A novel luminescence-based β-arrestin recruitment assay for unmodified receptors
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A novel luminescence-based β-arrestin recruitment assay for unmodified receptors. / Pedersen, Maria Hauge; Pham, Jennifer; Mancebo, Helena; Inoue, Asuka; Asher, Wesley B.; Javitch, Jonathan A.
In: Journal of Biological Chemistry, Vol. 296, 100503, 2021.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - A novel luminescence-based β-arrestin recruitment assay for unmodified receptors
AU - Pedersen, Maria Hauge
AU - Pham, Jennifer
AU - Mancebo, Helena
AU - Inoue, Asuka
AU - Asher, Wesley B.
AU - Javitch, Jonathan A.
N1 - Publisher Copyright: © 2021 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
U2 - 10.1016/j.jbc.2021.100503
DO - 10.1016/j.jbc.2021.100503
M3 - Journal article
C2 - 33684444
AN - SCOPUS:85104669798
VL - 296
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
M1 - 100503
ER -
ID: 278487992