Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor
Abstract
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.
Data availability
All data generated and analyzed during the study are included in the manuscript and supporting files. Source files have been provided for Fig 3.
Article and author information
Author details
Funding
National Institutes of Health (T32 DA007267)
- Kathryn E Livingston
American Heart Association (13PRE17110027)
- Jacob P Mahoney
National Institutes of Health (T32GM007767)
- Jacob P Mahoney
National Institutes of Health (R01 DA03339)
- John R Traynor
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Volker Dötsch, J.W. Goethe-University, Germany
Version history
- Received: October 5, 2017
- Accepted: May 26, 2018
- Accepted Manuscript published: June 22, 2018 (version 1)
- Version of Record published: July 12, 2018 (version 2)
Copyright
© 2018, Livingston et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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