Chemical Biology Methods for Investigating G Protein-Coupled Receptor Signaling

G protein-coupled receptors (GPCRs) are targets for a quarter of prescription drugs. Despite recent progress in structural biology of GPCRs, only few key conformational states in the signal transduction process have been elucidated. Agonist ligands frequently display functional selectivity where activated receptors are biased to either G protein- or arrestin-mediated downstream signaling pathways. Selective manipulation of individual steps in the GPCR activation scheme requires precise information about the kinetics of ligand binding and the dynamics of downstream signaling. One approach is to obtain time-resolved information using receptors tagged with fluorescent or structural probes. Recent advances allow for site-specific introduction of genetically encoded unnatural amino acids into expressed GPCRs. We describe how bioorthogonal functional groups on GPCRs enable the mapping of receptor-ligand interactions and how bioorthogonal chemical reactions can be used to introduce fluorescent labels for single-molecule fluorescence applications to study the kinetics and conformational dynamics of GPCR signaling complexes (“signalosomes”).