Unraveling allosteric signaling of G protein-coupled receptors (GPCRs) by single-molecule fluorescence.

G protein-coupled receptors (GPCRs) are the largest and most functionally diverse family of membrane receptors in eukaryotes. They play central roles in numerous physiological processes and are implicated in a wide range of diseases, making them prime targets for therapeutic intervention. Allostery is central to GPCR function, enabling the transmission of extracellular signals across the membrane into intracellular responses. Specifically, three key allosteric phenomena-ligand efficacy, biased signaling, and allosteric modulation-are fundamental to GPCR signaling and have been explored through various approaches. In this review, we summarize how single-molecule fluorescence techniques, particularly single-molecule Förster resonance energy transfer (smFRET) and single-molecule photoisomerization-related/protein-induced fluorescence enhancement (smPIFE), have deepened our understanding of these allosteric processes. We discuss existing gaps in our understanding of GPCR allostery and how these techniques could be leveraged to address these challenges, driving the development and design of more effective and selective therapeutics.