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Luminescence- and fluorescence-based complementation assays to screen for GPCR oligomerization : current state of the art

Elise Wouters (UGent) , Lakshmi Vasudevan (UGent) , René Crans (UGent) , Deepak K Saini and Christophe Stove (UGent)
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Abstract
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.
Keywords
G protein-coupled receptor (GPCR), dimerization, oligomerization, protein complementation assay, bimolecular fluorescence complementation (BiFC) assay, bimolecular luminescence complementation (BiLC) assay, PROTEIN-PROTEIN INTERACTIONS, ADENOSINE A(2A) RECEPTORS, HIGHER-ORDER OLIGOMERS, RESONANCE ENERGY-TRANSFER, M-1 MUSCARINIC RECEPTOR, PROXIMAL TUBULE CELLS, LIVING CELLS, DOPAMINE D2, IN-VIVO, NEURODEGENERATIVE DISEASES

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Chicago
Wouters, Elise, Lakshmi Vasudevan, René Crans, Deepak K Saini, and Christophe Stove. 2019. “Luminescence- and Fluorescence-based Complementation Assays to Screen for GPCR Oligomerization : Current State of the Art.” International Journal of Molecular Sciences 20 (12).
APA
Wouters, Elise, Vasudevan, L., Crans, R., Saini, D. K., & Stove, C. (2019). Luminescence- and fluorescence-based complementation assays to screen for GPCR oligomerization : current state of the art. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 20(12).
Vancouver
1.
Wouters E, Vasudevan L, Crans R, Saini DK, Stove C. Luminescence- and fluorescence-based complementation assays to screen for GPCR oligomerization : current state of the art. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. 2019;20(12).
MLA
Wouters, Elise et al. “Luminescence- and Fluorescence-based Complementation Assays to Screen for GPCR Oligomerization : Current State of the Art.” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 20.12 (2019): n. pag. Print.
@article{8619842,
  abstract     = {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.},
  articleno    = {2958},
  author       = {Wouters, Elise and Vasudevan, Lakshmi and Crans, René and Saini, Deepak K and Stove, Christophe},
  issn         = {1422-0067},
  journal      = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES},
  keywords     = {G protein-coupled receptor (GPCR),dimerization,oligomerization,protein complementation assay,bimolecular fluorescence complementation (BiFC) assay,bimolecular luminescence complementation (BiLC) assay,PROTEIN-PROTEIN INTERACTIONS,ADENOSINE A(2A) RECEPTORS,HIGHER-ORDER OLIGOMERS,RESONANCE ENERGY-TRANSFER,M-1 MUSCARINIC RECEPTOR,PROXIMAL TUBULE CELLS,LIVING CELLS,DOPAMINE D2,IN-VIVO,NEURODEGENERATIVE DISEASES},
  language     = {eng},
  number       = {12},
  pages        = {35},
  title        = {Luminescence- and fluorescence-based complementation assays to screen for GPCR oligomerization : current state of the art},
  url          = {http://dx.doi.org/10.3390/ijms20122958},
  volume       = {20},
  year         = {2019},
}

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