Image-Based cAMP Sensor Assay for Analyzing Odorant Receptor Activity

Odorant receptors (ORs), the largest subfamily of G-protein-coupled receptors (GPCRs), are transmembrane proteins essential for olfactory perception. Recent research has revealed their expression in nonolfactory tissues, highlighting their potential as novel drug targets. However, the functional analysis of ORs beyond nasal tissues is constrained by limited availability of suitable ligands. Conventional in vitro methods for studying OR–ligand interactions via cAMP often face challenges in large-scale applications. This protocol introduces an advanced cAMP assay employing the Pink Flamindo sensor, a cAMP fluorescence sensor that normally does not exhibit fluorescence but shows fluorescence when combined with cAMP. This enables the real-time visualization of OR–ligand interactions through an imaging-based method. This technique significantly aids in the deorphanization of ORs.