Role of Tyrosine 441 of Interferon-γ Receptor Subunit 1 in SOCS-1-mediated Attenuation of STAT1 Activation

Suppressor of cytokine signaling (SOCS)-1, the key negative regulator of interferon (IFN)-γ-dependent signaling, is induced in response to IFNγ. SOCS-1 binds to and inhibits the IFNγ receptor-associated kinase Janus-activated kinase (JAK) 2 and inhibits its function in vitro, but the mechanism by which SOCS-1 inhibits IFNγ-dependent signaling in vivo is not clear. Upon stimulation, mouse IFNγ receptor subunit 1 (IFNGR1) is phosphorylated on several cytoplasmic tyrosine residues, and Tyr419 is required for signal transducer and activator of transcription (STAT) 1 activation in mouse embryo fibroblasts. However, the functions of the other three cytoplasmic tyrosine residues are not known. Here we show that Tyr441 is required to attenuate STAT1 activation in response to IFNγ. Several tyrosine to phenylalanine mutants of IFNGR1, expressed at normal levels in stable pools of IFNGR1-null cells, were analyzed for the phosphorylation of STAT1 during a 48-h period, and antiviral activity in response to IFNγ was also measured. Stronger activation of STAT1 was observed in cells expressing all IFNGR1 variants mutated at Tyr441, and, consistently, stronger antiviral activity was also observed in these cells. Furthermore, constitutive overexpression of SOCS-1 inhibited IFNγ-dependent signaling only in cells expressing IFNGR1 variants that included the Tyr441 mutation. Mutation of Tyr441 also blocked the ability of SOCS-1 to bind to IFNGR1 and JAK2 in response to IFNγ and the normal down-regulation of STAT1 activation and antiviral activity. These results, together with data from the literature, suggest a model in which, in response to IFNγ, phosphorylation of Tyr441 creates a docking site for SOCS-1, which then binds to JAK2 within the receptor-JAK complex to partially inhibit JAK2 phosphorylation. Furthermore, the virtually complete blockade of STAT1 phosphorylation by overexpressed SOCS-1 in this experiment suggests that the binding of SOCS-1 to Tyr441 also blocks the access of STAT1 to Tyr419 and that this effect may be the principal mechanism of inhibition of downstream signaling.