Binding of Murine 125I-labelled Natural Interferon- to Murine Cell Receptors

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Abstract
Natural murine interferon-.gamma. (naMuIFN-.gamma.) produced by T-lymphoma cells (L12-R4) stimulated with phorbol myristic acetate was purified by use of an anti-MuIFN-.gamma. immunoadsorbent and was labelled with 125I to study its binding to murine cell receptors. All the cell lines examined bound naMuIFN-.gamma., although the binding affinity varied considerably. By adding increasing concentrations of unlabelled naMuIFN-.gamma. in competition binding assays we determined dissociation constants (KD) of 8.2 .times. 10-10 and 7.4 .times. 10-10 M for L1210 and TS/A cells, respectively, and of 6.5 .times. 10-9 M for L-929 cells. The numbers of receptors present per cell of these lines were 3000, 1000 and 2000, respectively. Highly purified naMuIFN-.gamma. as well as recombinant MuIFN-.gamma. competed for binding sites with 125I-labelled IFN-.gamma. on L1210 cells, although the latter displayed a KD greater than the former (5.8 .times. 10-9 M compared to 8.2 .times. 10-10 M). Moreover, protease, but not endoglycosidase, treatment of target cells prevented the subsequent binding of 125I-labelled IFN-.gamma., suggesting that a protein moiety is involved in the binding of the ligand. These studies demonstrate that naMuIFN-.gamma. binds in a specific manner and with high affinity to murine cell receptors.