Comment on "The use of BRET to study receptor-protein interactions"

International audienceThis Research Topic assembles for the first time a comprehensive selection of articles (mini review, review articles, original research, and opinion articles) on the bioluminescence resonance energy transfer (BRET) technology. BRET is a natural phenomenon known in several marine organisms. It relies on the non-radiative transfer of energy from an appropriate energy donor to an energy acceptor, provided that both are located at a distance lower than 10 nm [see Ref. (1) for a basic and theoretical introduction]. BRET has been first applied to the detection of protein–protein interac-tions (PPIs) in 1999 (2). Since then, the technology constantly evolved by combining new donor and acceptor couples and devel-oping various BRET assay formats as discussed in the Mini Review article of De et al. (3). Many BRET assays have been developed to study the oligomer-ization of seven-transmembrane-spanning G protein-coupled receptors (GPCRs). The BRET technology provides an attrac-tive way to study this phenomenon in intact cells without the need of solubilization of receptors from their natural membrane environment. The overwhelming majority of BRET-based studies conclude that GPCRs do indeed exist as dimers or higher-order oligomers when transfected into cells at physiological levels. Three of the articles of this Research Topic discuss BRET assays that have been developed to properly address the specificity of BRET sig-nals obtained upon expression of different GPCRs (1, 4, 5). The current consensus confirms that several different BRET assays are needed to evaluate the specificity of BRET signals. The precise role of each of these assays remains a source of controversy in the field (4). Further refinement of BRET control experiments and appli-cation of new techniques like single-molecule measurements and functional in vivo studies are likely to provide new insights to the existence and physiological relevance of GPCR oligomerization. Not surprisingly, GPCR oligomerization is the main issue of four articles of this Research Topic (4–7) ranging from studies on class A and B GPCR homo-and heteromers using BRET or alternative approaches like time-resolved FRET measurements. Apart from performing the proper control experiments, another important issue in the BRET field concerns the proper interpretation of stimulus-induced BRET signals. In the context of GPCR oligomerization, agonist-induced BRET signals can be gen-erated by an agonist-driven oligomerization or agonist-induced conformational changes within preassembled oligomers. Discrim-ination between these two possibilities is not trivial but has become possible due to the development of BRET donor saturation exper-iments in the absence and presence of receptor stimulation (8, 9). This issue, to which BRET has made a significant contribution, is obviously of general importance for the field of PPIs. This is illustrated by the articles describing the interaction of the protease-activated receptor 1 and 2 with its cognate G proteins as described by Ayoub and Pin (10) and Ayoub et al. (11). The BRET technology has been extended toward other recep-tor families like tyrosine kinase receptors and cytokine receptors. This diversification demonstrates the general feature of this tech-nique. These studies did not only address the question of receptor oligomerization but also monitored the real-time interaction of receptors with various effector molecules such as Grb2, PTP1-B, PLC-γ1, etc. This important aspect is discussed in the review arti-cle of Siddiqui et al. (12). Receptor–effector interactions have been also monitored by BRET for two privileged GPCR interacting part-ners, heterotrimeric G proteins and β-arrestins, as documented in articles of this Research Topic (6, 10, 11, 13). A more recent application of BRET concerns the development of biosensors to monitor downstream events of cellular signal-ing like the generation of second messengers and activation of intracellular kinases. These sensors are typically composed of the energy donor and acceptor separated by an assay-specific domain that changes its conformation upon second messenger binding or phosphorylation, thus modifying the position of the donor and acceptor and consequently the BRET signal. Similar sensors have been developed for FRET applications, which served as source of inspiration for the development of the BRET sensors. The articles from Salahpour et al. (13) and Xu et al. (2) describe the design and validation of cAMP and ERK sensors. The high reproducibility of BRET and the robustness of the measurements make BRET an interesting option for the design of high throughput screening assays. Two applications are discussed in this Research Topic. The first concerns the design of an assay for the identification of compounds that specifically activate GPCR heteromers (6) based on the recruitment of β-arrestin to GPCR heteromers. The second case concerns the identification of PPI inhibitors (14). Taken together, this Research Topic provides an illustrative overview of the principles and applications of the BRET tech-nology that should be of interest for any scientist interested in monitoring PPI in intact cells

Frontiers Commentary onTallet et al. Investigation of prolactin receptor activation and blockade using time-resolved fluorescence resonance energy transfer A commentary on Investigation of prolactin receptor acti- vation and blockade using time-resolved fluorescence resonance energy transfer

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THE MELATONIN RECEPTORS AND THEIR ASSOCIATED PROTEIN COMPLEXES

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Biological Significance of GPCR Heteromerization in the Neuro-Endocrine System

Clustering of proteins in higher order complexes is a common theme in biology and profoundly influences protein function. The idea that seven-transmembrane spanning G protein-coupled receptors (GPCRs) might form dimers or higher order oligomeric complexes has been formulated more than 20 years ago. Since then, this phenomenon has been investigated with many different biochemical and biophysical techniques. The more recent notion of GPCR heteromerization describes the specific association of two different GPCRs. GPCR heteromerization may be of primary importance in neuroendocrinology, as this may explain at least some of the functional crosstalks described between different hormonal systems. Importantly, many GPCR heteromers have distinct functional properties compared to their corresponding homomers. Heteromer-specific pharmacological profiles might be exploited for drug design and open new therapeutic options. GPCR heteromerization has been first studied in heterologous expression systems. Today, increasing evidence for the existence of GPCR heteromers in endogenous systems is emerging providing crucial evidence for the physiological function of GPCR heteromerization

Bitopic ligands: all-in-one orthosteric and allosteric

Natural ligands of G-protein-coupled receptors interact with the orthosteric ligand binding site, as do most of the classical synthetic ligands. The discovery of ligands targeting different, allosteric binding sites considerably expanded the repertoire of G-protein-coupled receptor ligands. More recently, bitopic ligands have been described that target both orthosteric and allosteric sites at the same time

New pharmacological perspectives for the leptin receptor in the treatment of obesity

International audienceAfter its discovery in 1994, leptin became the great hope as an anti-obesity treatment based on its ability to reduce food intake and increase energy expenditure. However, treat-ing obese people with exogenous leptin was unsuccessful in most cases since most of them present already high circulating leptin levels to which they do not respond anymore defining the so-called state of "leptin resistance." Indeed, leptin therapy is unsuccessful to lower body weight in commonly obese people but effective in people with rare single gene mutations of the leptin gene. Consequently, treatment of obese people with leptin was given less attention and the focus of obesity research shifted toward the prevention and reversal of the state of leptin resistance. Many of these new promising approaches aim to restore or sensitize the impaired function of the leptin receptor by pharmacological means. The current review will focus on the different emerging therapeutic strategies in obesity research that are related to leptin and its receptor

Understanding Melatonin Receptor Pharmacology: Latest Insights from Mouse Models, and Their Relevance To Human Disease

Melatonin, the neuro-hormone synthesized during the night, has recently seen an unexpected extension of its functional implications toward type 2 diabetes development, visual functions, sleep disturbances, and depression. Transgenic mouse models were instrumental for the establishment of the link between melatonin and these major human diseases. Most of the actions of melatonin are mediated by two types of G protein-coupled receptors, named MT1 and MT2, which are expressed in many different organs and tissues. Understanding the pharmacology and function of mouse MT1 and MT2 receptors, including MT1/MT2 heteromers, will be of crucial importance to evaluate the relevance of these mouse models for future therapeutic developments. This review will critically discuss these aspects, and give some perspectives including the generation of new mouse models

Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic β cells.

International audienceAcute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic β-cell dysfunction. However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human β-cell line EndoC-βH2, we demonstrate that β-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human β-cells. Using gain- and loss-of-function studies, we provide evidence that the β-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by β-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated β-cell response to acidic microenvironment, which could be a new target to protect β-cell against acidosis induced inflammation

G protein-coupled receptor-effector macromolecular membrane assemblies (GEMMAs)

G protein-coupled receptors (GPCRs) are the largest group of receptors involved in cellular signaling across the plasma membrane and a major class of drug targets. The canonical model for GPCR signaling involves three components the GPCR, a heterotrimeric G protein and a proximal plasma membrane effector that have been generally thought to be freely mobile molecules able to interact by 'collision coupling'. Here, we synthesize evidence that supports the existence of GPCR-effector macromolecular membrane assemblies (GEMMAs) comprised of specific GPCRs, G proteins, plasma membrane effector molecules and other associated transmembrane proteins that are pre-assembled prior to receptor activation by agonists, which then leads to subsequent rearrangement of the GEMMA components. The GEMMA concept offers an alternative and complementary model to the canonical collision-coupling model, allowing more efficient interactions between specific signaling components, as well as the integration of the concept of GPCR oligomerization as well as GPCR interactions with orphan receptors, truncated GPCRs and other membrane-localized GPCR-associated proteins. Collision-coupling and pre-assembled mechanisms are not exclusive and likely both operate in the cell, providing a spectrum of signaling modalities which explains the differential properties of a multitude of GPCRs in their different cellular environments. Here, we explore the unique pharmacological characteristics of individual GEMMAs, which could provide new opportunities to therapeutically modulate GPCR signaling

Melatonin receptors in GtoPdb v.2023.1

Melatonin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Melatonin Receptors [40]) are activated by the endogenous ligands melatonin and clinically used drugs like ramelteon, agomelatine and tasimelteon