Antibody-Based Fusion Proteins Allow Ca²⁺ Rewiring to Most Extracellular Ligands

The Ca²⁺ signaling toolkit is the set of proteins used by living systems to generate and respond to Ca²⁺ signals. The selective expression of these proteins in particular tissues, cell types and subcellular locations allows the Ca²⁺ signal to regulate a diverse set of cellular processes. Through synthetic biology, the Ca²⁺ signaling toolkit can be expanded beyond the natural repertoire to potentially allow a non-natural ligand to control downstream cellular processes. To realize this potential, we exploited the ability of an antibody to bind its antigen exclusively in combination with the ability of the cytoplasmic domain of vascular endothelial growth factor receptor 2 (VEGFR2) to generate a Ca²⁺ signal upon oligomerization. Using protein fusions between antibody variants (i.e., nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca²⁺ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumor necrosis factor alpha and soluble CD14. The Ca²⁺ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state, but instead only required an increase in the oligomeric state. The Ca²⁺ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca²⁺ indicator or with an engineered Ca²⁺ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.