HUMAN IFN-GAMMA RECEPTOR CYTOPLASMIC DOMAIN - EXPRESSION AND INTERACTION WITH HUIFN-GAMMA

To investigate the structural basis for human interferon gamma (huIFN gamma) binding to intracellular regions of the human IFN gamma recepto r (huIFN gamma R), we have subcloned and expressed the huIFN gamma R f ree of fusion proteins in the yeast strain Pichia pastoris. HuIFN gamm a bound to the cytoplasmic domain of the receptor via the IFN gamma C- terminus. Binding was inhibited by both human and mouse C-terminus pep tides. N-terminus peptides failed to inhibit cytoplasmic binding. Thus , while extracellular receptor domain binding is species specific, bin ding to the cytoplasmic domain of the receptor is species non-specific . In solid-phase binding assays, IFN gamma had a K-d of 3.7 x 10(-8) R I for the newly expressed cytoplasmic domain. Peptide competitions sho wed that IFN gamma bound to a receptor site corresponding to the membr ane proximal residues 253-287, which is adjacent to the site of bindin g of the tyrosine kinase JAK2. The cytoplasmic binding affinity and bi nding site specificity suggest that the huIFN gamma R cytoplasmic doma in can function independent of the extracellular domain to bind huIFN gamma and induce the biological activity previously associated with in ternalized huIFN gamma. (C) 1998 Academic Press.