C-terminal tagging enhances the detection sensitivity of interlekin receptor type 1.

Abstract

Substances released outside of the cells during cell necrosis are collectively called danger-associated molecular patterns (DAMPS) or alarmins. A pro-inflammatory cytokine, interleukin-1α (IL-1α) is known as a typical alarmin. IL-1α transmits signals by binding to IL-1 receptor 1 (IL-1R1), type I protein, expressed on the cell membrane of target cells, but detection of IL-1R1 at the protein and mRNA levels is difficult. Although the reasons are not elucidated, we attempted to add the HiBiT-tag to the N-terminus (N'-R1) or C-terminus (C'-R1) of IL-1R1 to examine whether the detection sensitivity can be augmented. Increase in detection sensitivity will allow the investigation of its function and subcellular localization much further. Using uterine cervical cancer-derived HeLa cells and its derivative CR-R1-4 cells lacking IL-1R1, C'-R1 was demonstrated to significantly increase the detection sensitivity of IL-1R1. Furthermore, the signal transduction function of neither N'-R1 nor C'-R1 was affected. Immunofluorescence cell staining revealed that wild-type IL-1R1 is mainly localized in the nucleus, whereas C'-R1 is localized both in the nucleus and the cytoplasm. The above results showed that adding a tag to the C-terminus of IL-1R1 increases detection sensitivity while maintaining its function. In the future, we would like to further investigate the relationship between changes in the intracellular localization of C'-R1 and increases in detection sensitivity.