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β(2)-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors
Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064670/ https://www.ncbi.nlm.nih.gov/pubmed/21464970 http://dx.doi.org/10.1371/journal.pone.0018226 |
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author | Caro, Lydia N. Moreau, Christophe J. Revilloud, Jean Vivaudou, Michel |
author_facet | Caro, Lydia N. Moreau, Christophe J. Revilloud, Jean Vivaudou, Michel |
author_sort | Caro, Lydia N. |
collection | PubMed |
description | Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K(+)-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K(+) channel Kir6.2 and muscarinic M(2) or dopaminergic D(2) receptors. Here, we extend the concept to the distinct, longer β(2)-adrenergic receptor which, unlike M(2) and D(2) receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of β(2) constructs. We then demonstrate how engineering of both receptor and channel can produce β(2)-Kir6.2 ICCRs. Specifically, removal of 62–72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the β(2) ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the β(2) receptor. |
format | Text |
id | pubmed-3064670 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-30646702011-04-04 β(2)-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors Caro, Lydia N. Moreau, Christophe J. Revilloud, Jean Vivaudou, Michel PLoS One Research Article Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K(+)-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K(+) channel Kir6.2 and muscarinic M(2) or dopaminergic D(2) receptors. Here, we extend the concept to the distinct, longer β(2)-adrenergic receptor which, unlike M(2) and D(2) receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of β(2) constructs. We then demonstrate how engineering of both receptor and channel can produce β(2)-Kir6.2 ICCRs. Specifically, removal of 62–72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the β(2) ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the β(2) receptor. Public Library of Science 2011-03-25 /pmc/articles/PMC3064670/ /pubmed/21464970 http://dx.doi.org/10.1371/journal.pone.0018226 Text en Caro et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Caro, Lydia N. Moreau, Christophe J. Revilloud, Jean Vivaudou, Michel β(2)-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors |
title | β(2)-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors |
title_full | β(2)-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors |
title_fullStr | β(2)-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors |
title_full_unstemmed | β(2)-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors |
title_short | β(2)-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors |
title_sort | β(2)-adrenergic ion-channel coupled receptors as conformational motion detectors |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064670/ https://www.ncbi.nlm.nih.gov/pubmed/21464970 http://dx.doi.org/10.1371/journal.pone.0018226 |
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