| Project Detail |
G protein-coupled receptors (GPCRs) are key to human physiology and disease, yet their signaling remains enigmatic. What we do know is that GPCRs signal by means of allosteric communication across the cell membrane, acting as logic gates that relay signals from extracellular ligands to various intracellular transducers. Nonetheless, the logic underlying GPCR signaling at the molecular level is not yet understood. This is particularly true of three key signaling phenomena: ligand efficacy, biased signaling, and allosteric modulation. In SignAlloMod, we will address this challenge and investigate the logic of transmembrane signaling through GPCRs at the single-molecule level. We will develop smMDSFRET, a ground-breaking method that integrates single-molecule microfluidic diffusional sizing (smMDS) with single-molecule Frster resonance energy transfer (smFRET). By simultaneously measuring binding (smMDS) and conformations (smFRET), this method will make rigorous, quantitative models of GPCR allostery experimentally accessible. This will allow us to separate, for the first time, ligand-specific from transducer-specific effects in GPCR signaling, thereby decoding the molecular logic of GPCR signaling. We will use the adenosine A2A receptor as a well-studied model GPCR to establish our approach. We will then study ligand efficacy through the 2 adrenergic receptor, explore biased signaling through the angiotensin II type 1 receptor, and examine allosteric modulation of all three receptors by ions, small molecules, and lipids. The quantitative insights into ligand-specific and transducer-specific effects afforded by SignAlloMod will redefine our understanding of GPCR signaling. SignAlloMod will provide pharmacological ligand profiles with unprecedented detail, opening new horizons for drug discovery and pharmacological intervention guided by physical principles. |
