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Title: Single-molecule analysis of ligand efficacy in β2AR–G-protein activation

Abstract

G-protein-coupled receptor (GPCR)-mediated signal transduction is vital to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). In this work, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β2AR–Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data further reveal transient nucleotide-bound β2AR–Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.

Authors:
 [1];  [2];  [2];  [1];  [1];  [1];  [1];  [3];  [4];  [5];  [5];  [6];  [2];  [1]
+ Show Author Affiliations
  1. Cornell Univ., Ithaca, NY (United States). Weill Medical College
  2. Stanford Univ., CA (United States). Dept. of Molecular and Cellular Physiology
  3. Cornell Univ., Ithaca, NY (United States). Weill Medical College; IBM, Yorktown Heights, NY (United States). Thomas J. Watson Research Center
  4. Columbia Univ., New York, NY (United States). Dept. of Psychiatry; New York State Psychiatric Inst., New York, NY (United States). Division of Molecular Therapeutics; Univ. of Copenhagen (Denmark). Lab. for Molecular Pharmacology and NNF Center for Basic Metabolic Research
  5. Columbia Univ., New York, NY (United States). Dept. of Psychiatry; New York State Psychiatric Inst., New York, NY (United States). Division of Molecular Therapeutics
  6. Cornell Univ., Ithaca, NY (United States). Weill Medical College and HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Inst. for Computational Biomedicine
Publication Date:
Research Org.:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Oak Ridge Leadership Computing Facility (OLCF)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH)
OSTI Identifier:
1565581
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Nature (London)
Additional Journal Information:
Journal Name: Nature (London); Journal Volume: 547; Journal Issue: 7661; Journal ID: ISSN 0028-0836
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Gregorio, G. Glenn, Masureel, Matthieu, Hilger, Daniel, Terry, Daniel S., Juette, Manuel, Zhao, Hong, Zhou, Zhou, Perez-Aguilar, Jose Manuel, Hauge, Maria, Mathiasen, Signe, Javitch, Jonathan A., Weinstein, Harel, Kobilka, Brian K., and Blanchard, Scott C. Single-molecule analysis of ligand efficacy in β2AR–G-protein activation. United States: N. p., 2017. Web. doi:10.1038/nature22354.
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Gregorio, G. Glenn, Masureel, Matthieu, Hilger, Daniel, Terry, Daniel S., Juette, Manuel, Zhao, Hong, Zhou, Zhou, Perez-Aguilar, Jose Manuel, Hauge, Maria, Mathiasen, Signe, Javitch, Jonathan A., Weinstein, Harel, Kobilka, Brian K., & Blanchard, Scott C. Single-molecule analysis of ligand efficacy in β2AR–G-protein activation. United States. https://doi.org/10.1038/nature22354
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Gregorio, G. Glenn, Masureel, Matthieu, Hilger, Daniel, Terry, Daniel S., Juette, Manuel, Zhao, Hong, Zhou, Zhou, Perez-Aguilar, Jose Manuel, Hauge, Maria, Mathiasen, Signe, Javitch, Jonathan A., Weinstein, Harel, Kobilka, Brian K., and Blanchard, Scott C. Wed . "Single-molecule analysis of ligand efficacy in β2AR–G-protein activation". United States. https://doi.org/10.1038/nature22354. https://www.osti.gov/servlets/purl/1565581.
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@article{osti_1565581,
title = {Single-molecule analysis of ligand efficacy in β2AR–G-protein activation},
author = {Gregorio, G. Glenn and Masureel, Matthieu and Hilger, Daniel and Terry, Daniel S. and Juette, Manuel and Zhao, Hong and Zhou, Zhou and Perez-Aguilar, Jose Manuel and Hauge, Maria and Mathiasen, Signe and Javitch, Jonathan A. and Weinstein, Harel and Kobilka, Brian K. and Blanchard, Scott C.},
abstractNote = {G-protein-coupled receptor (GPCR)-mediated signal transduction is vital to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). In this work, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β2AR–Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data further reveal transient nucleotide-bound β2AR–Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.},
doi = {10.1038/nature22354},
journal = {Nature (London)},
number = 7661,
volume = 547,
place = {United States},
year = {Wed Jun 07 00:00:00 EDT 2017},
month = {Wed Jun 07 00:00:00 EDT 2017}
}
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