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.

Characterization of antihuman IFNAR-1 monoclonal antibodies: epitope localization and functional analysis.

The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.

Mapping human interferon-alpha (IFN-alpha 2) binding determinants of the type I interferon receptor subunit IFNAR-1 with human/bovine IFNAR-1 chimeras.

Type I interferons bind to a common receptor (IFNAR), composed of two transmembrane polypeptides, IFNAR-1 and IFNAR-2. Although human IFNAR-1 has a weak intrinsic affinity for human Type I interferons (IFNs), bovine IFNAR-1 binds human Type I IFNs with moderate (nM) affinity, and can be conveniently used to investigate the regions of IFNAR-1 involved in ligand binding. We have constructed 14 bovine/human IFNAR-1 chimeras by exchanging homologous subdomains in the extracellular portion of the receptor. These chimeras were expressed at very high levels on COS cells, and their ability to bind HuIFN-alpha2 was measured. No single bovine subdomain substituted into human IFNAR-1 could confer moderate-affinity ligand binding on the resulting chimera. Simultaneous substitution of bovine IFNAR-1 subdomains 2 and 3 for the homologous human subdomains resulted in a dramatic increase in the binding of IFN-alpha2, suggesting that critical determinants for moderate-affinity ligand binding by BoIFNAR-1 reside in these two subdomains. Bovine subdomains 1 and/or 4 each further enhanced IFN-alpha2 binding in the presence of bovine subdomains 2 and 3. Thus, the binding interactions of BoIFNAR-1 with IFNs appears to be more complex than that of other class II cytokine receptors with their ligands.

Contributions of cloned type I interferon receptor subunits to differential ligand binding.

The human type I interferons, including at least 12 IFN-alphas, IFN-beta and IFN-omega, bind to a receptor (IFNAR) composed of at least two transmembrane subunits, IFNAR-1 and IFNAR-2. The contributions of the receptor subunits to ligand binding were investigated by measuring the binding properties of IFNAR-1 or IFNAR-2 alone, or when co-expressed. The affinity of IFNAR-2 for IFN-alpha2 was increased by the co-expression of IFNAR-1, which itself binds ligand very weakly. Most type I IFNs inhibited the binding of IFN-alpha2 to IFNAR-2 alone with IC50 values of 2-20 nM. For cells co-expressing IFNAR-1 and IFNAR-2, the IC50 values decreased 3-20-fold for various ligands, relative to their values on IFNAR-2 alone. Thus, while IFNAR-2 plays the major role in affinity determination and differential recognition of type I IFNs, IFNAR-1 modulates both the ligand affinity and selectivity of the IFNAR-1/IFNAR-2 receptor complex.