Identification of residues of the IFNAR1 chain of the type I human interferon receptor critical for ligand binding and biological activity

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.     

Identification of critical residues in bovine IFNAR-1 responsible for interferon binding

Interferons have antiviral, antigrowth and immunomodulatory effects. The human type I interferons, IFN-alpha, IFN-beta, and IFN-omega, induce somewhat different cellular effects but act through a common receptor complex, IFNAR, composed of subunits IFNAR-1 and IFNAR-2. Human IFNAR-2 binds all type I IFNs but with lower affinity and different specificity than the IFNAR complex. Human IFNAR-1 has low intrinsic binding of human IFNs but strongly affects the affinity and differential ligand specificity of the IFNAR complex. Understanding IFNAR-1 interactions with the interferons is critical to elucidating the differential ligand specificity and activation by type I IFNs. However, studies of ligand interactions with human IFNAR-1 are compromised by its low affinity. The homologous bovine IFNAR-1 serendipitously binds human IFN-alphas with nanomolar affinity. Exploiting its strong binding of human IFN-alpha2, we have identified residues important for ligand binding. Mutagenesis of any of five aromatic residues of bovine IFNAR-1 caused strong decreases in ligand binding, whereas mutagenesis of proximal neutral or charged residues had smaller effects. These residues were mapped onto a homology model of IFNAR-1 to identify the ligand-binding face of IFNAR-1, which is consistent with previous structure/function studies of human IFNAR-1. The topology of IFNAR-1/IFN interactions appears novel when compared with previously studied cytokine receptors.     

Ligand-induced association of the type I interferon receptor components.

Two transmembrane polypeptides, IFNAR and IFN-alpha/Beta R, were previously identified as essential components of the type I interferon (IFN) receptor, but their interrelationship and role in ligand binding were not clear. To study these issues, we stably expressed and characterized the two polypeptides in host murine cells. In human cells, native IFN-alpha/beta R is a 102-kDa protein but upon reduction only a 51-kDa protein is detected. In host murine cells human IFN-alpha/beta R was expressed as a 51-kDa protein. Host cells expressing IFN-alpha/beta R bound IFN-alpha 2 with a high affinity (Kd of 3.6 nM), whereas cells expressing IFNAR exhibited no ligand binding. Upon coexpression of IFNAR and the 51-kDa IFN-alpha/beta R, the affinity for IFN-alpha 2 was increased 10-fold, approaching that of the native receptor. We show by cross-linking that both the cloned (51-kDa) and native (102-kDa) IFN-alpha/beta R bind IFN-alpha 2 to form an intermediate product, while IFNAR associates with this product to form a ternary complex. Hence, IFNAR and IFN-alpha/beta R are components of a common type I IFN receptor, cooperating in ligand binding. Ligand-induced association of IFNAR and IFN-alpha/beta R probably triggers transmembrane signaling.     

Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer

The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.     

The human interferon receptor: NMR-based modeling, mapping of the IFN-alpha 2 binding site, and observed ligand-induced tightening

The human interferon receptor (IFNAR) mediates the antiviral and antiproliferative activities of type I interferons (IFNs). This receptor is comprised of subunits IFNAR1 and IFNAR2, the latter exhibiting nanomolar affinity for IFNs. Here the extracellular domain of IFNAR2 (IFNAR2-EC), a soluble 25 kDa IFN-binding polypeptide, and its complex with IFN-alpha 2 were studied using multidimensional NMR. IFNAR2-EC is comprised of two fibronectin-III (FN-III) domains connected by a helical hinge region. The deduced global fold was utilized to improve the alignment of IFNAR2-EC against structurally related receptors and to model its structure. A striking feature of IFNAR2-EC is the limited and localized deviations in chemical shifts exhibited upon ligand binding, observed for only 15% of its backbone (1)H and (15)N nuclei. Analysis of these deviations maps the IFN-alpha 2 binding site upon IFNAR2-EC to a contiguous surface on the N-terminal domain, including the S3-S4 loop (residues 44-53), the S5-S6 loop and S6 beta-strand (residues 74-82), and the S7 beta-strand and the hinge region (residues 95-105). The C-terminal domain contributes only marginally to ligand binding, and no change in the hypothesized interdomain interface is observed. The proposed binding domain encompasses all residues implicated by mutagenesis studies in IFN binding, and suggests adjacent residues cooperate in forming the binding surface. D(2)O-exchange experiments indicate that binding of IFN-alpha2 induces tightening of the N-terminal domain of IFNAR2-EC. This increase in receptor rigidity may play an important role in initiating the intracellular stage of the IFN signaling cascade.     

Effects of type I interferons on Friend retrovirus infection

The type I interferon (IFN) response plays an important role in the control of many viral infections. However, since there is no rodent animal model for human immunodeficiency virus, the antiviral effect of IFN-alpha and IFN-beta in retroviral infections is not well characterized. In the current study we have used the Friend virus (FV) model to determine the activity of type I interferons against a murine retrovirus. After FV infection of mice, IFN-alpha and IFN-beta could be measured between 12 and 48 h in the serum. The important role of type I IFN in the early immune defense against FV became evident when mice deficient in IFN type I receptor (IFNAR(-/-)) or IFN-beta (IFN-beta(-/-)) were infected. The levels of FV infection in plasma and in spleen were higher in both strains of knockout mice than in C57BL/6 wild-type mice. This difference was induced by an antiviral effect of IFN-alpha and IFN-beta and was most likely mediated by antiviral enzymes as well as by an effect of these IFNs on T-cell responses. Interestingly, the lack of IFNAR and IFN-beta enhanced viral loads during acute and chronic FV infection. Exogenous IFN-alpha could be used therapeutically to reduce FV replication during acute but not chronic infection. These findings indicate that type I IFN plays an important role in the immediate antiviral defense against Friend retrovirus infection.     

Formation of a uniquely stable type I interferon receptor complex by interferon beta is dependent upon particular interactions between interferon beta and its receptor and independent of tyrosine phosphorylation

Human type I interferons (IFN) require two receptor chains, IFNAR1 and IFNAR2c for high affinity (pM) binding and biological activity. Our previous studies have shown that the ligand dependent assembly of the type I IFN receptor chains is not identical for all type I IFNs. IFNbeta appears unique in its ability to assemble a stable complex of receptor chains, as demonstrated by the observation that IFNAR2c co-immunoprecipitates with IFNAR1 when cells are stimulated with IFNbeta but not with IFNalpha. The characteristics of such a receptor complex are not well defined nor is it understood if differential signaling events can be mediated by variations in receptor assembly. To further characterize the factors required for formation of such a stable receptor complex we demonstrate using IFN stimulated Daudi cells that (1) IFNAR2c co-immunoprecipitates with IFNAR1 even when tyrosine phosphorylation of receptor chains is blocked with staurosporine, and (2) IFNbeta1b but not IFNalpha2, is present in the immunoprecipitated receptor complex. These results demonstrate that the unique IFNbeta induced assembly of type I IFN receptor chains is independent of receptor tyrosine phosphorylation and the recruitment of additional proteins to the receptor by such events. Furthermore, the presence of IFNbeta1b in the immunoprecipitated IFN receptor complex suggests that IFNbeta interacts and binds differently to the receptor than IFNalpha2. These results suggest that the specific assembly of type I IFN receptor chains is ligand dependent and may represent an early event which leads to the differential biological responses observed among type I IFNs.     

Homology model of human interferon-alpha 8 and its receptor complex.

Human interferon-alpha 8 (HuIFN alpha 8), a type I interferon (IFN), is a cytokine belonging to the hematopoietic super-family that includes human growth hormone (HGH). Recent data identified two human type I IFN receptor components. One component (p40) was purified from human urine by its ability to bind to immobilized type I IFN. A second receptor component (IFNAR), consisting of two cytokine receptor-like domains (D200 and D200'), was identified by expression cloning. Murine cells transfected with a gene encoding this protein were able to produce an antiviral response to human IFN alpha 8. Both of these receptor proteins have been identified as members of the immunoglobulin superfamily of which HGH receptor is a member. The cytokine receptor-like structural motifs present in p40 and IFNAR were modeled based on the HGH receptor X-ray structure. Models of the complexes of HuIFN alpha 8 with the receptor subunits were built by superpositioning the conserved C alpha backbone of the HuIFN alpha 8 and receptor subunit models with HGH and its receptor complex. The HuIFN alpha 8 model was constructed from the C alpha coordinates of murine interferon-beta crystal structure. Electrostatic potentials and hydrophobic interactions appear to favor the model of HuIFN alpha 8 interacting with p40 at site 1 and the D200' domain of IFNAR at site 2 because there are regions of complementary electrostatic potential and hydrophobic interactions at both of the proposed binding interfaces. Some of the predicted receptor binding residues within HuIFN alpha 8 correspond to functionally important residues determined previously for human IFN alpha 1, IFN alpha 2, and IFN alpha 4 subtypes by site-directed mutagenesis studies. The models predict regions of interaction between HuIFN alpha 8 and each of the receptor proteins, and provide insights into interactions between other type I IFNs (IFN-alpha subtypes and IFN-beta) and their respective receptor components.     

Binding and activity of all human alpha interferon subtypes

Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5–5 μM and 0.4–5 nM respectively (except for IFN-alpha1 – 220 nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC₅₀ values differ significantly (1.5 nM versus 0.1 nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system.     

The stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities

Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNalpha2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC(50) values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNalpha2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.     

Kinetic analysis of cytokine‐mediated receptor assembly using engineered FC heterodimers

A method for analyzing ligand–receptor binding kinetics is described, which is based on an engineered FC domain (FChk) that forms a covalent heterodimer. To validate the system, the type I IFN receptors (IFNAR1 and IFNAR2) were expressed as IFNAR1‐FChk, IFNAR2‐FCkh, and IFNAR1/IFNAR2‐FChk fusion proteins. Surface plasmon resonance (SPR) analysis of binary IFNα2a/IFNAR interactions confirmed prior affinity measurements, while the affinity of the IFNα2a/IFNAR1/IFNAR2‐FChk interaction reproduced the affinity of IFNα2a binding to living cells. In cellular assays, IFNAR1/IFNAR2‐FChk potently neutralized IFNα2a bioactivity with an inhibitory concentration equivalent to the K_D measured by SPR. These studies suggest that FChk provides a simple reagent to evaluate the binding kinetics of multiple ligand–receptor signaling systems that control cell growth, development, and immunity.     

Receptor for activated C-kinase (RACK-1), a WD motif-containing protein, specifically associates with the human type I IFN receptor

The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-alphaRbetaL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-alphaRbetaL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-alphaRbetaL, the minimum site for RACK-1 binding was mapped to aa 300-346. RACK-1 binding to IFN-alphaRbetaL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2. The interaction between RACK-1 and IFN-alphaRbetaL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-alphaRalpha), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-alphaRbetaL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-beta1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-beta1b. However, stimulation of cells with either IFN-beta1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.     

Structural linkage between ligand discrimination and receptor activation by type I interferons

Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.     

Intracellular interferon triggers Jak/Stat signaling cascade and induces p53-dependent antiviral protection

Intracellular interferons (IFNs) exert biological functions similar to those of extracellular IFNs, but the signal transduction pathway triggered by the intracellular ligands has not been fully revealed. We investigated the signaling cascade by sequence-specific knockdown of signaling molecules by means of the RNA interference. Truncated IFN-beta gene was constructed so that the N-terminal secretory signal sequence was deleted (SD.IFN-beta). Cells transfected with this construct showed phosphorylation and activation of the STAT1 without any detectable secretion of the cytokine. The MHC class I expression was significantly augmented, while the augmentation was suppressed by short interfering RNA duplexes specific for JAK1, TYK2, and IFN-alpha/beta receptor (IFNAR) 1 and 2c chains. The SD.IFN-beta also induced p53 and phosphorylation of p53 at Ser(15). Specific silencing of p53 abrogated the antiviral effect of SD.IFN-beta, suggesting that the tumor suppressor is critically involved in antiviral defense mediated by intracellular IFN.     

Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-alpha, IFN-beta and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-lambda uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1(0/0)) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-lambda might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-lambda readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1(0/0) mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-lambda failed to induce Mx1 in the liver of IFNAR1(0/0) mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-lambda receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-alpha/beta and IFN-lambda were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1(0/0) mice. From these results we conclude that IFN-lambda contributes to inborn resistance against viral pathogens infecting the lung but not the liver.     

Determination of residues involved in ligand binding and signal transmission in the human IFN-alpha receptor 2.

The human IFN-alpha receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-alpha2/alpha1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-alpha2/alpha1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-alpha8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-alpha2, but remained responsive to hIFN-beta. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-alpha2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.