A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells

New technologies based on luminescence have been essential to monitor the organization, signaling, trafficking or ligand binding of G Protein-Coupled Receptors (GPCRs), but they rely on the overexpression of genetically modified receptors. As more and more studies indicate the importance of studying...

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Authors: Gómez-Santacana, Xavier, Boutonnet, Marin, Martínez-Juvés, Carles, Catena, Juan Lorenzo, Moutin, Enora, Roux, Thomas, Lamarque, Laurent, Perroy, Julie, Prézeau, Laurent, Zwier, Jurriaan M., Llebaria, Amadeu
Format: article
Status:Published version
Publication Date:2022
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/304936
Online Access:http://hdl.handle.net/10261/304936
Access Level:Open access
Keyword:ligand-directed labeling
protein labeling
GPCR
G protein-coupled receptors
Dopamine receptors
D1 receptor
Fluorescence
Endogenous proteins
Native protein
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
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spelling A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cellsGómez-Santacana, XavierBoutonnet, MarinMartínez-Juvés, CarlesCatena, Juan LorenzoMoutin, EnoraRoux, ThomasLamarque, LaurentPerroy, JuliePrézeau, LaurentZwier, Jurriaan M.Llebaria, Amadeuligand-directed labelingprotein labelingGPCRG protein-coupled receptorsDopamine receptorsD1 receptorFluorescenceEndogenous proteinsNative proteinhttp://metadata.un.org/sdg/3Ensure healthy lives and promote well-being for all at all agesNew technologies based on luminescence have been essential to monitor the organization, signaling, trafficking or ligand binding of G Protein-Coupled Receptors (GPCRs), but they rely on the overexpression of genetically modified receptors. As more and more studies indicate the importance of studying native receptors in their natural environment, it is essential to develop approaches allowing the specific labeling of native receptors. Here we report an innovative ligand directed approach to specifically label residues of native GPCRs upon ligand binding. To this end, we developed a ligand-directed toolbox based on a novel approach that uses molecular modules to build fluorescent ligand-directed probes that can label an archetypical aminergic GPCR (D1R). Our probes can be readily prepared before the labeling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific click reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labeling reaction occurs. Subsequently, the ligand unbinds the GPCR pocket, leaving the receptor fluorescently labeled and fully functional. This novel labeling approach allowed us to label both D1 receptor in transfected cells and native receptors in neuronal cell lines. This approach will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution, trafficking, activity or binding properties in their native environment.Funding Agence Nationale de la Recherche ANR-17-CE11-0046 Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe CTQ2017-89222-R Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe PCI2018-093047 Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe PID2020-120499RB-I00 Catalan government 2017 SGR 1604 the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement No. 801342 (TecniospringINDUSTRY) and the Government of Catalonia's Agency for Business Competitive-ness (ACCIÓ). TECSPR19-1-0062Peer reviewedAmerican Chemical SocietyEuropean CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202320232022info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/304936reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/EC/H2020/80134210.26434/chemrxiv-2022-mqqtz-v2Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3049362026-05-22T06:33:51Z
dc.title.none.fl_str_mv A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
title A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
spellingShingle A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
Gómez-Santacana, Xavier
ligand-directed labeling
protein labeling
GPCR
G protein-coupled receptors
Dopamine receptors
D1 receptor
Fluorescence
Endogenous proteins
Native protein
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
title_short A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
title_full A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
title_fullStr A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
title_full_unstemmed A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
title_sort A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells
dc.creator.none.fl_str_mv Gómez-Santacana, Xavier
Boutonnet, Marin
Martínez-Juvés, Carles
Catena, Juan Lorenzo
Moutin, Enora
Roux, Thomas
Lamarque, Laurent
Perroy, Julie
Prézeau, Laurent
Zwier, Jurriaan M.
Llebaria, Amadeu
author Gómez-Santacana, Xavier
author_facet Gómez-Santacana, Xavier
Boutonnet, Marin
Martínez-Juvés, Carles
Catena, Juan Lorenzo
Moutin, Enora
Roux, Thomas
Lamarque, Laurent
Perroy, Julie
Prézeau, Laurent
Zwier, Jurriaan M.
Llebaria, Amadeu
author_role author
author2 Boutonnet, Marin
Martínez-Juvés, Carles
Catena, Juan Lorenzo
Moutin, Enora
Roux, Thomas
Lamarque, Laurent
Perroy, Julie
Prézeau, Laurent
Zwier, Jurriaan M.
Llebaria, Amadeu
author2_role author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv ligand-directed labeling
protein labeling
GPCR
G protein-coupled receptors
Dopamine receptors
D1 receptor
Fluorescence
Endogenous proteins
Native protein
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
topic ligand-directed labeling
protein labeling
GPCR
G protein-coupled receptors
Dopamine receptors
D1 receptor
Fluorescence
Endogenous proteins
Native protein
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
description New technologies based on luminescence have been essential to monitor the organization, signaling, trafficking or ligand binding of G Protein-Coupled Receptors (GPCRs), but they rely on the overexpression of genetically modified receptors. As more and more studies indicate the importance of studying native receptors in their natural environment, it is essential to develop approaches allowing the specific labeling of native receptors. Here we report an innovative ligand directed approach to specifically label residues of native GPCRs upon ligand binding. To this end, we developed a ligand-directed toolbox based on a novel approach that uses molecular modules to build fluorescent ligand-directed probes that can label an archetypical aminergic GPCR (D1R). Our probes can be readily prepared before the labeling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific click reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labeling reaction occurs. Subsequently, the ligand unbinds the GPCR pocket, leaving the receptor fluorescently labeled and fully functional. This novel labeling approach allowed us to label both D1 receptor in transfected cells and native receptors in neuronal cell lines. This approach will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution, trafficking, activity or binding properties in their native environment.
publishDate 2022
dc.date.none.fl_str_mv 2022
2023
2023
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/304936
url http://hdl.handle.net/10261/304936
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/EC/H2020/801342
10.26434/chemrxiv-2022-mqqtz-v2
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv American Chemical Society
publisher.none.fl_str_mv American Chemical Society
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15.811543

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