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Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introd...

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Autores principales: Stoeber, Miriam, Jullié, Damien, Li, Joy, Chakraborty, Soumen, Majumdar, Susruta, Lambert, Nevin A, Manglik, Aashish, von Zastrow, Mark
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041944/
https://www.ncbi.nlm.nih.gov/pubmed/32096468
http://dx.doi.org/10.7554/eLife.54208
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author Stoeber, Miriam
Jullié, Damien
Li, Joy
Chakraborty, Soumen
Majumdar, Susruta
Lambert, Nevin A
Manglik, Aashish
von Zastrow, Mark
author_facet Stoeber, Miriam
Jullié, Damien
Li, Joy
Chakraborty, Soumen
Majumdar, Susruta
Lambert, Nevin A
Manglik, Aashish
von Zastrow, Mark
author_sort Stoeber, Miriam
collection PubMed
description G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric ‘bias’ into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.
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spelling pubmed-70419442020-02-27 Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells Stoeber, Miriam Jullié, Damien Li, Joy Chakraborty, Soumen Majumdar, Susruta Lambert, Nevin A Manglik, Aashish von Zastrow, Mark eLife Biochemistry and Chemical Biology G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric ‘bias’ into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists. eLife Sciences Publications, Ltd 2020-02-25 /pmc/articles/PMC7041944/ /pubmed/32096468 http://dx.doi.org/10.7554/eLife.54208 Text en © 2020, Stoeber et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Stoeber, Miriam
Jullié, Damien
Li, Joy
Chakraborty, Soumen
Majumdar, Susruta
Lambert, Nevin A
Manglik, Aashish
von Zastrow, Mark
Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells
title Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells
title_full Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells
title_fullStr Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells
title_full_unstemmed Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells
title_short Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells
title_sort agonist-selective recruitment of engineered protein probes and of grk2 by opioid receptors in living cells
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041944/
https://www.ncbi.nlm.nih.gov/pubmed/32096468
http://dx.doi.org/10.7554/eLife.54208
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