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Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation
[Image: see text] G protein-coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here, we describe the design of a generalizable and cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10588934/ https://www.ncbi.nlm.nih.gov/pubmed/37868356 http://dx.doi.org/10.1021/acsmeasuresciau.3c00021 |
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author | Miller, Ruby M. Sescil, Jennifer Sarcinella, Marina C. Bailey, Ryan C. Wang, Wenjing |
author_facet | Miller, Ruby M. Sescil, Jennifer Sarcinella, Marina C. Bailey, Ryan C. Wang, Wenjing |
author_sort | Miller, Ruby M. |
collection | PubMed |
description | [Image: see text] G protein-coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here, we describe the design of a generalizable and cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific binder to reconstitute split nanoluciferase (NanoLuc) in vitro. This tool, In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), has broad applications. We have demonstrated IGNiTR’s use with three G(s)-coupled GPCRs, two G(i)-coupled GPCRs and three classes of conformation-specific binders: nanobodies, miniG proteins, and G protein peptidomimetics. As an in vitro method, IGNiTR enables the use of synthetic G protein peptidomimetics and provides easily scalable and portable reagents for characterizing GPCRs and ligands. We tested three diverse applications of IGNiTR: (1) proof-of-concept GPCR ligand screening using dopamine receptor D1 IGNiTR; (2) detection of opioids for point-of-care testing; and (3) characterizing GPCR functionality during Nanodisc-based reconstitution processes. Due to IGNiTR’s unique advantages and the convenience of its cell lysate-based format, this tool will find extensive applications in GPCR ligand detection, screening, and GPCR characterization. |
format | Online Article Text |
id | pubmed-10588934 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-105889342023-10-21 Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation Miller, Ruby M. Sescil, Jennifer Sarcinella, Marina C. Bailey, Ryan C. Wang, Wenjing ACS Meas Sci Au [Image: see text] G protein-coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here, we describe the design of a generalizable and cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific binder to reconstitute split nanoluciferase (NanoLuc) in vitro. This tool, In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), has broad applications. We have demonstrated IGNiTR’s use with three G(s)-coupled GPCRs, two G(i)-coupled GPCRs and three classes of conformation-specific binders: nanobodies, miniG proteins, and G protein peptidomimetics. As an in vitro method, IGNiTR enables the use of synthetic G protein peptidomimetics and provides easily scalable and portable reagents for characterizing GPCRs and ligands. We tested three diverse applications of IGNiTR: (1) proof-of-concept GPCR ligand screening using dopamine receptor D1 IGNiTR; (2) detection of opioids for point-of-care testing; and (3) characterizing GPCR functionality during Nanodisc-based reconstitution processes. Due to IGNiTR’s unique advantages and the convenience of its cell lysate-based format, this tool will find extensive applications in GPCR ligand detection, screening, and GPCR characterization. American Chemical Society 2023-07-07 /pmc/articles/PMC10588934/ /pubmed/37868356 http://dx.doi.org/10.1021/acsmeasuresciau.3c00021 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Miller, Ruby M. Sescil, Jennifer Sarcinella, Marina C. Bailey, Ryan C. Wang, Wenjing Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation |
title | Accessible and Generalizable in Vitro Luminescence
Assay for Detecting GPCR Activation |
title_full | Accessible and Generalizable in Vitro Luminescence
Assay for Detecting GPCR Activation |
title_fullStr | Accessible and Generalizable in Vitro Luminescence
Assay for Detecting GPCR Activation |
title_full_unstemmed | Accessible and Generalizable in Vitro Luminescence
Assay for Detecting GPCR Activation |
title_short | Accessible and Generalizable in Vitro Luminescence
Assay for Detecting GPCR Activation |
title_sort | accessible and generalizable in vitro luminescence
assay for detecting gpcr activation |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10588934/ https://www.ncbi.nlm.nih.gov/pubmed/37868356 http://dx.doi.org/10.1021/acsmeasuresciau.3c00021 |
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