A novel luminescence-based β-arrestin recruitment assay for unmodified receptors

Pedersen, Maria; Pham, Jennifer; Mancebo, Helena; Inoue, Asuka; Asher, Wesley B.; Javitch, Jonathan A.

doi:10.1016/j.jbc.2021.100503

Cited by 13 publications

(8 citation statements)

References 34 publications

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“…Researchers were able to visualize the shifting of cytosolic β‐arrs to activated receptors at the cell surface using luminescent tags on β‐arrs through recently established technologies through confocal microscopic study. This intracellular visualization supports the previously demonstrated distinct patterns of β‐arr recruitment, which are found to be receptor type‐dependent (Pedersen et al, 2021). These are named Class A and Class B patterns.…”

Section: Introductionsupporting

confidence: 90%

Deciphering the signaling mechanisms of β‐arrestin1 and β‐arrestin2 in regulation of cancer cell cycle and metastasis

Aamna

Dan

Sahu

et al. 2022

Journal Cellular Physiology

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β‐Arrestins are ubiquitously expressed intracellular proteins with many functions which interact directly and indirectly with a wide number of cellular partners and mediate downstream signaling. Originally, β‐arrestins were identified for their contribution to GPCR desensitization to agonist‐mediated activation, followed by receptor endocytosis and ubiquitylation. However, current investigations have now recognized that in addition to GPCR arresting (hence the name arrestin). β‐Arrestins are adaptor proteins that control the recruitment, activation, and scaffolding of numerous cytoplasmic signaling complexes and assist in G‐protein receptor signaling, thus bringing them into close proximity. They have participated in various cellular processes such as cell proliferation, migration, apoptosis, and transcription via canonical and noncanonical pathways. Despite their significant recognition in several physiological processes, these activities are also involved in the onset and progression of various cancers. This review delivers a concise overview of the role of β‐arrestins with a primary emphasis on the signaling processes which underlie the mechanism of β‐arrestins in the onset of cancer. Understanding these processes has important implications for understanding the therapeutic intervention and treatment of cancer in the future.

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Section: Introductionsupporting

confidence: 90%

Deciphering the signaling mechanisms of β‐arrestin1 and β‐arrestin2 in regulation of cancer cell cycle and metastasis

Aamna

Dan

Sahu

et al. 2022

Journal Cellular Physiology

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“…To note, based on non-imaging-based β-arrestin recruitment assays, the degree of biased agonism of herkinorin has shown dependence on experimental conditions. The presence of fusion tags directly attached to the C terminus of GPCRs may explain the differences observed in these experiments [36,37]. For clarity, the systems proposed in this work as predominant are highlighted in italics throughout the text.…”

Section: Resultsmentioning

confidence: 99%

Bias-inducing allosteric binding site in mu-opioid receptor signaling

2021

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G-protein-biased agonism of the mu-opioid receptor (μ-OR) is emerging as a promising strategy in analgesia. A deep understanding of how biased agonists modulate and differentiate G-protein-coupled receptors (GPCR) signaling pathways and how this is transferred into the cell are open questions. Here, using extensive all-atom molecular dynamics simulations, we analyzed the binding recognition process and signaling effects of three prototype μ-OR agonists. Our suggested structural mechanism of biased signaling in μ-OR involves an allosteric sodium ion site, water networks, conformational rearrangements in conserved motifs and collective motions of loops and transmembrane helices. These analyses led us to highlight the relevance of a bias-inducing allosteric binding site in the understanding of μ-OR’s G-protein-biased signaling. These results also suggest a competitive equilibrium between the agonists and the allosteric sodium ion, where the bias-inducing allosteric binding site can be modulated by this ion or an agonist such as herkinorin. Notably, herkinorin arises as the archetype modulator of μ-OR and its interactive pattern could be used for screening efforts via protein–ligand interaction fingerprint (PLIF) studies. Article highlights Agonists and a sodium ion compete for the bias-inducing allosteric binding site that modulates signaling in mu-opioid receptors. Molecular dynamics simulations of the prototype μ-OR agonist suggest a competitive equilibrium involving the agonist and an allosteric sodium ion. Analysis of experimental data from the literature and molecular models provides the structural bases of biased agonism on μ-OR.

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“…To overcome these limitations, an alternative approach called indirect NanoBiT or membrane-tethered NanoBiT, has been developed (Hauge Pedersen et al, 2021;Spillmann et al, 2020). In the indirect NanoBiT assay, one of the NanoBiT fragments is separately anchored to the plasma membrane instead of being attached to a GPCR.…”

Section: Nanobit Assay To Measure Ligand-induced Recruitment Of Arres...mentioning

confidence: 99%

In‐Cell Arrestin‐Receptor Interaction Assays

Zheng,

Javitch,

Lambert

et al. 2023

Current Protocols

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G protein‐coupled receptors (GPCRs) represent ∼30% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which function in different signaling pathways. Given that functionally selective or biased ligands preferentially activate one of these two groups of pathways, they may be superior medications for certain disease states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for assays that monitor reversible arrestin recruitment to GPCRs in living cells using either bioluminescence resonance energy transfer (BRET) or nanoluciferase complementation (NanoLuc). Two types of assays can be used: one configuration directly measures arrestin recruitment to a GPCR fused to a protein tag at its intracellular C‐terminus, whereas the other configuration detects arrestin translocation to the plasma membrane in response to activation of an unmodified GPCR. Together, these assays are powerful tools for studying dynamic interactions between GPCRs and arrestins. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Receptor‐arrestin BRET assay to measure ligand‐induced recruitment of arrestin to receptorsBasic Protocol 2: Receptor‐arrestin NANOBIT assay to measure ligand‐induced recruitment of arrestin to receptorsAlternative Protocol 1: BRET assay to measure ligand‐induced recruitment of arrestin to the plasma membraneAlternative Protocol 2: NANOBIT assay to measure ligand‐induced recruitment of arrestin to the plasma membraneSupport Protocol 1: Optimization of polyethylenimine (PEI) concentration for transfection

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A novel luminescence-based β-arrestin recruitment assay for unmodified receptors

Cited by 13 publications

References 34 publications

Deciphering the signaling mechanisms of β‐arrestin1 and β‐arrestin2 in regulation of cancer cell cycle and metastasis

Deciphering the signaling mechanisms of β‐arrestin1 and β‐arrestin2 in regulation of cancer cell cycle and metastasis

Bias-inducing allosteric binding site in mu-opioid receptor signaling

In‐Cell Arrestin‐Receptor Interaction Assays

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