Type I Interferon Signal Transduction

grantor: University of TorontoThe Type I interferons (IFNs), including IFN-alpha, -beta and -omega (IFN-$\alpha/\beta/\omega),$ bind to a receptor consisting of the IFNAR1 and IFNAR2 transmembrane subunits and the intracellular kinases, Tyk2 and Jak1. Following ligand binding, the kinases activate the signal transducer and activator of transcription (STAT) proteins. STAT complexes formed in response to Type I IFNs regulate transcription by binding to enhancer elements in the promoters of interferon stimulated genes (ISGs). The interferon stimulated gene factor-3 (ISGF3), which consists of Stat2, Stat1 and an adapter protein, p48, binds to the interferon stimulated response element (ISRE). In addition, several other STAT complexes are formed that bind to the palindromic interferon response element (pIRE). The glycosphingolipids (GSLs) globotriaosylceramide (Gb$\sb3$) and galabiosylceramide (Gb$\sb2$) are receptors for the Escherichia coli derived subunit toxin, Verocytotoxin (VT-1). Interestingly, the Gb$\sb2$/Gb$\sb3$ binding subunit of VT-1 exhibits sequence identity with the extracellular region of IFNAR1. Consistent with earlier studies, evidence is provided that low levels of Gb$\sb2$/Gb$\sb3$ correlate with both a decreased responsiveness and binding capacity of cells for IFN-$\alpha.$ Using a fusion protein containing the extracellular domain of IFNAR1, preferential binding of IFNAR1 to Gb$\sb2$ was demonstrated. These results suggest that an association of IFNAR1 with Gb$\sb2$/Gb$\sb3$ facilitates receptor-mediated signaling. Exploiting the species specificity of the ligand-receptor interaction identified for Type I IFNs, experiments were conducted to elucidate the role of IFNAR1 in IFN-induced signaling. Murine cells expressing human IFNAR1 are deficient in the human Type I IFN response. However, murine cells containing human chromosome 21, encoding IFNAR1, are responsive to all human subtypes. The increased response may be explained in part by the chromosomal location of the second receptor chain, IFNAR2, on chromosome 21. Nevertheless, studies with human MRC-5 cells, expressing all known components of the Type I IFN signaling pathway, reveal that an additional factor(s) that modulates ligand binding, may be required for a full biological response. In an attempt to identify all DNA-binding STAT complexes formed in response to Type I IFNs, a biochemical procedure was developed, based on genomic DNA affinity chromatography (GDAC). Using this approach, an IFN-$\alpha$-inducible ISGF3-independent Stat2 containing STAT complex was identified. In parallel, coimmunoprecipitation studies revealed the IFN-dependent formation of both Stat2:1 and Stat2:3 complexes. The Stat2:1 complex exhibited pIRE binding activity in the mobility shift/Western assay. In studies that employed cyclic amplification and selection of targets (CASTing), the data indicate that Stat2:1 complexes bind with highest affinity to the consensus pIRE. Therefore, in addition to ISGF3, IFN-$\alpha$ activates multiple Stat2-containing STAT complexes that may regulate gene expression by binding to the pIRE.Ph.D