Joined Signatures of Erythropoietin and Interleukin-6 Signaling by Synthetic Heterodimeric Gp130:Epo Receptor Complexes

Synthetic engineering has shown that cytokine receptors from different families can be functionally combined to a large extent. We and others have developed fully synthetic cytokine ligand-receptor systems. Here, our synthetic cytokine receptor (SyCyR) system has been used to generate highly effective non-natural cytokine receptor complexes for the interleukin (IL)-6-type signaling receptors gp130, LIFR, and the erythropoietin receptor (EpoR). Epo promotes the formation of red blood cells in the bone marrow, while IL-6 plays a role in the immune response and regulation of inflammation. Their synergistic effects in combination could contribute to effectiveness of anemia treatment in future in vivo studies. Activation of these SyCyRs is based on homo- and heterodimeric GFP-mCherry fusion proteins as synthetic cytokine ligands and VHH nanobodies (variable domain of single-domain antibodies) against GFP and mCherry fused to the transmembrane and intracellular domains of engineered cytokine receptors. Although synthetic cytokine signaling was achieved for most designs, gp130:LIFR and gp130:EpoR designs were more efficient compared to LIFR:EpoR designs. The gp130:EpoR signaling combines typical signaling signature of both pathways. Taken together, our data show that synthetic cytokine receptors for members of the IL-6 family and EpoR are generally feasible.