Receptor binding and G-protein-dependent assays. Schematic representation of receptor binding and major pathways activated by different G proteins. Red indicates the detection points of commonly used HTS assays: GTPγS binding, cAMP detection, IP₃/IP₁ detection, Ca²⁺ flux and reporter expression.

Transfluor^™ and receptor internalization assays. Schematic representation of Transfluor^™ β-arrestin recruitment assay multiplexed with a receptor internalization assay. The receptor is tagged at the C-terminus with RFP, and β-arrestin is tagged with GFP. In the resting state, the receptor is located on the cell membrane and β-arrestin is localized in the cytoplasm (Phase 0). A few seconds to several minutes after stimulation, the receptor is phosphorylated by GRK and β-arrestin is recruited to the cell membrane (Phase 1). β-arrestin binding leads to the internalization of the receptor-β-arrestin complex, initially forming internalization pits (Phase 2). For fast-recycling GPCRs, the receptor dissociates with β-arrestin and returns to the plasma membrane. For slow-recycling GPCRs, the receptor-β-arrestin complex traffics towards the endosome and forms large vesicles (Phase 3).

Non-imaging-based β-arrestin recruitment assays. (A) BRET assay. The GPCR is tagged with a RLuc, and β-arrestin is tagged with GFP, or vice versa. Upon β-arrestin recruitment, the two tags come into close proximity and the light emitted from the RLuc reaction excites the GFP, which then emits a detectable signal at a higher wavelength. (B) Tango^™ assay. β-arrestin is fused to a protease, while GPCR is extended at its C-terminus with a protease cleavage site followed by a transcription factor (TF). Upon β-arrestin recruitment, the TF fused to the receptor is cleaved and enters the nucleus to regulate the transcription of a reporter gene. (C) PathHunter^™ assay. β-arrestin is fused to a deletion mutant of β-galactosidase that is catalytically inactive, and GPCR is tagged with a small fragment derived from the deleted sequence of the enzyme (ProLink^™). Upon GPCR-β-arrestin interaction, the two parts of β-galactosidase are brought into close proximity, which results in cleavage of the substrate and generation of a chemiluminescent signal.

Principles of the biosensors for label-free, whole-cell detection. (A) Schematic representation of an electrical biosensor. Cells are cultured on the surface of arrayed gold microelectrodes. A low AC voltage at variable frequencies is applied to the cell layer and both the extracellular current and transcellular current are measured. (B) Schematic representation of an optical biosensor. Cells are cultured on the surface of the biosensor with an embedded grating structure. Only the mass redistribution within the bottom portion of cells is directly measured.

Receptor dimerization assays. (A) BRET dimerization assay. GPCRs are tagged with a RLuc donor and a GFP acceptor. Upon receptor dimerization, the two tags come into close proximity and energy transfer occurs. (B) Tag-lite^™ dimerization assay. GPCRs are tagged with either a SNAP- or CLIP-tag at the N-terminus, which can be subsequently labeled with their corresponding cell-impermeable substrates carrying appropriate TR-FRET-compatible fluorophores. (C) PathHunter^™heterodimerization assay. β-arrestin is fused to the larger portion of a β-galactosidase enzyme acceptor, and the ProLink^™ tag is attached to one of the GPCR targets. A second untagged GPCR can be introduced into the cells, and the transactivation effects of the untagged GPCR on the ProLink^™-tagged GPCR can be measured by the recruitment of β-arrestin to the tagged-GPCR using PathHunter detection reagents.