Cargando…

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor

The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the bi...

Descripción completa

Detalles Bibliográficos
Autores principales: Livingston, Kathryn E, Mahoney, Jacob P, Manglik, Aashish, Sunahara, Roger K, Traynor, John R
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042960/
https://www.ncbi.nlm.nih.gov/pubmed/29932421
http://dx.doi.org/10.7554/eLife.32499
_version_ 1783339247212691456
author Livingston, Kathryn E
Mahoney, Jacob P
Manglik, Aashish
Sunahara, Roger K
Traynor, John R
author_facet Livingston, Kathryn E
Mahoney, Jacob P
Manglik, Aashish
Sunahara, Roger K
Traynor, John R
author_sort Livingston, Kathryn E
collection PubMed
description The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs.
format Online
Article
Text
id pubmed-6042960
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher eLife Sciences Publications, Ltd
record_format MEDLINE/PubMed
spelling pubmed-60429602018-07-16 Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor Livingston, Kathryn E Mahoney, Jacob P Manglik, Aashish Sunahara, Roger K Traynor, John R eLife Biochemistry and Chemical Biology The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs. eLife Sciences Publications, Ltd 2018-06-22 /pmc/articles/PMC6042960/ /pubmed/29932421 http://dx.doi.org/10.7554/eLife.32499 Text en © 2018, Livingston 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
Livingston, Kathryn E
Mahoney, Jacob P
Manglik, Aashish
Sunahara, Roger K
Traynor, John R
Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
title Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
title_full Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
title_fullStr Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
title_full_unstemmed Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
title_short Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
title_sort measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042960/
https://www.ncbi.nlm.nih.gov/pubmed/29932421
http://dx.doi.org/10.7554/eLife.32499
work_keys_str_mv AT livingstonkathryne measuringligandefficacyatthemuopioidreceptorusingaconformationalbiosensor
AT mahoneyjacobp measuringligandefficacyatthemuopioidreceptorusingaconformationalbiosensor
AT manglikaashish measuringligandefficacyatthemuopioidreceptorusingaconformationalbiosensor
AT sunahararogerk measuringligandefficacyatthemuopioidreceptorusingaconformationalbiosensor
AT traynorjohnr measuringligandefficacyatthemuopioidreceptorusingaconformationalbiosensor