Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art
G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the...
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doaj-19e896cc99f641e885182f9f95285eb42020-11-24T21:53:26ZengMDPI AGInternational Journal of Molecular Sciences1422-00672019-06-012012295810.3390/ijms20122958ijms20122958Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the ArtElise Wouters0Lakshmi Vasudevan1René A. J. Crans2Deepak K. Saini3Christophe P. Stove4Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumLaboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumLaboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumDepartment of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, IndiaLaboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, BelgiumG protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR−GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein−protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR−GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR−GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.https://www.mdpi.com/1422-0067/20/12/2958G protein-coupled receptor (GPCR), dimerizationoligomerizationprotein complementation assaybimolecular fluorescence complementation (BiFC) assaybimolecular luminescence complementation (BiLC) assay |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Elise Wouters Lakshmi Vasudevan René A. J. Crans Deepak K. Saini Christophe P. Stove |
spellingShingle |
Elise Wouters Lakshmi Vasudevan René A. J. Crans Deepak K. Saini Christophe P. Stove Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art International Journal of Molecular Sciences G protein-coupled receptor (GPCR), dimerization oligomerization protein complementation assay bimolecular fluorescence complementation (BiFC) assay bimolecular luminescence complementation (BiLC) assay |
author_facet |
Elise Wouters Lakshmi Vasudevan René A. J. Crans Deepak K. Saini Christophe P. Stove |
author_sort |
Elise Wouters |
title |
Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art |
title_short |
Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art |
title_full |
Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art |
title_fullStr |
Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art |
title_full_unstemmed |
Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art |
title_sort |
luminescence- and fluorescence-based complementation assays to screen for gpcr oligomerization: current state of the art |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2019-06-01 |
description |
G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR−GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein−protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR−GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR−GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays. |
topic |
G protein-coupled receptor (GPCR), dimerization oligomerization protein complementation assay bimolecular fluorescence complementation (BiFC) assay bimolecular luminescence complementation (BiLC) assay |
url |
https://www.mdpi.com/1422-0067/20/12/2958 |
work_keys_str_mv |
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