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The δ-opioid receptor (δ-OR) couples to Gi/Go proteins to modulate a variety of responses in the nervous system. Τhe regulator of G protein signalling 4 (RGS4) was previously shown to directly interact within the C-terminal region of δ-OR using its N-terminal domain to negatively modulate opioid receptor signalling. Herein, using molecular dynamics simulations and in vitro pull-down experiments we delimit this interaction to 12 helix 8 residues of δ-ΟR and to the first 17 N-terminal residues (NT) of RGS4. Monitoring the complex arrangement and stabilization between RGS4 and δ-OR by molecular dynamics simulations combined with mutagenesis studies, we defined that two critical interactions are formed: one between Phe329 of helix8 of δ-ΟR and Pro9 of the NT of RGS4 and the other a salt bridge between Glu323 of δ-ΟR and Lys17 of RGS4. Our observations allow drafting for the first time a structural model of a ternary complex including the δ-opioid receptor, a G protein and a RGS protein. Furthermore, the high degree of conservation among opioid receptors of the RGS4-binding region, points to a conserved interaction mode between opioid receptors and this important regulatory protein.

Summary The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, β-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy., Graphical abstract, Highlights • Obesity-associated MC4R mutations that do not reduce cAMP disrupt other processes • MC4R mutations impact receptor homodimerization, endocytosis, and trafficking • Obesity-protecting mutations increase plasma membrane MC4Rs and enhance signaling • Multiple mechanisms regulate melanocortin tone to a physiologically relevant level, Using mutations in the human Melanocortin-4 Receptor (MC4R), Brouwers et al. identify receptor trafficking and endocytosis, coupling to Gαs/β-arrestins, and homodimerization as mechanisms involved in the regulation of body weight that may be targeted for weight loss therapy.

The serotonin 2A (5-HT2A) receptor is a G-protein coupled receptor (GPCR) with a conserved disulfide bridge formed by Cys148 (transmembrane helix 3, TM3) and Cys227 (extracellular loop 2, ECL-2). We hypothesized that disulfide bridges may determine serotonin 5-HT2A receptor functions such as receptor activation, functional selectivity and ligand recognition. We used the reducing agent dithiothreitol (DTT) to determine how the reduction of disulfide bridges affects radioligand binding, second messenger mobilization and receptor dimerization. A DTT-induced decrease in the number of binding sites (1190 ± 63.55 fmol/mg protein for control cells compared with 921.2 ± 60.84 fmol/mg protein for DTT-treated cells) as well as in the efficacy of both signalling pathways characterized was observed, although the affinity and potency were unchanged. Bioluminiscence resonance energy transfer (BRET) assays revealed the DTT treatment did not modify the homodimeric nature of serotonin 5-HT2A receptors. In molecular dynamic simulations, the ECL-2 of the receptor with a broken cysteine bond adopts a wider variety of conformations, some of which protrude deeper into the receptor orthosteric binding pocket leading to collapse of the pocket. A shrunken binding pocket would be incapable of accommodating lysergic acid diethylamide (LSD). Our findings suggest that the decrease of efficacy may be due to disruption of disulfide bridge between TM3 and ECL-2. This reveals the integrity of the ECL-2 epitope, which should be explored in the development of novel ligands acting as allosteric modulators of serotonin 5-HT2A receptors. his work was supported by Ministerio de Ciencia e Innovacion (SAF2009-13609-C04-01), Ministerio de Economia y Competitividad (SAF2014-57138-C2-2R), and Innopharma project (PI12/00742; Ministerio de Economía y Competitividad -FEDER). A.I. was recipient of a FPI fellowship from Ministerio de Ciencia e Innovacion. M. Cimadevila is recipient of a financial support from the Xunta de Galicia (ED481A-2016/096) and the European Union (European Social Fund - ESF).

Summary Astrocytes tile the central nervous system and are widely implicated in brain diseases, but the molecular mechanisms by which astrocytes contribute to brain disorders remain incompletely explored. By performing astrocyte gene expression analyses following 14 experimental perturbations of relevance to the striatum, we discovered that striatal astrocytes mount context-specific molecular responses at the level of genes, pathways, and upstream regulators. Through data mining, we also identified astrocyte pathways in Huntington’s disease (HD) that were reciprocally altered with respect to the activation of striatal astrocyte G protein-coupled receptor (GPCR) signaling. Furthermore, selective striatal astrocyte stimulation of the Gi-GPCR pathway in vivo corrected several HD-associated astrocytic, synaptic, and behavioral phenotypes, with accompanying improvement of HD-associated astrocyte signaling pathways, including those related to synaptogenesis and neuroimmune functions. Overall, our data show that astrocytes are malleable, using context-specific responses that can be dissected molecularly and used for phenotypic benefit in brain disorders.

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs., G-protein-coupled receptors trigger several signalling pathways and their activity was proposed to be allosteric modulated by cholesterol. Here the authors use molecular dynamics simulations and ligand binding assays to show that membrane cholesterol can bind to adenosine A2A receptor orthosteric site.

G-protein coupled receptors (GPCRs) are the most important class of current pharmacological targets. However, it is now widely acknowledged that their regulation is more complex than previously thought: the evidence that GPCRs can couple to several effector pathways, and the existence of biased agonists able to activate them differentially, has introduced a new level of complexity in GPCR drug research. Considering bias represents a challenge for the research of new GPCR modulators, because it demands a detailed characterization of compound properties for several effector pathways. Still, biased ligands could provide an opportunity to modulate GPCR function in a finer way and to separate therapeutic from side effects. Nowadays, a variety of agonists for GPCRs have been described, which differ in their ability to promote receptor coupling to different Gprotein families or even subunits, recruit signal transducers such as arrestins, activate a variety of downstream molecular pathways and induce certain phosphorylation signatures or gene expression patterns. In this review, we will cover some of the experimental techniques currently used to understand and characterize biased agonism and discuss their strengths and limitations. Additionally, we will comment on the computational efforts that are being devoted to study ligand-induced bias and on the potential they hold for rationalizing its structural determinants. Finally, we will discuss which of these strategies could be used for the rational design of biased ligands and give some examples of the potential therapeutic value of this class of compounds.

Detection of biased agonists for the serotonin 5-HT2A receptor can guide the discovery of safer and more efficient antipsychotic drugs. However, the rational design of such drugs has been hampered by the difficulty detecting the impact of small structural changes on signaling bias. To overcome these difficulties, we characterized the dynamics of ligand-receptor interactions of known biased and balanced agonists using molecular dynamics simulations. Our analysis revealed that interactions with residues S5.46 and N6.55 discriminate compounds with different functional selectivity. Based on our computational predictions, we selected three derivatives of the natural balanced ligand serotonin and experimentally validated their ability to act as biased agonists. Remarkably, our approach yielded compounds promoting an unprecedented level of signaling bias at the 5-HT2A receptor, which could help interrogate the importance of particular pathways in conditions like schizophrenia.

G protein-coupled receptors (GPCRs) constitute one of the largest classes of cell surface receptors. GPCR biology has been a subject of widespread interest owing to the functional relevance of these receptors and their potential importance in the development of new drugs. At present, over 30% of all launched drugs target these receptors. GPCRs have been considered for a long time to function as monomeric entities and the idea of GPCR dimerization and oligomerization was initially accepted with disbelief. However, a significant amount of experimental and molecular modeling evidence accumulated during the last several years suggests that the process of GPCRs dimer or oligomer formation is a general phenomenon, in some cases even essential for receptor function. Among the many methods to study GPCR dimerization and oligomerization, modern biophysical techniques such as those based on resonance energy transfer (RET) and particularly bioluminescence resonance energy transfer (BRET) have played a leading role. RET methods are commonly applied as non-destructive indicators of specific protein-protein interactions (PPIs) in living cells. Data from numerous BRET experiments support the idea that the process of GPCR oligomerization may be relevant in many physiological and pathological conditions. The application of BRET to the study of GPCRs is not only limited to the assessment of receptor oligomerization but also expands to the investigation of the interactions of GPCRs with other proteins, including G proteins, G protein-coupled receptor kinases, β-arrestins or receptor tyrosine kinases, as well as to the characterization of GPCR activation and signaling. In this review, we briefly summarize the fundaments of BRET, discuss new trends in this technology and describe the wide range of applications of BRET to study GPCRs.

Sphingosine-1-phosphate (S1P) is an immunomodulatory lipid mediator that plays an important role in lymphocyte trafficking. Elevated levels of S1P are found in bronchoalveolar lavage (BAL) fluid of patients with asthma; however, its role in disease is not known. FTY720, a synthetic analog of S1P, has been shown to abrogate allergic inflammation and airway hyperresponsiveness following acute allergen challenge. However, its effects on asthmatic airway remodeling induced by repeated allergen exposure are unknown. Ovalbumin (OVA)-sensitized rats were challenged on days 14, 19, and 24 after sensitization. FTY720 or vehicle (PBS) therapy was administered 1 h prior to each challenge. BAL fluid and quantitative histological analysis were performed 48 h after the last challenge. FTY720 inhibited OVA-induced features of airway remodeling including increased airway smooth muscle mass and bronchial neovascularization, without affecting lymphocyte numbers in secondary lymphoid organs. Furthermore, CD3+ cells adjacent to airway smooth muscle bundles were increased in OVA-challenged rats but the increase was inhibited by FTY720. There was an expansion of bronchus-associated lymphoid tissue following FTY720 treatment of OVA-challenged animals. Real-time quantitative PCR revealed that Th2-associated transcription factors were inhibited following FTY720 therapy. Airway remodeling is a cardinal feature of severe asthma. These results demonstrate that allergen-driven airway remodeling can be inhibited by FTY720, offering potential new therapies for the treatment of severe asthma.

Crosstalk between G protein-coupled receptors (GPCRs) is one of the key mechanisms used by the cell for integrating multiple signaling pathways. Functional crosstalk at the level of signaling pathways was initially thought to regulate receptor function. Importantly, the existence of GPCR heteromers demonstrates that direct physical interactions between GPCRs could also be behind the crosstalk phenomenon. Neurological disorders such as Parkinson's disease (PD) and schizophrenia have been linked to a dysfunctional communication between certain GPCRs. In this review, we discuss functional and physical crosstalk of the main GPCR families involved in the aforementioned disorders. In addition, we analyze the available structural information on physical crosstalk and highlight some strategies in drug discovery based on these crosstalk mechanisms.