1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor

  1. Kathryn E Livingston
  2. Jacob P Mahoney
  3. Aashish Manglik
  4. Roger Sunahara
  5. John R Traynor  Is a corresponding author
  1. University of Michigan, United States
  2. University of California, San Francisco, United States
  3. University of California, San Diego, United States
Research Article
  • Cited 12
  • Views 3,682
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Cite this article as: eLife 2018;7:e32499 doi: 10.7554/eLife.32499

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

  1. Kathryn E Livingston

    Department of Pharmacology, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Jacob P Mahoney

    Department of Pharmacology, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. 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.

  4. Roger Sunahara

    Department of Pharmacology, University of California, San Diego, San Diego, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. 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.
    ORCID icon "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.

Reviewing Editor

  1. Volker Dötsch, J.W. Goethe-University, Germany

Publication history

  1. Received: October 5, 2017
  2. Accepted: May 26, 2018
  3. Accepted Manuscript published: June 22, 2018 (version 1)
  4. 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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