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Novel Bioluminescent Tools to Study GPCR Pharmacology in Living Cells
Author(s) -
Edenson Steven
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02628
Subject(s) - luciferase , g protein coupled receptor , internalization , bioluminescence , chemistry , fusion protein , receptor , multiplex , computational biology , microbiology and biotechnology , biology , biochemistry , bioinformatics , transfection , gene , recombinant dna
G‐protein coupled receptors (GPCRs) continue to be prominent targets for new therapeutics. To support these efforts, we have developed a suite of bioluminescent assays to study GPCR pharmacology in real time in living cells. Quantification of ligand binding and receptor internalization relies on N‐terminal fusion to High BiT (HiBiT), an 11 a.a. peptide. HiBiT binds with high affinity to Large BiT (LgBiT) to form NanoBiT Luciferase. For both applications, a non‐lytic detection reagent is added containing cell‐impermeable LgBiT and furimazine substrate. Ligand binding dynamics at the cell surface can be measured in real time through bioluminescent resonance energy transfer (BRET) between NanoBiT Luciferase and a fluorescent tracer. Receptor internalization and recycling are monitored in real time by measuring changes in the cell surface density of HiBiT‐tagged GPCRs. Notably, the bright signal of NanoBiT Luciferase allows both assays to be run at endogenous levels of expression using CRISPR/Cas9 to introduce the HiBiT tag. Similarly, fusion proteins to LgBiT and Small BiT (SmBiT), an 11 a.a. peptide with low affinity binding to LgBiT, can be used to measure β‐arrestin‐1/2 recruitment to activated GPCRs in real time. Furthermore, changes in intracellular cAMP concentration can be sensitively measured using endpoint (cAMP‐Glo) or real time formats (GloSensor cAMP) using thermal stable or circularly permuted forms of luciferase, respectively. These homogenous, cell‐based assays, which are readily adaptable to laboratory automation, should be immensely useful in the study of GPCR pharmacology. Support or Funding Information Promega Corporation