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

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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.

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All data generated and analyzed during the study are included in the manuscript and supporting files. Source files have been provided for Fig 3.

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Kathryn E Livingston

Department of Pharmacology, University of Michigan, Ann Arbor, United States

Competing interests
The authors declare that no competing interests exist.
Jacob P Mahoney

Department of Pharmacology, University of Michigan, Ann Arbor, United States

Competing interests
The authors declare that no competing interests exist.
Aashish Manglik

Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Roger Sunahara

Department of Pharmacology, University of California, San Diego, San Diego, United States

Competing interests
The authors declare that no competing interests exist.
John R Traynor

Department of Pharmacology, University of Michigan, Ann Arbor, United States

For correspondence
jtraynor@umich.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1849-8316

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.

Copyright

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.