Characterization of the human type I interferon receptor by ligand blotting.
The human type I interferon (IFN) receptor was characterized by ligand blotting. In this method, plasmalemma proteins or detergent-lysed whole-cell extracts from human Burkitt lymphoma cell lines were separated on polyacrylamide gels and subsequently transferred onto nitrocellulose sheets. Probing the blots with 3 x 10(-10) M 125I-labeled recombinant IFN-alpha A (125I-rIFN-alpha A) revealed an IFN-alpha-binding protein with an apparent molecular mass of 95 kDa (p95). Performing the electrophoretic run under reducing conditions completely abrogated the signal on the blot, indicating that the type I IFN receptor contains a disulfide bond essential for IFN binding. Optimal binding of 125I-rIFN-alpha A occurred at pH 9. The specificity of the binding reaction was established by simultaneously adding an excess of unlabeled IFN during incubation of the blots with 125I-rIFN-alpha A. The addition of either unlabeled IFN-alpha or IFN-beta, but not IFN-gamma, abolished the binding of 125I-rIFN-alpha A to p95. 125I-IFN-gamma at 1.25 x 10(-11) M bound to two proteins distinct from p95 with apparent molecular mass of 92 and 87 kDa, respectively. Saturability of 125I-rIFN-alpha A binding was demonstrated by probing a constant amount of membrane proteins with increasing amounts of 125I-rIFN-alpha A. Scatchard analysis of the binding data yielded an apparent Kd of 5.4 x 10(-10) M for the immobilized type I IFN receptor. The expression of p95 on IFN-alpha-resistant and -sensitive cells was indistinguishable. We conclude that p95 is the IFN-alpha/beta receptor and that two proteins (p92 and p87) can specifically bind IFN-gamma. These results indicate that ligand blotting is a versatile method for characterization of unmodified IFN receptors and IFN-receptor interaction and could also provide a new investigational approach for other cytokine receptor systems.
Schwabe
M
M
Laboratory of Biochemical Physiology, National Cancer Institute, Frederick, MD 21701.
Princler
G L
GL
Faltynek
C R
CR
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007370:Interferon Type I / Q000378:metabolism*; D007371:Interferon-gamma / Q000378:metabolism; D008024:Ligands; D008854:Microscopy, Electron; D008970:Molecular Weight; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000378:metabolism*; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D007370:Interferon Type I; D008024:Ligands; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma