide bonds, presumably of the receptor. [32P]HuIFN-gamma also specifically binds to human placental membranes. Each placenta has about 170 ng of IFN-gamma receptors. Covalent attachment of [32P]HuIFN-gamma to placental plasma membranes via DSS produced 2 crosslinked complexes with the molecular sizes of 100-120 kD and 60-70 kD. The IFN-gamma.receptor complex of placental membranes was solubilized with NP-40 after DSS treatment and partially purified with immobilized antibody to the carboxyl terminus of IFN-gamma. Treatment of the receptor complex with trypsin and papain was used to demonstrate its differential proteolytic sensitivity.