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A novel luminescence-based β-arrestin recruitment assay for unmodified receptors
- Source :
- The Journal of Biological Chemistry, Pedersen, M H, Pham, J, Mancebo, H, Inoue, A, Asher, W B & Javitch, J A 2021, ' A novel luminescence-based β-arrestin recruitment assay for unmodified receptors ', Journal of Biological Chemistry, vol. 296, 100503 . https://doi.org/10.1016/j.jbc.2021.100503
- Publication Year :
- 2021
-
Abstract
- G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by β-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or β-arrestinsignaling pathways. However, nearly all screening techniques for measuring β-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure β-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to β-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, β-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of β-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring β-arrestin recruitment to diverse GPCR types in heterologous or native cells.
- Subjects :
- 0301 basic medicine
AT1R, angiotensin II type 1 receptor
genetic structures
KOR, κ-opioid receptor
Ligands
HTS, high-throughput screening
Biochemistry
Receptors, G-Protein-Coupled
Luciferases
Hek, human embryonic kidney
Cells, Cultured
beta-Arrestins
NanoLuc
DAMGO, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin
Chemistry
bioluminescence
Cell biology
Biological Assay
Signal transduction
signaling
Protein Binding
Signal Transduction
Research Article
signaling pathway
Cell signaling
Endosome
G protein
GRK, G protein-coupled receptor kinase
G protein-coupled receptor (GPCR)
DOR, δ-opioid receptor
CHO, Chinese hamster ovary
RLuc8, Renilla luciferase 8
03 medical and health sciences
Arrestin
Humans
cell signaling
NOP, nociceptin opioid receptor
BRET, bioluminescence resonance energy transfer
Molecular Biology
GPCR, G protein-coupled receptor
G protein-coupled receptor
G protein-coupled receptor kinase
Ang II, angiotensin II
030102 biochemistry & molecular biology
β-arrestin
Cell Membrane
D2R, dopamine D2 receptor
Cell Biology
MOR, μ-opioid receptor
Angiotensin II
030104 developmental biology
complementation-based assay
Subjects
Details
- ISSN :
- 1083351X
- Volume :
- 296
- Database :
- OpenAIRE
- Journal :
- The Journal of biological chemistry
- Accession number :
- edsair.doi.dedup.....953cfc6071b8c13a92b3add8ed756a95