Mapping Human Interferon-alpha (IFN-α2) Binding Determinants of the Type I Interferon Receptor Subunit IFNAR-1 with Human/Bovine IFNAR-1 Chimeras

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Abstract
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-α2 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-α2, 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-α2 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.