Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β₂AR
Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3...
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Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 46; pp. 14254 - 14259 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
National Academy of Sciences
17.11.2015
National Acad Sciences |
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Abstract | Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β₂-adrenergic receptor (β₂AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs. |
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AbstractList | Activation of G protein-coupled receptors (GPCRs) by agonists is the first step of eukaryotic cellular signal transduction. Because GPCRs are expressed in almost all human tissues and play a key role in human physiology, they are the targets for more than 30% of pharmaceutical drugs. Binding of ligands on the extracellular surface of a GPCR induces a conformational change on the cytoplasmic surface, which is recognized by G proteins or other cellular effectors. Here we show that the β
2
-adrenergic receptor, a prototypical GPCR, naturally fluctuates between inactive and active conformations, and that agonist or inverse agonist ligands modulate the conformational exchange kinetics in distinct ways, explaining their different pharmacological efficacies. These insights should assist in the design of improved GPCR-targeting drugs.
Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β
2
-adrenergic receptor (β
2
AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs. Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs. Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β₂-adrenergic receptor (β₂AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs. |
Author | van der Schans, Edwin Millar, David P. Stevens, Raymond C. Pljevaljcic, Goran Lamichhane, Rajan White, Kate L. Wüthrich, Kurt Liu, Jeffrey J. Katritch, Vsevolod |
Author_xml | – sequence: 1 givenname: Rajan surname: Lamichhane fullname: Lamichhane, Rajan organization: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037 – sequence: 2 givenname: Jeffrey J. surname: Liu fullname: Liu, Jeffrey J. organization: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037 – sequence: 3 givenname: Goran surname: Pljevaljcic fullname: Pljevaljcic, Goran organization: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037 – sequence: 4 givenname: Kate L. surname: White fullname: White, Kate L. organization: Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089 – sequence: 5 givenname: Edwin surname: van der Schans fullname: van der Schans, Edwin organization: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037 – sequence: 6 givenname: Vsevolod surname: Katritch fullname: Katritch, Vsevolod organization: Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089 – sequence: 7 givenname: Raymond C. surname: Stevens fullname: Stevens, Raymond C. organization: Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089 – sequence: 8 givenname: Kurt surname: Wüthrich fullname: Wüthrich, Kurt organization: Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland – sequence: 9 givenname: David P. surname: Millar fullname: Millar, David P. organization: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037 |
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Keywords | signal transduction mechanisms phospholipid nanodiscs conformational polymorphism agonists and inverse agonists single-molecule fluorescence spectroscopy |
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Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 1R.L. and J.J.L. contributed equally to this work. 3Present Address: BioNano Genomics, San Diego, CA 92121. Contributed by Kurt Wüthrich, October 9, 2015 (sent for review August 16, 2015; reviewed by W. E. Moerner and David Rueda) Author contributions: R.C.S., K.W., and D.P.M. designed research; R.L., J.J.L., G.P., K.L.W., E.v.d.S., and V.K. performed research; J.J.L., G.P., K.L.W., E.v.d.S., and V.K. contributed new reagents/analytic tools; R.L., K.L.W., and D.P.M. analyzed data; and R.L., K.L.W., V.K., R.C.S., K.W., and D.P.M. wrote the paper. Reviewers: W.E.M., Stanford University; and D.R., Imperial College London. 2Present Address: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. |
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Title | Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β₂AR |
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