Part:BBa_K5466007

EpoR-D2-TM

D2 and transmembrane domain from mammalian erythropoietin receptor (residues 118-272).

The scaffold of the GEMS sensing platform. Also, for yeasts the scaffold of a receptor inspired in GEMS platform: Patrol Yeast, using a split-ubiquitin yeast two hybrid as signaling module.

In the N-terminal there is a short flexible peptide (SGEF), allowing movement on the ligand binding domain (LBD).

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]

EpoR

Cytokine receptors possess a modular structure that allows the fusion of intracellular and extracellular domains from different receptors, enabling the formation of functional chimeras. In the case of inactive EpoR dimers, transmembrane helix interactions keep them in a conformation that blocks downstream signaling. Upon ligand binding, it is believed that each receptor subunit undergoes rotation around its axis, likely increasing the distance between the intracellular domains. These combined structural changes activate downstream signaling pathways.

The receptor no longer responds to erythropoietin because it only has the transmembrane domain and D2. To construct GEMS, they fused the EpoR transmembrane domain to affinity domains, such as antibody fragments that dimerize in the presence of target molecules, a linker region between antibody and EpoR was added with the objective to provide rotational freedom to facilitate antibody binding. This modified receptor was also fused to various intracellular signal transduction domains.

Fuse with any intracellular domain described in Scheller et al. (2018), and with a LBD domain (that dimerize in the presence of the ligand or two different LBD that dimerize in the presence of the same ligand in different EpoR) of interest to construct a GEMS receptor in mammalian cells.

Since the transmembrane (TM) signaling capability of sensor proteins is crucial for detecting molecules outside of cells, Su et al., (2022) utilized the EpoR D2 and TM regions in Yeast Patrol to link the LBD and split ubiquitin within the cells. So, to build a receptor in yeast, you can fuse one with NubG (BBa_K5466008), another one with Cub (BBa_K5466009) and a LBD (with the same criteria mentioned above) to construct a receptor inspired in GEMS platform, using as signaling module a yeast two hybrid, based in split-ubiquitin system.

You can fuse it with a golden gate assembly without scars and in frame.

References

Scheller, L., Strittmatter, T., Fuchs, D., Bojar, D., & Fussenegger, M. (2018). Generalized extracellular molecule sensor platform for programming cellular behavior. Nature Chemical Biology, 14(7), 723-729. https://doi.org/10.1038/s41589-018-0046-z

Su, J., Zhu, B., Inoue, A., Oyama, H., Morita, I., Dong, J., Yasuda, T., Sugita-Konishi, Y., Kitaguchi, T., Kobayashi, N., Miyake, S., & Ueda, H. (2022). The Patrol Yeast: A new biosensor armed with antibody-receptor chimera detecting a range of toxic substances associated with food poisoning. Biosensors And Bioelectronics, 219, 114793. https://doi.org/10.1016/j.bios.2022.114793