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 utilizing the same JAK-STAT signaling cascade, type I and III IFN receptors produce different magnitudes and dynamics in the phosphorylation of signal transducers and activators of transcription (STATs), gene transcription and antiviral responses. Prior studies have tested the role of ligand binding affinity and receptor expression level on IFN receptor signaling strength; however, the ability of intracellular domains to differentiate type I and III IFN signaling has not been previously examined. Therefore, we engineered synthetic, heterodimeric type I and III IFN receptors that can be stably expressed at similar levels in human cells and respond to the same ligand. Even when controlling for ligand binding and receptor expression, we find that our synthetic type I receptors produce a greater magnitude of STAT phosphorylation and gene expression than the corresponding type III receptors. Furthermore, our results show that short <55 amino acid “box motifs” known to bind JAK1 are sufficient to encode differences between type I and III receptors. Altogether, our work indicates that specific regions within the intracellular domains of IFNAR2 and IFNLR1 encode different downstream signaling strengths that allow type I and III receptors to produce distinct signaling outcomes. Comparative gene expression profiling analysis of RNA-seq data for U2OS cells expressing chimeric EPOR-interferon receptors, treated with EPO or IFNs, at multiple time points.