Artificial signaling in mammalian cells enabled by prokaryotic two-component system

Augmenting live cells with new signal transduction capabilities is a key objective in genetic engineering and synthetic biology. We showed earlier that two-component signaling pathways could function in mammalian cells, albeit while losing their ligand sensitivity. Here, we show how to transduce small-molecule ligands in a dose-dependent fashion into gene expression in mammalian cells using two-component signaling machinery. First, we engineer mutually complementing truncated mutants of a histidine kinase unable to dimerize and phosphorylate the response regulator. Next, we fuse these mutants to protein domains capable of ligand-induced dimerization, which restores the phosphoryl transfer in a ligand-dependent manner. Cytoplasmic ligands are transduced by facilitating mutant dimerization in the cytoplasm, while extracellular ligands trigger dimerization at the inner side of a plasma membrane. These findings point to the potential of two-component regulatory systems as enabling tools for orthogonal signaling pathways in mammalian cells. Bacterial two-component signaling machinery has been reprogrammed for orthogonal signaling in mammalian cells that is triggered by small-molecule-mediated dimerization or ligand-induced GPCR/beta-arrestin signaling.