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.
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
Publication 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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