Creating a Synthetic, Extracellular EGFR Circuit to Explore RTK Signaling

Abstract

EGF presents on the cell surface as a pro-ligand, which can be cleaved by proteases such as ADAM10/17. Free EGF ligands can then bind to the EGFR, which allows for EGFR dimerization that induces signal transduction and cellular responses in receiving cells. EGFR activation can be detected via an ERK-KTR biosensor that changes its localization in response to the activation of ERK a downstream EGFR signaling transduction. On a tissue scale, ERK activation has been observed in waves or pulses that can travel tens of micrometers per hour across a sheet of cells. A synthetic extracellular signaling circuit was designed to model a potential mechanism for how these dynamic ERK waves form. To do this, three key signaling components were designed – surface expressed TEVp, synthetic pro-ligand with two receptor binding sites, and an engineered EGFR-like receptor with a synthetic extracellular binding domain. Each component was cloned and expressed in HEK293T cells, where proper localization to the cell surface was verified via confocal microscopy. Surface expressed TEVp was shown to maintain its ability to cleave substrates using the eNRGies biosensor, which serves as a generalized indicator for extracellular shedding via translocation of a fluorescent protein. Additionally, synthetic receptors properly dimerize in response to the addition of purified ligand. Signal transduction in response to receptor dimerization was detected using the ERK-KTR. Ultimately, with the key components successfully constructed, their integration into a complete synthetic signaling circuit would establish a model for the analysis of ERK wave propagation.