Part:BBa_K4803002

IFNGR1 extracellular domain

Usage and Biology

Interferon gamma receptor 1 (IFNGR1) also known as CD119 (Cluster of Differentiation 119) is a type of cytokine receptor that functions in mammalian cells. In vivo, this protein binds to interferon gamma (IFN-γ). Receptors for interferon gamma in humans include IFNGR1 and IFNGR2. In the presence of IFN-γ, IFNGR1 and IFN-γ bind first, and IFNGR2 binds to these complexes, resulting in signal transduction [1].

This part is codon-optimized for sequences outside the plasma membrane from the IFNGR1 cDNA [2] listed in GenBank.

The UTokyo2023 project used this receptor as part of the MESA system [3]. In MESA, this part works as the receptor domain for MESA system in which IFNGR1s form homodimers with each other in the presence of IFN-γ, which allows intracytoplasmic proteases to act and transmit signals.

Figure 1: Overview of MESA system

Characterization

Figure 2: Stereoscopically predicted IFNGR1 in AlphaFold2

Wet Experiment

We checked the responsiveness of the MESA system by varying the concentration of IFN-γ. HEK293A was co-transfected with the MESA Target Chain(BBa_K4803106), the MESA Protease Chain (BBa_K4803105) and the plasmid encoding coordinated expression of the green fluorescent protein under the TRE3G promoter along with tTA (BBa_K4803100). Three days after transfection, DW only, IFN-γ 1pM, 1nM, 10nM and 100nM were added and incubated at 37°C for 24 h. Fluorescence intensity was measured using BD FACS Melody. Results are as follows.

Analysis by flow cytometry(BD FACS Melody) was performed 24 h after IFN-γ addition.

Figure 3: Percentage of cells with fluorescence stronger than the maximum fluorescence intensity of non-transfected HEK293A (293a) for each IFN-γ concentration at 24 h after IFN-γ addition.

Figure 4: Mean fluorescence intensity for each IFN-γ concentration at 24 h after IFN-γ addition. The error bars indicate standard error.

Figure 5: Histogram of fluorescence intensity by IFN-γ concentration at 24 h after IFN-γ addition. Orange →NC, Blue →IFN-γ 0 nM, Red → IFN-γ 10 nM

Although tTA leaks to some extent when MESA parts are added, the amount of tTA that migrates into the nucleus increases with the addition of IFN-γ.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1] Schroder, K., Hertzog, P. J., Ravasi, T., & Hume, D. A. (2004). Interferon-γ: an overview of signals, mechanisms and functions. Journal of Leucocyte Biology, 75(2), 163-189. https://doi.org/10.1189/jlb.0603252

[2]https://www.ncbi.nlm.nih.gov/nuccore/NM_000416.3?report=genbank

[3] Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N. (2014). Modular extracellular sensor architecture for engineering mammalian cell-based devices. ACS synthetic biology, 3(12), 892-902. https://doi.org/10.1021/sb400128g