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* This minireview will be reprinted in the 2007 Minireview Compendium, which will be available in January, 2008. This work was supported by the NHMRC, Australia, and the Cooperative Research Centre for Chronic Inflammatory Diseases. This is the second article of four in the Interferon Minireview Series.
The abbreviations used are: IFN, interferon; IFNAR, type I IFN receptor; IFNGR, type II IFNγ receptor; hu, human; hCR, helical cytokine receptor; ECD, extracellular domain; FBN, fibronectin; TF, tissue factor; SD, subdomain; PDB, Protein Data Bank; IL, interleukin; aa, amino acid(s); s (sIL, sIFN), soluble; JAK, Janus kinase; STAT, signal transducers and activators of transcription.
2The abbreviations used are: IFN, interferon; IFNAR, type I IFN receptor; IFNGR, type II IFNγ receptor; hu, human; hCR, helical cytokine receptor; ECD, extracellular domain; FBN, fibronectin; TF, tissue factor; SD, subdomain; PDB, Protein Data Bank; IL, interleukin; aa, amino acid(s); s (sIL, sIFN), soluble; JAK, Janus kinase; STAT, signal transducers and activators of transcription.
receptor (IFNAR) is comprised, as other cytokine receptors, of multiple components, in this case designated IFNAR1 and IFNAR2. However it is unique among cytokine receptors in the number of cognate ligands, including 13 IFNα subtypes, β, ω, ∊, κ, and others in some species. The type I IFN receptors are distinct from those required for the type II IFNγ (IFNGR1 and IFNGR2) and type III IFNs (IFNLR and IL10Rβ). Nevertheless, genes encoding a component of each type of IFN receptor, namely IFNAR1, IFNAR2, IFNGR2, and IL10Rβ, are located on human chromosome 21q22.1 in a cytokine receptor gene cluster, as typical of functionally related genes.
Although IFNs were identified 50 years ago and the existence of IFN receptors 10 years later, it was in 1990 when the first type I IFN receptor, now designated IFNAR1, was cloned. This was achieved utilizing human gene libraries expressed in murine cells and rescue of the definitive, species specific antiviral activity of human IFNα8 (
). It was subsequently discovered that the original cDNA encoded only one isoform of the IFNAR2 gene, which also encoded a long transmembrane isoform that transduced a signal, a truncated transmembrane isoform, and a soluble/secreted isoform (
Subsequently, the functions of the type I IFN receptors have been elucidated with respect to ligand interaction, mechanisms of signal transduction, and biological responses. The pioneering studies that discovered IFNARs and their mechanisms of actions in vitro have been largely validated in vivo using genetargeted mice. This body of work has highlighted the important roles of IFNARs in mediating type I IFN responses in hemopoiesis and innate and acquired immunity to infection and cancer. However, IFNs elicit many biological effects that can even be opposite in different cell types. For example, type I IFN inhibits proliferation and is proapoptotic for many cell types (
) (Fig. 1A) These transcripts encode a long trans-membrane IFNAR2c, a short transmembrane IFNAR2b chain, and a soluble sIFNAR2a chain. Transfection of human IFNAR1 and IFNAR2c, but not IFNAR2b, reconstituted the antiviral IFN response (
The mouse has been the primary model for pathophysiological studies of IFNs due to the capability of generating knockout mice, which can demonstrate cause-and-effect associations in vivo. The mouse has a comparable type I IFN system to human with multiple ligands (α, β, ∊, etc.) and Ifnar1 and Ifnar2 genes (
). No IFNAR2b chain has been identified in mouse, but two transcripts capable of encoding soluble isoforms (sIfnar2a and sIfnar2a′) are generated by differential splicing. The more abundant 1.5-kb sIfnar2a transcript encodes the complete IFNAR2 extracellular domain and reads through the splice site on the exon 7–7′ boundary producing a transcript encoding 12 unique and mostly hydrophobic C-terminal residues (
). The sIfnar2a′ minor transcript is generated from a transcript missing 128 nucleotides after codon 236 of the Ifnar2 cDNA. This isoform originates by skipping the transmembrane-encoding exon 8, leading to a frameshift forming a stop codon that generates a transcript capable of producing a soluble receptor with 11 unique C-terminal amino acids (Fig. 1A).
The transmembrane and soluble Ifnar2 transcripts are differentially regulated based on Northern blot analyses of expression in murine tissues. Ratios of tmIfnar2c:sIfnar2a range from >10:1 in some tissues to ∼1:1 in hemopoietic tissues (
). Analysis of the 5′ flanking region of Ifnar2 using promoter reporter constructs identified three regulatory regions that confer basal expression, inducible expression by IFNα + IFNγ, and a negative regulatory region (
Structure-Function Relationships—Studies of type I IFN receptors prior to their cloning have indicated that most cell types bind IFNs, with large variation in the number of binding sites (200–10,000/cell) and binding affinities. Scatchard analyses of binding usually identify two types of binding sites of low (μm) and high affinity (nm–pm) (
). This pattern of binding is consistent with a multicomponent receptor containing a high affinity binding chain (often called the α or primary binding chain) and a β or signal-transducing chain that has low intrinsic ligand binding affinity and converts the affinity of interaction of ligand with α chain from moderate (nm) to high affinity (pm) (
). Recently, elegant studies using recombinant IFNAR extracellular domains (ECDs) tethered to lipid membranes have clearly demonstrated that various type I IFNs bind to IFNAR2 with Kd values mostly in the nm range (from 0.1 to 1000 nm) and bind to IFNAR1 with Kd mostly in the μm range (from 0.05 to 10 μm) (
). The ECD of huIFNAR1, comprising 409 aa (403 in the murine form) contains four subdomains, referred to as SD1–SD4, each housing one fibronectin (FBN-III-like) domain (Fig. 2). SD1 contains conserved residues implicated in binding membrane glycosphingolipids (
) and on information regarding the locations of amino acids implicated in ligand binding on both IFNAR1 and IFNAR2, a three-dimensional model for the ligand-bound human type I IFN receptor complex has been proposed (
) (Fig. 2), albeit the latter may be inaccurate because of steric hindrances. Residues involved in ligand binding are found on the three membrane-distal FBN-III SD domains. Residues 69VY70 have been identified as the key residues in IFNAR1 recognition by a neutralizing monoclonal antibody (
) (Fig. 2). Forming the core of the IFNAR2 ligand binding domain are three highly conserved residues Thr44, Met46, and Lys48, whereas residues, His76, Glu77, Tyr81, Trp100, Ile103, and Asp106 also facilitate IFNα2 binding (
), but there is no definitive in vivo evidence for this effect. Nevertheless, there is a precedent in the IL6 receptor system where the soluble IL6Rα is generated both by alternative splicing and by cleavage by ADAM 10 and 17 proteases (
). Soluble IFNAR2a can inhibit IFN signaling in normal cells, whereas in primary thymocytes from Ifnar2–/– mice, sIFNAR2a can bind IFNα or -β and generate an antiproliferative signal (Fig. 1B). In another study, ovine soluble IFNAR2 was able to mediate antiviral activity in vitro (
). Transgenic mice over expressing the soluble receptor are more susceptible to LPS-induced, IFNβ-mediated septic shock, suggesting that high levels of the sIFNAR2a receptor can potentiate IFN signaling in vivo.
Signal Transduction Domains and Pathways—The type I IFN receptor, typical of class II hCR, lack intrinsic kinase activity and thus rely on associated Janus kinases (JAKs) to phosphorylate receptors and signal transducing molecules, such as STAT proteins, after ligand-induced receptor clustering. IFNAR1 is preassociated with Tyk2 (
). These data suggest that the intracellular domains and signal-transducing molecules such as STATs may form multimolecular signal transduction complexes in which each molecule has multiple interactions (
Negative Regulation of Signal Transduction—The diversity of signals generated through IFNARs can protect the host against infection and cancer and mount controlled immune responses, whereas excessive or deregulated signaling can lead to toxicity, leucopenia, autoimmunity, and even death. Thus, IFNARs also interact with a number of negative regulatory molecules, including SOCS-1 (suppressor of cytokine signaling), UBP43, and SHP (
Trans-signaling—Soluble cytokine receptors such as sIL6R, sCNTFR, sIL11R, and sIL15R can mediate cytokine biological effects by a mechanism known as trans-signaling (Fig. 1B). Trans-signaling occurs when the soluble receptor bound ligand interacts with a complementary transmembrane receptor chain of the receptor complex (
). In the case of IL6Rα and the signaling gp130 chain, this is the major mechanism of IL6 signaling because gp130 is expressed on most cells, but the transmembrane form of IL6Rα shows restricted expression (
). Furthermore, our experiments using mice overexpressing sIFNAR2a suggest that high levels of sIFNAR2a may act as trans-signaling molecules in vivo.4 Although more direct evidence of trans-signaling in the IFN system is required, it provides a compelling mechanism for transducing alternative signals.
In Vivo Function
Expression Patterns—All tissues and organs and most cell lines express transcripts for IFNAR1 and both soluble and transmembrane isoforms of IFNAR2 (
) and thus bind and respond to IFNs. Microarray analysis of Ifnar expression in different tissues and cells indicate that there is some differential expression of IFNAR1 and IFNAR2, with IFNAR1 being more widespread and IFNAR2 more restricted. These studies need to be validated by other techniques, with reagents that enable the measurement of IFNAR protein levels, particularly at the cell surface, to allow a clearer picture of the composition of the IFNAR receptor on different cell types during homeostasis and disease.
Knock-out Studies—Mice with null mutations in Ifnar1 have demonstrated that this component is essential for responses to multiple IFNα as well as IFNβ (
). Extensive use of these mice has demonstrated the necessity of Ifnar1 for survival against most viral infections, myelopoiesis, and B and T cell-mediated immune responses and as a potent proinflammatory cytokine.
We have also generated mice with a null mutation in the Ifnar2 gene that had a phenotype similar to Ifnar1 null mice in their susceptibility to viral and bacterial infections but were distinct in their abnormal thymic T cell development.
In Down syndrome, where HSA 21 containing the IFNAR1 and IFNAR2 genes is trisomic, cells are more sensitive to IFNα treatment; the aberrant immune response in this condition has been associated with aberrant IFN signaling (
). Interestingly, other studies have not found a correlation between increased sIFNAR2a serum levels and lack of response to IFN therapy. Although the importance of soluble IFNAR2 in human disease will be clearer once it has been ascertained whether it participates mostly in agonist or antagonist activities, the above studies support the former.
Not only do host cells produce IFNAR proteins during viral infections, but certain viruses have evolved a form of soluble type I IFN receptor as a means of evading the immune response. Poxvirus encode a soluble IFN receptor homologue that neutralizes all type I IFNs tested, which is essential for virulence (
). This is an unusual protein in that it has low amino acid homology to IFNAR1 or IFNAR2 but has a tertiary structural similarity, based on modeling studies, and is a potent inhibitor of the antiviral activity of a broad range of type I IFNs without the species specificity of mammalian IFN-IFNAR interactions.
Summary and Perspective
The IFNAR complex is novel among cytokine receptors in mediating signaling by more than 15 different but related type I IFN ligands. This system has been instrumental in the discovery of the JAK/STAT signaling pathway, which is necessary for regulating genes involved in the characteristic antiviral response. However emerging data indicate that many more so-called alternative pathways are activated by IFNAR activation. This diversity of signals may explain how IFNs generate complex biological responses. There has been considerable advance in understanding the structure of the receptor signaling complex. However, more detailed structural studies and confirmation of the potential difference in receptor configurations, like that which would elicit trans-signaling, are necessary to elucidate how the different biological activities of type I IFNs can be regulated by the receptor.