Agonist-induced Conformational Changes Research Articles

Structural basis of G protein-Coupled receptor CMKLR1 activation and signaling induced by a chemerin-derived agonist.

Chemokine-like receptor 1 (CMKLR1), also known as chemerin receptor 23 (ChemR23) or chemerin receptor 1, is a chemoattractant G protein-coupled receptor (GPCR) that responds to the adipokine chemerin and is highly expressed in innate immune cells, including macrophages and neutrophils. The signaling pathways of CMKLR1 can lead to both pro- and anti-inflammatory effects depending on the ligands and physiological contexts. To understand the molecular mechanisms of CMKLR1 signaling, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of the CMKLR1-Gi signaling complex with chemerin9, a nanopeptide agonist derived from chemerin, which induced complex phenotypic changes of macrophages in our assays. The cryo-EM structure, together with molecular dynamics simulations and mutagenesis studies, revealed the molecular basis of CMKLR1 signaling by elucidating the interactions at the ligand-binding pocket and the agonist-induced conformational changes. Our results are expected to facilitate the development of small molecule CMKLR1 agonists that mimic the action of chemerin9 to promote the resolution of inflammation.

Structural basis for ligand recognition and signaling of hydroxy-carboxylic acid receptor 2

Hydroxycarboxylic acid receptors (HCAR1, HCAR2, and HCAR3) transduce Gi/o signaling upon biding to molecules such as lactic acid, butyric acid and 3-hydroxyoctanoic acid, which are associated with lipolytic and atherogenic activity, and neuroinflammation. Although many reports have elucidated the function of HCAR2 and its potential as a therapeutic target for treating not only dyslipidemia but also neuroimmune disorders such as multiple sclerosis and Parkinson’s disease, the structural basis of ligand recognition and ligand-induced Gi-coupling remains unclear. Here we report three cryo-EM structures of the human HCAR2–Gi signaling complex, each bound with different ligands: niacin, acipimox or GSK256073. All three agonists are held in a deep pocket lined by residues that are not conserved in HCAR1 and HCAR3. A distinct hairpin loop at the HCAR2 N-terminus and extra-cellular loop 2 (ECL2) completely enclose the ligand. These structures also reveal the agonist-induced conformational changes propagated to the G-protein-coupling interface during activation. Collectively, the structures presented here are expected to help in the design of ligands specific for HCAR2, leading to new drugs for the treatment of various diseases such as dyslipidemia and inflammation.

WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction.

Frizzleds (FZDs) are G protein-coupled receptors (GPCRs) that bind to WNT family ligands. FZDs signal through multiple effector proteins, including Dishevelled (DVL), which acts as a hub for several downstream signaling pathways. To understand how WNT binding to FZD stimulates intracellular signaling and influences downstream pathway selectivity, we investigated the dynamic changes in the FZD5-DVL2 interaction elicited by WNT-3A and WNT-5A. Ligand-induced changes in bioluminescence resonance energy transfer (BRET) between FZD5 and DVL2 or the isolated FZD-binding DEP domain of DVL2 revealed a composite response consisting of both DVL2 recruitment and conformational dynamics in the FZD5-DVL2 complex. The combination of different BRET paradigms enabled us to identify ligand-dependent conformational dynamics in the FZD5-DVL2 complex and distinguish them from ligand-induced recruitment of DVL2 or DEP to FZD5. The observed agonist-induced conformational changes at the receptor-transducer interface suggest that extracellular agonist and intracellular transducers cooperate through transmembrane allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs.

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR. Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488). These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α). EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes.

Determination of binding affinity and efficacy for NMDA receptor agonists and GluN2A selective allosteric modulators

NMDA-type ionotropic glutamate receptors are composed of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Simultaneous occupation of all 4 agonist binding domains allows the receptor to undergo all the conformational changes that lead to opening of the ion channel. Agonist-induced conformational changes in turn alter agonist affinity. Thus, the efficacy by which agonists induce conformational changes and channel gating influences the apparent affinity. Allosteric modulators influence the agonist-induced conformational changes and can therefore alter agonist binding affinity and efficacy.

An ionic lock and a hydrophobic zipper mediate the coupling between an insect pheromone receptor BmOR3 and downstream effectors

Pheromone receptors (PRs) recognize specific pheromone compounds to guide the behavioral outputs of insects, which are the most diverse group of animals on earth. The activation of PRs is known to couple to the calcium permeability of their coreceptor (Orco) or putatively with G proteins; however, the underlying mechanisms of this process are not yet fully understood. Moreover, whether this transverse seven transmembrane domain (7TM)-containing receptor is able to couple to arrestin, a common effector for many conventional 7TM receptors, is unknown. Herein, using the PR BmOR3 from the silk moth Bombyx mori and its coreceptor BmOrco as a template, we revealed that an agonist-induced conformational change of BmOR3 was transmitted to BmOrco through transmembrane segment 7 from both receptors, resulting in the activation of BmOrco. Key interactions, including an ionic lock and a hydrophobic zipper, are essential in mediating the functional coupling between BmOR3 and BmOrco. BmOR3 also selectively coupled with Gi proteins, which was dispensable for BmOrco coupling. Moreover, we demonstrated that trans-7TM BmOR3 recruited arrestin in an agonist-dependent manner, which indicates an important role for BmOR3–BmOrco complex formation in ionotropic functions. Collectively, our study identified the coupling of G protein and arrestin to a prototype trans-7TM PR, BmOR3, and provided important mechanistic insights into the coupling of active PRs to their downstream effectors, including coreceptors, G proteins, and arrestin.

Probing The Structural Basis of Metabotropic Glutamate Receptor 5 (mGlu 5 ) Activation and Positive Allosteric Modulation

The metabotropic glutamate receptor 5 (mGlu5) is a class C G protein-coupled receptor (GPCR) that has emerged as a promising drug target for multiple central nervous system disorders. Structurally, mGlu5 forms obligate dimers. Each protomer possesses a 7-transmembrane (7TM) domain which is connected through a cysteine-rich domain (CRD) to a large extracellular domain (ECD), encompassing the orthosteric ligand-binding Venus flytrap domain (VFD). Recent spectroscopic studies on mGlu5 signaling demonstrated large-scale conformational changes underlie signal transmission from the ECD to the G protein coupling 7TM domain. Although these studies reported the necessity of extracellular loops (ECL) for activation of mGlu5 by orthosteric agonists, specific residues related to transmission of receptor activation remain unknown. We used a combination of site-directed mutagenesis, Ca2+ mobilization assays and radioligand binding studies to identify residues within the ECLs that aid in the propagation of orthosteric agonist-induced conformational changes in the VFD to the 7TM. Multiple ECL2 residues markedly reduced activation by glutamate but not allosteric agonists. These same mutations had no effect on receptor expression and thus likely contribute to the mGlu­5 activation network. Select mutations that compromised glutamate activation could be rescued with positive allosteric modulators (PAMs). mGlu5 PAMs are promising potential new therapeutic strategies for schizophrenia and cognition, however, structure-activity relationships for mGlu5 PAMs are notoriously difficult to interpret. A deeper understanding of the structural basis of dynamic receptor activation and modulation processes will aid future rational structure-based drug discovery efforts

Mechanism of gating and partial agonist action in the glycine receptor

Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.

Spinal Inhibition of GABAB Receptors by the Extracellular Matrix Protein Fibulin-2 in Neuropathic Rats.

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). Receptor interaction with partner proteins has emerged as a novel mechanism to alter GPCR signaling in pathophysiological conditions. We propose here that GABAB activity is inhibited through the specific binding of fibulin-2, an extracellular matrix protein, to the B1a subunit in a rat model of neuropathic pain. We demonstrate that fibulin-2 hampers GABAB activation, presumably through decreasing agonist-induced conformational changes. Fibulin-2 regulates the GABAB-mediated presynaptic inhibition of neurotransmitter release and weakens the GABAB-mediated inhibitory effect in neuronal cell culture. In the dorsal spinal cord of neuropathic rats, fibulin-2 is overexpressed and colocalized with B1a. Fibulin-2 may thus interact with presynaptic GABAB receptors, including those on nociceptive afferents. By applying anti-fibulin-2 siRNA in vivo, we enhanced the antinociceptive effect of intrathecal baclofen in neuropathic rats, thus demonstrating that fibulin-2 limits the action of GABAB agonists in vivo. Taken together, our data provide an example of an endogenous regulation of GABAB receptor by extracellular matrix proteins and demonstrate its functional impact on pathophysiological processes of pain sensitization.

NMDA receptor channel gating control by the pre-M1 helix.

The NMDA receptor (NMDAR) is an ionotropic glutamate receptor formed from the tetrameric assembly of GluN1 and GluN2 subunits. Within the flexible linker between the agonist binding domain (ABD) and the M1 helix of the pore-forming transmembrane helical bundle lies a two-turn, extracellular pre-M1 helix positioned parallel to the plasma membrane and in van der Waals contact with the M3 helix thought to constitute the channel gate. The pre-M1 helix is tethered to the bilobed ABD, where agonist-induced conformational changes initiate activation. Additionally, it is a locus for de novo mutations associated with neurological disorders, is near other disease-associated de novo sites within the transmembrane domain, and is a structural determinant of subunit-selective modulators. To investigate the role of the pre-M1 helix in channel gating, we performed scanning mutagenesis across the GluN2A pre-M1 helix and recorded whole-cell macroscopic and single channel currents from HEK293 cell-attached patches. We identified two residues at which mutations perturb channel open probability, the mean open time, and the glutamate deactivation time course. We identified a subunit-specific network of aromatic amino acids located in and around the GluN2A pre-M1 helix to be important for gating. Based on these results, we are able to hypothesize about the role of the pre-M1 helix in other NMDAR subunits based on sequence and structure homology. Our results emphasize the role of the pre-M1 helix in channel gating, implicate the surrounding amino acid environment in this mechanism, and suggest unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.

Effect of thermostable mutations on the neurotensin receptor 1 (NTSR1) activation state

Neurotensin (NTS) is a 13-amino acid neuropeptide with neuroendocrine and vasoactive functions that is widely expressed in the central nervous system and gastrointestinal tract. NTS is sensed by a multiple cell surface proteins including two G protein-coupling receptors (GPCRs): NTS receptors 1 and 2 (NTSR1 and NTSR2). Crystal structures of NTSR1 have successfully elucidated agonist binding within the orthosteric pocket of receptor but have not revealed the full activation state of the receptor. Recent studies have attempted to address this challenge by improving NTSR1 crystal formation via thermostable mutants; unfortunately, these mutations exhibit functional defects in the G protein coupling of NTSR1. Here, we have used molecular dynamics simulations to gain greater insights into how the amino acid substitutions used in these thermostable mutants (E166A, L310A and F358A) impact receptor activation. Our simulations indicate that wild-type NTSR1 in complex with NTS8-13 shows more active-like features including a 17.7 Å shift in TM6, reflecting a network of polar and aromatic interactions orchestrating agonist-induced receptor conformational changes. We also provide evidence indicating that F358 is a precursor to the rotamer change observed in W321, and our collective analysis also suggests that mutations E166A and F358A are less impactful to G protein coupling than L310A. Furthermore, we believe that our findings can be used to design future NTSR1 mutants that do not interfere with agonist-induced conformational changes and downstream G protein coupling and thus produce structures that will allow visualization of the fully activated receptor conformation.

Role of palmitoylation of cysteine 415 in functional coupling CB1 receptor to Gαi2 protein.

In this study, we investigated the role of CB1 palmitoylation in modulating the functional interaction with G proteins both in the absence and presence of agonist binding. Our data show that the nonpalmitoylated CB1 receptor significantly reduced its association with Gαi2 . The agonist stimulation induced a partial dissociation of Gαi2 proteins from the wild-type receptor, while on the C415A mutant the agonist binding was not able to induce a significant dissociation of Gαi2 from the receptor. The lack of palmitoyl chain seems to hamper the ability of the receptor to functionally interact with the Gαi2 and indicate that the palmitoyl chain is responsible for the functional transmission of the agonist-induced conformational change in the receptor of the G protein. These data were further corroborated by molecular dynamics simulations. Overall these results suggest that palmitoylation of the CB1 receptor finely tunes its interaction with G proteins and serves as a targeting signal for its functional regulation. Of note, the possibility to reversibly modulate the palmitoylation of CB1 receptor may offer a coordinated process of regulation and could open new therapeutic approaches.

Sigma-1 Receptor Plays a Negative Modulation on N-type Calcium Channel.

The sigma-1 receptor is a 223 amino acids molecular chaperone with a single transmembrane domain. It is resident to eukaryotic mitochondrial-associated endoplasmic reticulum and plasma membranes. By chaperone-mediated interactions with ion channels, G-protein coupled receptors and cell-signaling molecules, the sigma-1 receptor performs broad physiological and pharmacological functions. Despite sigma-1 receptors have been confirmed to regulate various types of ion channels, the relationship between the sigma-1 receptor and N-type Ca2+ channel is still unclear. Considering both sigma-1 receptors and N-type Ca2+ channels are involved in intracellular calcium homeostasis and neurotransmission, we undertake studies to explore the possible interaction between these two proteins. In the experiment, we confirmed the expression of the sigma-1 receptors and the N-type calcium channels in the cholinergic interneurons (ChIs) in rat striatum by using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) and immunofluorescence staining. N-type Ca2+ currents recorded from ChIs in the brain slice of rat striatum was depressed when sigma-1 receptor agonists (SKF-10047 and Pre-084) were administrated. The inhibition was completely abolished by sigma-1 receptor antagonist (BD-1063). Co-expression of the sigma-1 receptors and the N-type calcium channels in Xenopus oocytes presented a decrease of N-type Ca2+ current amplitude with an increase of sigma-1 receptor expression. SKF-10047 could further depress N-type Ca2+ currents recorded from oocytes. The fluorescence resonance energy transfer (FRET) assays and co-immunoprecipitation (Co-IP) demonstrated that sigma-1 receptors and N-type Ca2+ channels formed a protein complex when they were co-expressed in HEK-293T (Human Embryonic Kidney -293T) cells. Our results revealed that the sigma-1 receptors played a negative modulation on N-type Ca2+ channels. The mechanism for the inhibition of sigma-1 receptors on N-type Ca2+ channels probably involved a chaperone-mediated direct interaction and agonist-induced conformational changes in the receptor-channel complexes on the cell surface.

Activation mechanism of endothelin ETB receptor by endothelin-1.

Endothelin, a 21-amino-acid peptide, participates in various physiological processes, such as regulation of vascular tone, humoral homeostasis, neural crest cell development and neurotransmission. Endothelin and its G-protein-coupled receptor are involved in the development of various diseases, such as pulmonary arterial hypertension, and thus are important therapeutic targets. Here we report crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6 and 7 move and envelop the entire endothelin peptide, in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G-protein coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A G-protein-coupled receptors.

Activation and modulation of the nicotinic receptor

Nicotinic acetylcholine receptors (AChRs) are the best characterized ion channels representing the Cys-loop receptor superfamily. AChRs have all the machinery to recognize the neurotransmitter ACh and other agonists such as nicotine, and to transduce the agonist-induced conformational changes into the opening of the intrinsic cation channel. The gating machinery couples ligand binding, located at the extracellular portion, to the opening of the ion channel, located at the transmembrane region. The interface between the extracellular and the transmembrane domains is considered one of the most important structural and functional features for the process of gating. And finally, in the prolonged presence of agonists, the AChR becomes desensitized. Several drugs affect the functioning of these receptors. Among them, positive allosteric modulators (PAM) have acquired importance since are novel drugs for several neurological diseases. PAMs do not bind to the orthostetic binding sites but allosterically enhance the activity elicited by agonists by increasing the gating process and/or by decreasing desensitization. Instead, negative allosteric modulators (NAMs) produce the opposite effects. Interestingly, this negative effect is similar to that found for another class of allosteric drugs, i.e. non competitive antagonists (NCAs). However, the main difference between both categories of drugs is based on their distinct binding site locations. Although both NAMs and NCAs do not bind to the agonist sites, NACs bind to sites located in the ion channel, whereas NAMs bind to nonluminal sites. Interestingly, PAMs and NAMs might be developed as potential medications for the treatment of several diseases involving AChRs including, dementia-, skin-, and immunological-related diseases, drug addiction, and cancer. More exciting is the potential combination of specific agonists with PAMs. However, we are still in the beginning of understanding how these compounds act and how these drugs can be used therapeutically.

Non-ionotropic signaling by the NMDA receptor: controversy and opportunity.

Provocative emerging evidence suggests that the N-methyl-d-aspartate (NMDA) receptor can signal in the absence of ion flux through the receptor. This non-ionotropic signaling is thought to be due to agonist-induced conformational changes in the receptor, independently of channel opening. Non-ionotropic NMDA receptor signaling has been proposed to be sufficient to induce synaptic long-term depression (LTD), directly challenging the decades-old model that prolonged low-level calcium influx is required to induce LTD. Here, we briefly review these recent findings, focusing primarily on the potential role of non-ionotropic signaling in NMDA receptor-mediated LTD. Further reports concerning additional roles of non-ionotropic NMDA receptor signaling are also discussed. If validated, this new view of NMDA receptor-mediated signaling will usher in an exciting new era of exploring synapse function and dysfunction.

Structural Determinants for the Binding of Morphinan Agonists to the μ-Opioid Receptor.

Atomistic descriptions of the μ-opioid receptor (μOR) noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP) and hydromorphone (HMP), are investigated using molecular dynamics (MD) simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor’s activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation.

Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex.

Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for GαiGDP independent of Gβγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the GαGPR complex in a manner analogous to receptor coupling to the Gαβγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α2A adrenergic receptor (α2AAR-Gαi2YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oβγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of α2AAR-Gαi2YFP(C352I):AGS3Rluc and α2AAR-Gαi2YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or Gβγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oβγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαβγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.

Heterotrimeric G protein-mediated signaling and its non-canonical regulation in the heart.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) regulate a multitude of signaling pathways in mammalian cells by transducing signals from G protein-coupled receptors (GPCRs) to effectors, which in turn regulate cellular function. In the myocardium, G protein signaling occurs in all cardiac cell types and is centrally involved in the regulation of heart rate, pump function, and vascular tone and in the response to hemodynamic stress and injury. Perturbations in G protein-mediated signaling are well known to contribute to cardiac hypertrophy, failure, and arrhythmias. Most of the currently used drugs for cardiac and other diseases target GPCR signaling. In the canonical G protein signaling paradigm, G proteins that are located at the cytoplasmic surface of the plasma membrane become activated after an agonist-induced conformational change of GPCRs, which then allows GTP-bound Gα and free Gβγ subunits to activate or inhibit effector proteins. Research over the past two decades has markedly broadened the original paradigm with a GPCR-G protein-effector at the cell surface at its core by revealing novel binding partners and additional subcellular localizations for heterotrimeric G proteins that facilitate many previously unrecognized functional effects. In this review, we focus on non-canonical and epigenetic-related mechanisms that regulate heterotrimeric G protein expression, activation, and localization and discuss functional consequences using cardiac examples where possible. Mechanisms reviewed involve microRNAs, histone deacetylases, chaperones, alternative modes of G protein activation, and posttranslational modifications. Some of these newly characterized mechanisms may be further developed into novel strategies for the treatment of cardiac disease and beyond.

Comment on “The Use of BRET to Study Receptor-Protein Interactions”

This 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 interactions (PPIs) in 1999 (2). Since then, the technology constantly evolved by combining new donor and acceptor couples and developing 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 oligomerization of seven-transmembrane-spanning G protein-coupled receptors (GPCRs). The BRET technology provides an attractive 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 signals 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 application 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 generated by an agonist-driven oligomerization or agonist-induced conformational changes within preassembled oligomers. Discrimination between these two possibilities is not trivial but has become possible due to the development of BRET donor saturation experiments 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 receptor families like tyrosine kinase receptors and cytokine receptors. This diversification demonstrates the general feature of this technique. 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 article of Siddiqui et al. (12). Receptor–effector interactions have been also monitored by BRET for two privileged GPCR interacting partners, 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 signaling 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 technology that should be of interest for any scientist interested in monitoring PPI in intact cells.