Fig. 7. The IFNAR2 box motif region confers type I signaling strength to the chimeric type III IFN receptor.
(A) The “IFNAR2BOX1/2” triple chimera was generated by fusing the EPOR ECD (containing the mutations H114K, E117K, and M150E) to the TMD-ICD of IFNLR1, where amino acid residues 264 to 318 were replaced with residues 277 to 314 of IFNAR2. (B) The “IFNLR1BOX1/2” triple chimera was generated by fusing the EPOR ECD (containing the mutations H114K, E117K, and M150E) to the TMD-ICD of IFNAR2, where amino acid residues 277 to 314 were replaced with residues 264 to 318 of IFNRL1. All of the chimeras were expressed under an SFFV promoter and contained an IRES for concurrent expression of YFP. (C) Left: U2OS cells were transduced to express equivalent amounts of the canonical IFN receptor chimeras (type I or III) or “BOX1/2” chimeras paired with either EPORM150E-IFNAR1 or EPORM150E-IL10RB (each with the mutation M150E in EPOR). Cells were treated with EPO (100 ng/ml) for 30 min before being analyzed by Western blotting for phosphorylated (p) or total (t) STAT1. β-actin was used as a loading control. Western blots are representative of three independent experiments. Right: The relative abundances of pSTAT1 to tSTAT1 in the indicated samples from three independent experiments were determined by densitometry analysis and are expressed as a percentage of the pSTAT1 abundance in EPO-treated “type I” cells, which was set at 100%. (D) The “IFNAR2TMD/BOX1” triple chimera was generated by fusing the EPOR ECD (containing the mutations H114K, E117K, and M150E) to amino acid residues 243 to 295 of IFNAR2 and 282 to 520 of IFNLR1. (E) The “IFNLR1TMD/BOX1” triple chimera was generated by fusing the EPOR ECD (containing the mutations H114K, E117K, and M150E) to amino acid residues 229 to 281 of IFNLR1 and 296 to 515 of IFNAR2. All of the IFN receptor chimeras were expressed under an SFFV promoter and contained an IRES for concurrent expression of YFP. (F) Left: U2OS cells were transduced to express equivalent amounts of the canonical IFN receptor chimeras (type I or III) or the “TMD/BOX1” chimeras paired with either EPORM150E-IFNAR1 or EPOM150E-IL10RB. Cells were treated with EPO (100 ng/ml) for 30 min before being analyzed by Western blotting for pSTAT1 and tSTAT1. β-actin was used as a loading control. Western blots are representative of three independent experiments. Right: The relative abundances of pSTAT1 to tSTAT1 in the indicated samples from three independent experiments were determined by densitometry analysis and are expressed as a percentage of the pSTAT1 abundance in EPO-treated “type I” cells, which was set at 100%. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.