PT  - JOURNAL ARTICLE
AU  - Hailey I. Edelstein
AU  - Patrick S. Donahue
AU  - Joseph J. Muldoon
AU  - Anthony K. Kang
AU  - Taylor B. Dolberg
AU  - Lauren M. Battaglia
AU  - Everett R. Allchin
AU  - Mihe Hong
AU  - Joshua N. Leonard
TI  - Elucidation and refinement of synthetic receptor mechanisms
AID  - 10.1101/2020.04.16.045039
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.04.16.045039
4099  - http://biorxiv.org/content/early/2020/04/18/2020.04.16.045039.short
4100  - http://biorxiv.org/content/early/2020/04/18/2020.04.16.045039.full
AB  - Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors confer unique capabilities for detecting environmental ligands and transducing signals to control downstream gene expression events such as therapeutic protein production. For many applications, it would be useful to understand how receptor design choices impart desirable performance metrics and trade-offs. Towards this goal, we employed the existing modular extracellular sensor architecture (MESA) and systematically characterized biosensors with previously unexamined protein domain choices. A key finding that might extend to other receptor systems is that choice of transmembrane domain (TMD) is highly consequential. To provide mechanistic insights, we adopted and employed a FRET-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry, and we apply this understanding to rationally tune receptors. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.Competing Interest StatementJ.N.L. is an inventor on related intellectual property: United States Patent 9,732,392; WO2013022739.