The Ligand Binding Site on IFNAR1

IFNAR1 is a member of the cytokine receptor superfamily, sharing conserved structural FNIII building blocks that form the extracellular ligand-binding domain. In IFNGR1, IFNGR2, growth hormone receptor, tissue factor, and IFNAR2, there are two FNIII domains, each containing 100 amino acids with seven P-strands and connecting loops. The extracellular domain of IFNAR1 is atypical, consisting of a tandem array of four FNIII domains, here denoted subdomains 1 through 4 (SD1-4; beginning from the N terminus) (Kotenko and Langer 2004; Mogensen et al. 1999; Pestka 1997). The four-domain structure of IFNAR1 appears to represent a tandem duplication of the more common two-domain structure.

The low intrinsic affinity of huIFNAR1 for IFNs has previously hampered studies seeking to identify residues involved in ligand binding and specificity. The bovine IFNAR1 (bo-IFNAR1) homolog was found as an attractive target for mutagenesis and analysis of the IFN binding site, as human IFNs display uniformly high binding and biological activity on bovine cells. This reflects the ability of bo-IFNAR1 to bind human type I IFNs with moderately high affinity (Cutrone and Langer 1997). In vitro studies have shown that the three N-terminal FNIII domains of the ectodomain of IFNAR1 (IFNAR1-EC) are required for ligand recognition, which is very atypical for cytokine receptors (Lamken et al. 2005). Recent studies by Piehler et al. have indicated substantial conformational changes of IFNAR1-EC upon ligand binding, which are propagated to the membrane-proximal FNIII domain. Strikingly, the membrane-proximal domain of IFNAR1 is not involved in ligand binding, but is absolutely critical for formation of a functional signaling complex (Lamken et al. 2005). Modeling the mutagenesis results from bo-IFNARl on the human homolog suggested that four aromatic residues located on SD2 and SD3 (W129, F136, Y157, and W253) constitute an important part of the IFN binding epitope (Cutrone and Langer 2001). On the other hand, residues 62-70, which are recognized by the 64G12 monoclonal Ab may participate in binding, but are not crucial (Cutrone and Langer 2001). However, a report published in 2004 claimed that these residues are essential for IFN binding; therefore this issue will await further investigations (Cajean-Feroldi et al. 2004). The importance of both SD2 and 3 was corroborated by fragment studies of IFNAR1, showing that subunits SD1-2 and SD3-4 did not bind IFN (Lamken et al. 2005). Fragment SD1-2-3 retained almost normal binding affinity, while SD2-3-4 had no binding activity. Binding competition experiments have shown that IFNa and P bind the same epitope on IFNAR1. The comparison of the energetics of the mutual binding epitopes on IFNa 2 and IFNAR1 is somewhat perplexing. As mentioned, no hotspots for IFNAR1 binding were found on IFNa 2, contrary to a number of hotspot mutations found on IFNAR1 (particularly W129, F136, Y157, and W253). Additional structural knowledge is needed to explain this discrepancy.

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