Characterization of Interferon-α Binding Sites on Human Cell Lines

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Abstract
The binding sites for human interferon-α (IFN-α) have been characterized on human lymphoblastoid, melanoma, rhabdomyosarcoma, and cervical carcinoma cells. Crosslinking of iodinated-recombinant DNA-derived IFN-α-Con1, an analog of the known IFN-α subtypes, to the cell surface with disuccinimidyl suberate yielded four IFN-receptor complexes of 118, 138, 159, and 260 kD on all cell lines that specifically bind IFN-α. Since IFN-α exists in solution as monomers, dimers, and trimers, and the three lower molecular weight IFN-α—receptor complexes differ by the molecular weight of IFN-α (20 kD), this suggests that the human IFN-α receptor of 100 kD binds more than one molecule of IFN-α. The higher molecular weight complex of 260 kD may result from dimerization of the receptor. None of these complexes was observed in a rhabdomyosarcoma subclone that does not specifically bind IFN-α. Pretreatment of cells with trypsin abolished the formation of these complexes. Pretreatment of cells with neuraminidase did not reduce IFN-α binding, but increased the electrophoretic mobility of all four IFN-α-receptor complexes. Other glycosidases (i.e., mannosidase, β-galactosidase, and endoglycosidase F) had no effects on IFN-α binding or mobility of complexes. Thus, although the IFN-α receptor is a glycoprotein, the glycosylated portion is apparently not part of the IFN-aα-binding domain. The formation of IFN-α—receptor complexes is independent of the duration of incubation with IFN (from 5 min to 1 h at 15°C). Subclones of Dandi lymphoblastoid cells that are sensitive (Cl-1) or resistant (Cl-2) to the antiproliferative effect of IFN-α have similar IFN-α binding capacity. The association of the IFN—receptor complexes with Triton-insoluble cytoskeletal matrix is markedly diminished in resistant Cl-2 cells relative to that in sensitive Cl-1 cells. However, IFN—receptor complexes, including those noncovalently associated with the cytoskeleton, are structurally similar in sensitive and resistant subclones. Thus, a post-binding defect may be responsible for the resistance of Cl-2 cells to the antiproliferative effect of IFN-α.