Identification of Residues of the IFNAR1 Chain of the Type I Human Interferon Receptor Critical for Ligand Binding and Biological Activity

Abstract

The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.

Palmitoylation of Interferon-α (IFN-α) Receptor Subunit IFNAR1 Is Required for the Activation of Stat1 and Stat2 by IFN-α

“…2A), with a stronger signal for IFNAR2. The difference of band intensity was probably due to the higher level of IFNAR2 than IFNAR1 expression in this cell line, as shown on the corresponding whole cell extract (28). A similar experiment with a Jurkat cell line expressing a FLAG-tagged IFNAR1 showed that IFNAR1 was palmitoylated in this human lymphoid cell line also (data not shown).…”

“…Parental Plasmids and Mutagenesis-Wild-type human IFNAR1 was expressed in pEFIREShyg derived from pIREShyg as described previously (28). Mutagenesis of the IFNAR1 receptor chain was performed using the QuickChange site-directed mutagenesis kit (Stratagene, Amsterdam).…”

“…Cell Culture and Transfection-L929R2 murine fibroblasts stably expressing human IFNAR2 (28) were transfected by wild-type or mutated forms of human IFNAR1 using FuGENE 6 (Roche Applied Science). Mixed populations of transfected cells were selected under hygromycin selective pressure.…”

The protein kinase IKKε can inhibit HCV expression independently of IFN and its own expression is downregulated in HCV-infected livers

“…To determine whether the antiviral effect of IKK⑀ requires IFN induction, we then examined its effect on HCV expression in the presence of anti-IFNAR1 antibodies known to compete specifically for the binding of type I IFNs to the IFNAR chain of the IFN receptor. 31,32 Analysis of STAT1 phosphorylation in response to IFN␣ or IFN␥ allowed to determine that the IFN receptor was functional in the HCV replicon cells as in the parental Huh-7 cells (Fig. 2B).…”

“…For neutralization of IFN-␣ binding, we used a purified preparation of anti-IFNAR1 monoclonal antibody 64G12 as described. 31,32 For the detection of total levels and phosphorylated levels of Stat1, we used polyclonal antibodies anti-Stat1, anti phosphoSTAT1 Ser727, and anti phosphoSTAT1 Tyr701 (Cell Signaling Technology, Danvers, MA). For the detection of flag STAT1␣ Y701F, flag STAT1␣, and flag (IKK⑀wt), we used anti-flag monoclonal antibody (anti-FLAG M2; Sigma, St. Louis, MO).…”