Off-target-free chemogenetic platform that decodes physiological roles of target GPCRs

G-protein-coupled receptors (GPCRs) are essential mediators of cellular processes, and their dysfunction is implicated in various diseases. Although pharmacological approaches are powerful for elucidating GPCR functions, careful consideration of potential off-target effects remains crucial. Here, we present an off-target-free chemogenetic platform to dissect the physiological roles of target GPCRs. As a proof-of-concept, we focused on the adenosine A _2A receptor (A _2A R), a prototypical GPCR involved in neurodegenerative diseases. Leveraging the ligand recognition mechanisms of A _2A R, we designed a prominent chemogenetic pair comprising a clinical A _2A R antagonist and an engineered A _2A R mutant that is insensitive to the antagonist but retains responsiveness to adenosine. Using this pair, we uncovered the critical role of A _2A R in neurite outgrowth under hypoxic conditions in neuron-like cells, while avoiding potential off-target effects. Furthermore, we demonstrate the generalizability of this chemogenetic approach to other class A GPCRs, offering a versatile platform for receptor-specific dissection of GPCR signaling.