Cytosolic β-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or β-arrestin recruitment makes β-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor β-arrestin2 recruitment to the human histamine H1 receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H1 receptor.
|Title of host publication||G Protein-Coupled Receptor Screening Assays|
|Subtitle of host publication||Methods and Protocols|
|Editors||Sofia Aires M. Martins, Duarte Miguel F. Prazeres|
|Publisher||Humana Press Inc|
|Number of pages||16|
|ISBN (Print)||9781071612200, 9781071612231|
|Publication status||Published - 2021|
|Name||Methods in Molecular Biology|
Bibliographical noteFunding Information:
The NanoBiT starter kit plasmids were kindly provided by Promega Corporation (Madison, Wisconsin, USA). The ?-arrestin2 Nano-BiT constructs were kindly provided by Dr. J.Y. Seong (Korea University, Seoul, Republic of Korea). The Venus-?-arrestin2-pcDNA3 construct was kindly provided by Dr. V.V. Gurevich (Van-derbilt University School of Medicine, Nashville, Tennessee, USA). X.M. is supported by a CSC Chinese scholarship grant (201703250074).
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- Bioluminescence resonance energy transfer (BRET)
- Enzyme-fragment complementation (EFC)
- Protein-protein interaction (PPI)