Unveiling the complexity of G protein-coupled receptor heteromers: advances in live cell imaging technologies and biochemical methods

G protein-coupled receptors (GPCRs), crucial for diverse physiological responses, have traditionally been investigated in their monomeric form. However, some GPCRs can form heteromers, revealing complexity in their functional characteristics such as ligand binding properties, downstream signaling pathways, and trafficking. Understanding GPCR heteromers is crucial in both physiological contexts and drug development. Here, we review the methodologies for investigating physical interactions in GPCR heteromers, including co-immunoprecipitation, proximity ligation assays, interfering peptide approaches, and live cell imaging techniques based on resonance energy transfer and bimolecular fluorescence complementation. In addition, we discuss recent advances in live cell imaging techniques for exploring functional features of GPCR heteromers, for example, circularly permuted fluorescent protein-based GPCR biosensors, TRUPATH, and nanobody-based GPCR biosensors. These advanced biosensors and live cell imaging technologies promise a deeper understanding of GPCR heteromers, urging a reassessment of their physiological importance and pharmacological relevance.