Resolving Signal Transduction in Complex Biological Environments

Abstract

Cell surface receptors reside in complex biological environments along with their numerous extracellular ligands to transduce information across the membrane bilayer. These receptors regulate major functions including cell proliferation, facilitation of neuronal transmission, cell growth and metabolism, but their aberrant activity leads to debilitating disorders such as cancer, diabetes and paralysis. While signal transduction has been understood at a great detail for ion channels and G-protein coupled receptors (GPCRs), transmembrane signaling in enzyme-linked receptors have so far remained elusive due to the presence of only a single transmembrane helix in their structure for signal propagation. In this work, we explore the signal transduction in the epidermal growth factor receptor (EGFR), the most prominent member of the enzyme-linked category of cell surface receptors. By isolating full-length receptors using nanodiscs produced with cell-free expression, we discovered ligand-induced conformational coupling between the EGFR extracellular and intracellular domain using single-molecule Förster Resonance Energy Transfer measurements. Furthermore, we disentangle the role of the complex environment around EGFR in its transmembrane signaling by (1) uncovering the specific task of the active components of the plasma membrane; (2) ascertaining ligand-specific EGFR conformations which determine its downstream signaling and cellular process; and (3) illustrating the effect of EGFR phosphorylation in mediating interaction with intracellular signaling proteins.