Membrane-proximal motifs are the primary determinants of signaling strength differences between type I and III interferon receptors

Interferons (IFNs) play crucial roles in antiviral defenses. Despite using the same Janus-activated kinase (JAK)–signal transducer and activator of transcription (STAT) signaling cascade, type I and III IFN receptors differ in the magnitude and dynamics of their signaling in terms of STAT phosphorylation, gene transcription, and antiviral responses. These differences are not due to ligand-binding affinity and receptor abundance. Here, we investigated the ability of the intracellular domains (ICDs) of IFN receptors to differentiate between type I and III IFN signaling. We engineered synthetic, heterodimeric type I and III IFN receptors that were stably expressed at similar amounts in human cells and responded to a common ligand. We found that our synthetic type I IFN receptors stimulated STAT phosphorylation and gene expression to greater extents than did the corresponding type III IFN receptors. Furthermore, we identified short, “box motifs” within ICDs that bind to JAK1 that were sufficient to encode differences between the type I and III IFN receptors. Together, our results indicate that specific regions within the ICDs of IFN receptor subunits encode different downstream signaling strengths that enable type I and III IFN receptors to produce distinct signaling outcomes.