Engineering Synthetic Proteins to Generate Ca²⁺ Signals in Mammalian Cells

The versatility of Ca²⁺ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca²⁺ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca²⁺ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca²⁺ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca²⁺). By coupling these chimeric proteins that generate Ca²⁺ signals with proteins that respond to Ca²⁺ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca²⁺. Thus, using this design strategy, it is possible to engineer proteins to generate a Ca²⁺ signal to rewire a wide range of extracellular stimuli to a wide range of Ca²⁺-activated processes.