Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Caro, Lydia N., Moreau, Christophe J., Revilloud, Jean, Vivaudou, Michel
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2011
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
_version_ 1782200911803711488
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
work_keys_str_mv AT carolydian b2adrenergicionchannelcoupledreceptorsasconformationalmotiondetectors
AT moreauchristophej b2adrenergicionchannelcoupledreceptorsasconformationalmotiondetectors
AT revilloudjean b2adrenergicionchannelcoupledreceptorsasconformationalmotiondetectors
AT vivaudoumichel b2adrenergicionchannelcoupledreceptorsasconformationalmotiondetectors