ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells

Abstract FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the othe...

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Autores principales:	Rabia U. Malik, Matthew Dysthe, Michael Ritt, Roger K. Sunahara, Sivaraj Sivaramakrishnan
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/246552c8efa54c19bc36a88af7a47811
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spelling	oai:doaj.org-article:246552c8efa54c19bc36a88af7a478112021-12-02T12:32:46ZER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells10.1038/s41598-017-08029-32045-2322https://doaj.org/article/246552c8efa54c19bc36a88af7a478112017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-08029-3https://doaj.org/toc/2045-2322Abstract FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the β2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A1R), tethered to Gαs-XL, Gαi2, or Gαq subunits. Agonist stimulation of β2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.Rabia U. MalikMatthew DystheMichael RittRoger K. SunaharaSivaraj SivaramakrishnanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rabia U. Malik Matthew Dysthe Michael Ritt Roger K. Sunahara Sivaraj Sivaramakrishnan ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells
description	Abstract FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the β2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A1R), tethered to Gαs-XL, Gαi2, or Gαq subunits. Agonist stimulation of β2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.
format	article
author	Rabia U. Malik Matthew Dysthe Michael Ritt Roger K. Sunahara Sivaraj Sivaramakrishnan
author_facet	Rabia U. Malik Matthew Dysthe Michael Ritt Roger K. Sunahara Sivaraj Sivaramakrishnan
author_sort	Rabia U. Malik
title	ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells
title_short	ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells
title_full	ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells
title_fullStr	ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells
title_full_unstemmed	ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells
title_sort	er/k linked gpcr-g protein fusions systematically modulate second messenger response in cells
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/246552c8efa54c19bc36a88af7a47811
work_keys_str_mv	AT rabiaumalik erklinkedgpcrgproteinfusionssystematicallymodulatesecondmessengerresponseincells AT matthewdysthe erklinkedgpcrgproteinfusionssystematicallymodulatesecondmessengerresponseincells AT michaelritt erklinkedgpcrgproteinfusionssystematicallymodulatesecondmessengerresponseincells AT rogerksunahara erklinkedgpcrgproteinfusionssystematicallymodulatesecondmessengerresponseincells AT sivarajsivaramakrishnan erklinkedgpcrgproteinfusionssystematicallymodulatesecondmessengerresponseincells
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ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells

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