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.
Highlights
Membrane receptors are the key players in mediating communication between the cell and the extracellular space
This review aims at offering insight into the progress that has been made in the field of protein complementation assays to study G protein-coupled receptors (GPCRs) dimerization
Increasing evidence supports the existence of GPCR–GPCR interactions in vitro and in vivo [159], the sophisticated characteristics of these interactions, such as their variation in dynamics, complexity, binding affinities, and the lack of a consensus sequence in the interfaces, make GPCR–GPCR interactions a challenging research field
Summary
Membrane receptors are the key players in mediating communication between the cell and the extracellular space. Notwithstanding the broad collection of ligands to activate specific GPCRs, they all share a common structure, namely, an N-terminal domain, seven transmembrane α-helices, extra- (ECL1–3) and intracellular loops (ICL1–3) and a C-terminal domain. The motile domains, such as the ICL3 and the C-terminus, are crucial for interaction with intracellular signaling partners, i.e., G-proteins, GPCR kinases (GRKs) and β-arrestins. Given their crucial role in cellular signaling, altered activity or deviating expression levels of GPCRs have frequently been correlated with diseases, including neurodegenerative disorders, depression, and cancer [5–10]
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