G Protein-coupled Research Articles

Fast-diffusing receptor collisions with slow-diffusing peptide ligand assemble the ternary parathyroid hormone–GPCR–arrestin complex

The assembly of a peptide ligand, its receptor, and β-arrestin (βarr) into a ternary complex within the cell membrane is a crucial aspect of G protein-coupled receptor (GPCR) signaling. We explore this assembly by attaching fluorescent moieties to the parathyroid hormone (PTH) type 1 receptor (PTH1R), using PTH as a prototypical peptide hormone, along with βarr and clathrin, and recording dual-color single-molecule imaging at the plasma membrane of live cells. Here we show that PTH1R exhibits a near-Brownian diffusion, whereas unbound hormone displays limited mobility and slow lateral diffusion at the cell surface. The formation of the PTH–PTH1R–βarr complex occurs in three sequential steps: (1) receptor and ligand collisions, (2) phosphoinositide (PIP3)-dependent recruitment and conformational change of βarr molecules at the plasma membrane, and (3) collision of most βarr molecules with the ligand-bound receptor within clathrin clusters. Our results elucidate the non-random pathway by which PTH–PTH1R–βarr complex is formed and unveil the critical role of PIP3 in regulating GPCR signaling.

Structural plasticity of arrestin-G protein coupled receptor complexes as a molecular determinant of signaling

G protein coupled receptors (GPCRs) are critically regulated by arrestins. In this study, high-resolution data was combined with molecular dynamics simulations to infer the determinants of β-arrestin 1 (βarr1)-GPCR coupling, using the V2 vasopressin receptor (V2R) as a model system.The study highlighted the extremely high plasticity of βarr1-GPCR complexes, dependent on receptor type, state, and membrane environment. The multiple functions of receptor-bound βarr1 are likely determined by the interplay of intrinsic flexibility and collective motions both as a bi-domain protein and as a whole. The two major collective motions of the whole βarr1, consisting in rotation parallel to the membrane plane and inclination with respect to the receptor main axis, are distinctly linked to the two intermolecular interfaces involved in tail and core interactions.The intermolecular dynamic coupling between βarr1 and V2R depends on the allosteric effect of the agonist arginine-vasopressin (AVP). In the absence of AVP the dynamic coupling concerns only tail interactions, while in the presence of AVP it involves both tail and core interactions. This suggests that constitutive and agonist-induced arrestin-receptor dynamic coupling is linked to distinct arrestin functions.

Role of chemokines in aging and age-related diseases

Chemokines (chemotactic cytokines) play essential roles in developmental process, immune cell trafficking, inflammation, immunity, angiogenesis, cellular homeostasis, aging, and tumorigenesis. Chemokines also modulate response to immunotherapy, and consequently influence the therapeutic outcome. The mechanisms underlying these processes are accomplished by interaction of chemokines with their cognate cell surface G protein-coupled receptors (GPCRs) and subsequent cellular signaling pathways. Chemokines play crucial role in influencing aging process and age-related diseases across various tissues and organs, primarialy through inflammatory responses (inflammaging), recruitment of macrophages, and orchestrated trafficking of other immune cells. Chemokines are categorized in four distinct groups based on the position and number of the N-terminal cysteine residues; namely, the CC, CXC, CX3C, and (X)C. They mediate inflammatory responses, and thereby considerably impact aging process across multiple organ-systems. Therefore, understanding the underlying mechanisms mediated by chemokines may be of crucial importance in delaying and/or modulating the aging process and preventing age-related diseases. In this review, we highlight recent progress accomplished in understanding the role of chemokines and their cellular signaling pathways involved in aging and age-relaed diseases of various organs. Moreover, we explore potential therapeutic strategies involving chemokines and chemokine receptor antagonists aimed at reducing aging and mitigating age-related diseases. One of the modern methods in this direction involves use of chemokine receptor antagonists and anti-chemokines, which suppress the pro-inflammatory response, thereby helping in resolution of inflammation. Considering the wide-spectrum of functional involvements of chemokines in aging and associated diseases, several clinical trials are being conducted to develop therapeutic approaches using chemokine receptor antagonists to improve life span and promote healthy aging.

The Current Landscape in the Development of Small-molecule Modulators Targeting Sphingosine-1-phosphate Receptors to Treat Neurodegenerative Diseases.

Sphingosine 1-phosphate (S1P) is extensively researched as a lysophospholipid and is crucial in various physiological and pathological processes. It achieves this via signalling through five different subtypes of G protein-coupled receptors (GPCRs), namely S1PR1 to S1PR5. S1PR modulators possess the ability to traverse the blood-brain barrier, potentially leading to direct ac-tions within the Central Nervous System (CNS). S1PR modulators specifically bind to receptors located on the surface of naive and central memory lymphocytes, causing these cells to be trapped or confined within the lymph node. The investigation of the S1P pathway has resulted in the ap-proval of three S1PR modulators, namely fingolimod, siponimod, and ozanimod, as medications for the treatment of patients suffering from Multiple Sclerosis (MS). Additionally, new S1PR modulators, such as ponesimod and etrasimod, are currently being developed and tested in clini-cal trials. Research on the creation of S1P modulators in neurodegenerative illnesses is ongoing as scientists continue to explore novel possibilities for selective S1P modulators. This study provides a concise overview of sphingolipid metabolism, the mechanism by which S1P receptors are af-fected, and the structural characteristics of several small molecule S1P modulators, with a particu-lar focus on their structure-activity connections.

Dorsal raphe to basolateral amygdala corticotropin-releasing factor circuit regulates cocaine-memory reconsolidation.

Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocaine-related contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocaine-memory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. Next, they were briefly re-exposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 µL/hemisphere) immediately after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed the glutamatergic neuronal marker, vesicular glutamate transporter 3. Together, these findings suggest that the DRCRF → BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF and/or glutamate release in the BLA.

Exploring Bias in GPCR Signaling and its Implication in Drug Development: A One-Sided Affair.

G protein-coupled receptors (GPCRs) play a pivotal role in regulating numerous physiological processes through their interactions with two key effectors: G proteins and β-arrestins (βarrs). This makes them crucial targets for therapeutic drug development. Interestingly, the evolving concept of biased signaling where ligands selectively activate either the G proteins or the βarrs has not only refined our understanding of segregation of physiological responses downstream of GPCRs but has also revolutionized drug discovery, offering the potential for treatments with enhanced efficacy and minimal side effects. This Review explores the mechanisms behind biased agonism, exploring it through various lenses, including ligand, receptor, cellular systems, location, and tissue-specific biases. It also offers structural insights into both orthosteric and allosteric ligand-binding pockets, structural rearrangements associated with the loops, and how ligand-engineering can contribute to biased signaling. Moreover, we also discuss the unique conformational signature in an intrinsically biased GPCR, which currently remains relatively less explored and adds a new dimension in biased signaling. Lastly, we address the translational challenges and practical considerations in characterizing bias, emphasizing its therapeutic potential and the latest advancements in drug development. By designing ligands that target specific signaling pathways, biased signaling presents a transformative approach to creating safer and more effective therapies. This Review focuses on our current understanding of GPCR-biased signaling, discussing potential mechanisms that lead to bias, the effect of bias on GPCR structures at a molecular level, recent advancements, and its profound potential to drive innovation in drug discovery.

The membrane domains of mammalian adenylyl cyclases are lipid receptors.

The biosynthesis of cyclic 3',5'-adenosine monophosphate (cAMP) by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here, we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1-7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo. Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5-mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR-Gsα signaling regulating the biosynthesis of cAMP.

GPTrans: A Biological Language Model-Based Approach for Predicting Disease-Associated Mutations in G Protein-Coupled Receptors.

Accurately predicting mutations in G protein-coupled receptors (GPCRs) is critical for advancing disease diagnosis and drug discovery. In response to this imperative, GPTrans has emerged as a highly accurate predictor of disease-related mutations in GPCRs. The core innovation of GPTrans resides in the design of a novel feature extraction network, that is capable of integrating features from both wildtype and mutant protein variant sites, utilizing multifeature connections within a transformer framework to ensure comprehensive feature extraction. A key aspect of GPTrans's effectiveness is our introduction of an innovative deep feature integration strategy, which merges embeddings and class tokens from multiple protein language models, including evolutionary scale modeling and ProtTrans, thus shedding light on the biochemical properties of proteins. Leveraging transformer components and a self-attention mechanism, GPTrans captures higher-level representations of protein features. Employing both wildtype and mutation site information for feature fusion not only enriches the predictive feature set but also avoids the common issue of overestimation associated with sequence-based predictions. This approach distinguishes GPTrans, enabling it to significantly outperform existing methods. Our evaluations across diverse GPCR data sets, including ClinVar and MutHTP, demonstrate GPTrans's superior performance, with average AUC values of 0.874 and 0.590 in 10-fold cross-validation. Notably, compared to the AlphaMissense method, GPTrans exhibited a remarkable 38.03% improvement in accuracy when predicting disease-associated mutations in the MutHTP data set. A thorough analysis of the predicted results further validates the model's effectiveness. The source code, data sets, and prediction results for GPTrans are available for academic use at https://github.com/EduardWang/GPTrans.

GPR88 impairs the signaling of kappa opioid receptors in a heterologous system and in primary striatal neurons

The physiological role of GPR88, an orphan G protein-coupled receptor (GPCR) predominantly expressed in the striatum, remains unclear, despite its altered expression in parkinsonian animal models. GPR88 is known to interact with other GPCRs. Specifically, GPR88 expression inhibits signaling mediated by the μ-opioid receptor in cells coexpressing both receptors. The effect of GPR88 on the kappa-opioid receptor (KOR) is less understood. In this study, we examine the interaction between GPR88 and KOR, and the impact of GPR88 expression on KOR-mediated signaling in heterologous cells and primary striatal neurons. Bioluminescence resonance energy transfer and proximity ligation assays revealed an interaction between GPR88 and KOR. Functional assays showed that GPR88 antagonized the effects of U69,593, a selective KOR agonist, on forskolin-stimulated cAMP levels, β-arrestin-2 recruitment, and phosphorylation of extracellular signal-regulated kinases (ERK1/2) in HEK-293T cells coexpressing both receptors. In primary striatal neurons, GPR88 and KOR complexes were observed, with KOR activation effects enhanced when GPR88 expression was suppressed using RNA interference. These results suggest that GPR88 and KOR are coexpressed in striatal neurons, where GPR88 inhibits KOR activation. Notably, the GPR88-KOR heteromer was more prevalent in dopamine D1-receptor-containing neurons of the direct pathway of the basal ganglia. Given the roles of KORs in dopamine release, motor function regulation, and pain and reward perception, the GPR88-KOR interaction warrants further investigation in the context of neuropathic pain, Parkinson's disease, and neuropsychiatric disorders.

High-affinity ELR+ chemokine ligands show G protein bias over β-arrestin recruitment and receptor internalization in CXCR1 signalling

The human CXC chemokine receptor 1 (CXCR1), a G protein-coupled receptor (GPCR), plays significant roles in inflammatory diseases and cancer. While CXCL8 is a well-established high-affinity ligand for CXCR1, there is no consensus regarding the binding ability of the other ELR+ chemokines (CXCL1-3 and CXCL5-8). Since research has predominantly focused on CXCL8-mediated CXCR1 signalling, insight into potential signalling bias induced by different CXCR1 ligands is lacking. Therefore, in this study we first compared and clarified the binding ability of all ELR+ chemokines using a competition binding assay. In this assay CXCL1-3 and CXCL5 behaved as low-affinity ligands while CXCL6-8 were high affinity ligands. We further investigated potential ligand bias within the CXCR1 signalling system. Using NanoBRET-based assays heterotrimeric G protein dissociation, β-arrestin recruitment and receptor internalisation induced by chemokine binding to CXCR1 were investigated. A quantitative and qualitative investigation of ligand bias showed that the high-affinity ELR+ chemokines were biased towards G protein activation over β-arrestin recruitment and receptor internalisation, when CXCL8 was used as a reference ligand.

RNAi library screening reveals Gβ1, Casein Kinase 2 and ICAP-1 as novel regulators of LFA-1-mediated T cell polarity and migration.

The αLβ2 integrin LFA-1 plays a key role in T-cell adhesion to the endothelial vasculature and migration into both secondary lymphoid organs and peripheral tissues via interactions with its target protein ICAM-1, but the pathways that regulate LFA-1-mediated T-cell polarity and migration are not fully understood. In this study we screened two RNAi libraries targeting G protein-coupled receptors (GPCR)/GPCR-associated proteins and kinases in a HuT 78 T cell line model of LFA-1-stimulated T-cell migration. Based on staining of the actin cytoskeleton, multiple parameters to measure cell morphology were used to assess the contribution of 1109 genes to LFA-1-mediated T-cell polarity and migration. These RNAi screens identified a number of both novel and previously identified genes that either increased or decreased the polarity and migratory capacity of these cells. Following multiparametric analysis, hierarchical clustering and pathway analysis, three of these genes were characterized in further detail using primary human T cells, revealing novel roles for the heterotrimeric G protein subunit Gβ1 and Casein Kinase 2 in LFA-1-mediated T-cell polarity and migration in vitro. Our studies also highlighted a new role for ICAP-1, an adaptor protein previously described to be associated with β1 integrins, in β2 integrin LFA-1-directed migration in T cells. Knockdown of ICAP-1 expression in primary T cells revealed a role in cell polarity, cell velocity and transmigration towards SDF-1 for this adaptor protein. This study therefore uncovers new roles for GPCR/GPCR-associated proteins and kinases in T-cell migration and provides potential novel targets for modulation of the T-cell immune response.

Structural basis of orientated asymmetry in a mGlu heterodimer

The structural basis for the allosteric interactions within G protein-coupled receptors (GPCRs) heterodimers remains largely unknown. The metabotropic glutamate (mGlu) receptors are complex dimeric GPCRs important for the fine tuning of many synapses. Heterodimeric mGlu receptors with specific allosteric properties have been identified in the brain. Here we report four cryo-electron microscopy structures of mGlu2-4 heterodimer in different states: an inactive state bound to antagonists, two intermediate states bound to either mGlu2 or mGlu4 agonist only and an active state bound to both glutamate and a mGlu4 positive allosteric modulator (PAM) in complex with Gi protein. In addition to revealing a unique PAM binding pocket among mGlu receptors, our data bring important information for the asymmetric activation of mGlu heterodimers. First, we show that agonist binding to a single subunit in the extracellular domain is not sufficient to stabilize an active dimer conformation. Single-molecule FRET data show that the monoliganded mGlu2-4 can be found in both intermediate states and an active one. Second, we provide a detailed view of the asymmetric interface in seven-transmembrane (7TM) domains and identified key residues within the mGlu2 7TM that limits its activation leaving mGlu4 as the only subunit activating G proteins.

A massively parallel reporter assay library to screen short synthetic promoters in mammalian cells

Cellular responses to stimuli underpin discoveries in drug development, synthetic biology, and general life sciences. We introduce a library comprising 6144 synthetic promoters, each shorter than 250 bp, designed as transcriptional readouts of cellular stimulus responses in massively parallel reporter assay format. This library facilitates precise detection and amplification of transcriptional activity from our promoters, enabling the systematic development of tunable reporters with dynamic ranges of 50−100 fold. Our library proved functional in numerous cell lines and responsive to a variety of stimuli, including metabolites, mitogens, toxins, and pharmaceutical agents, generating robust and scalable reporters effective in screening assays, biomarkers, and synthetic circuits attuned to endogenous cellular activities. Particularly valuable in therapeutic development, our library excels in capturing candidate reporters to signals mediated by drug targets, a feature we illustrate across nine diverse G-protein coupled receptors (GPCRs), critical targets in drug development. We detail how this tool isolates and defines discrete signaling pathways associated with specific GPCRs, elucidating their transcriptional signatures. With its ease of implementation, broad utility, publicly available data, and comprehensive documentation, our library will be beneficial in synthetic biology, cellular engineering, ligand exploration, and drug development.

GPR180 is a new member of the Golgi-dynamics domain seven-transmembrane helix protein family

GOLD domain seven-transmembrane helix (GOST) proteins form a new protein family involved in trafficking of membrane-associated cargo. They share a characteristic extracellular/luminal Golgi-dynamics (GOLD) domain, possibly responsible for ligand recognition. Based on structural homology, GPR180 is a new member of this protein family, but little is known about the cellular role of GPR180. Here we show the X-ray structure of the N-terminal domain of GPR180 (1.9 Å) and can confirm the homology to GOLD domains. Using cellular imaging we show the localization of GPR180 in intracellular vesicular structures implying its exposure to acidic pH environments. With Hydrogen/Deuterium Exchange-Mass Spectrometry (HDX-MS) we identify pH-dependent conformational changes, which can be mapped to a putative ligand binding site in the transmembrane region. The results reveal GPR180’s role in intracellular vesicles and offer insights into the pH-dependent function of this conserved GOST protein.

Signaling by neutrophil G protein-coupled receptors that regulate the release of superoxide anions.

In human peripheral blood, the neutrophil granulocytes (neutrophils) are the most abundant white blood cells. These professional phagocytes are rapidly recruited from the bloodstream to inflamed tissues by chemotactic factors that signal danger. Neutrophils, which express many receptors that are members of the large family of G protein-coupled receptors (GPCRs), are critical for the elimination of pathogens and inflammatory insults, as well as for the resolution of inflammation leading to tissue repair. Danger signaling molecular patterns such as the N-formylated peptides that are formed during bacterial and mitochondrial protein synthesis and recognized by formyl peptide receptors (FPRs) and free fatty acids recognized by free fatty acid receptors (FFARs) regulate neutrophil functions. Short peptides and short-chain fatty acids activate FPR1 and FFA2R, respectively, while longer peptides and fatty acids activate FPR2 and GPR84, respectively. The activation profiles of these receptors include the release of reactive oxygen species (ROS) generated by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Activation of the oxidase and the production of ROS are processes that are regulated by proinflammatory mediators, including tumor necrosis factor α and granulocyte/macrophage colony-stimulating factor. The receptors have signaling and functional similarities, although there are also important differences, not only between the two closely related neutrophil FPRs, but also between the FPRs and the FFARs. In neutrophils, these receptors never walk alone, and additional mechanistic insights into the regulation of the GPCRs and the novel regulatory mechanisms underlying the activation of NADPH oxidase advance our understanding of the role of receptor transactivation in the regulation of inflammatory reactions.

Integrated bioinformatics analysis reveals that CCBP2 and GPR87 are new GPCR-associated biomarkers for preeclampsia

BackgroundPreeclampsia (PE) is a multifaceted pregnancy syndrome marked by multiple system involvement and a significant contributor to maternal mortality. This condition is characterized by a critical lack of early diagnostic measures and viable therapeutic options, underscoring an urgent need for the identification of reliable markers with both diagnostic and therapeutic potential.MethodsThis study utilized Weighted Gene Co-expression Network Analysis (WGCNA) to explore the role of G protein-coupled receptors (GPCRs) in the pathogenesis of PE.ResultsOur analysis pinpointed CCBP2 (ACKR2) and GPR87 as central PE-associated GPCRs. Experimental validation of these findings revealed that both CCBP2 and GPR87 significantly inhibit the proliferation, migration, and invasion of trophoblast cells—core phenomena underlying the pathology of PE.ConclusionThus, our findings add valuable candidates to the growing list of biomarkers for preeclampsia and offer promising targets for future therapeutic development.

ZFP36 Regulates Vascular Smooth Muscle Contraction and Maintains Blood Pressure.

Hypertension remains a major risk factor for cardiovascular diseases, but the underlying mechanisms are not well understood. Zinc finger protein 36 (ZFP36) is an RNA-binding protein that regulates mRNA stability by binding to adenylate-uridylate-rich elements in the mRNA 3'-untranslated region. This study reveals that ZFP36 expression is highly elevated in the arteries of hypertensive patients and rodents. In cultured vascular smooth muscle cell (VSMC), angiotensin II (AngII) activates poly (ADP-ribose) polymerases1 (PARP1) to stimulate Zfp36 expression at the transcriptional level. VSMC-specific ZFP36 deletion reduces vessel contractility and blood pressure levels in mice. Mechanistically, ZFP36 regulates G protein-coupled receptors (GPCRs)-mediated increases in intracellular calcium levels through impairing the mRNA stability of regulator of G protein signaling 2 (RGS2). Moreover, the VSMC-specific ZFP36 deficiency attenuates AngII-induced hypertension and vascular remodeling in mice. AAV-mediated ZFP36 knockdown ameliorates spontaneous hypertension in rats. These findings elucidate that ZFP36 plays an important role in the regulation of smooth muscle contraction and blood pressure through modulating RGS2 expression. ZFP36 inhibition may represent a new therapeutic strategy for the treatment of hypertension.

Unraveling GPCRs Allosteric Modulation. Cannabinoid 1 Receptor as a Case Study.

G-protein-coupled receptors (GPCRs) constitute one of the most prominent families of integral membrane receptor proteins that mediate most transmembrane signaling processes. Malfunction of these signal transduction processes is one of the underlying causes of many human pathologies (Parkinson's, Huntington's, heart diseases, etc), provoking that GPCRs are the largest family of druggable proteins. However, these receptors have been targeted traditionally by orthosteric ligands, which usually causes side effects due to the simultaneous targeting of homologous receptor subtypes. Allosteric modulation offers a promising alternative approach to circumvent this problematic and, thus, comprehending its details is a most important task. Here we use the Cannabinoid type-1 receptor (CB1R) in trying to shed light on this issue, focusing on positive allosteric modulation. This is done by using the protein-dipole Langevin-dipole (PDLD) within the linear response approximation (LRA) framework (PDLD/S-2000) along with our coarse-grained (CG) model of membrane proteins to evaluate the dissociation constants (KBs) and cooperativity factors (αs) for a diverse series of CB1R positive allosteric modulators belonging to the 2-phenylindole structural class, considering CP55940 as an agonist. The agreement with the experimental data evinces that significantly populated allosteric modulator:CB1R and allosteric modulator:CP55940:CB1R complexes have been identified and characterized successfully. Analyzing them, it has been determined that CB1R positive allosteric modulation lies in an outwards displacement of transmembrane α helix (TM) 4 extracellular end and in the regulation of the range of motion of a compound TM7 movement for binary and ternary complexes, respectively. In this respect, we achieved a better comprehension of the molecular architecture of CB1R positive allosteric site, identifying Lys1923.28 and Gly1943.30 as key residues regarding electrostatic interactions inside this cavity, and to rationalize (at both structural and molecular level) the exhibited stereoselectivity in relation to positive allosteric modulation activity by considered CB1R allosteric modulators. Additionally, putative/postulated allosteric binding sites have been screened successfully, identifying the real CB1R positive allosteric site, and most structure-activity relationship (SAR) studies of CB1R 2-phenylindole allosteric modulators have been rationalized. All these findings point out towards the predictive value of the methodology used in the current work, which can be applied to other biophysical systems of interest. The results presented in this study contribute significantly to understand GPCRs allosteric modulation and, hopefully, will encourage a more thorough exploration of the topic.

Naltrexone blocks alcohol-induced effects on kappa-opioid receptors in the plasma membrane

Naltrexone (NTX), a homolog of the opiate antidote naloxone, is an orally active long-acting general opioid receptor antagonist used in the treatment of opiate dependence. NTX is also found to relieve craving for alcohol and is one of few FDA-approved medications for treatment of alcohol use disorder (AUD). While it was early on established that NTX acts by blocking the binding of endogenous opioid peptide ligands released by alcohol, experimental evidence emerged that could not be fully accounted for by this explanation alone, suggesting that NTX may have additional modes of action. Mu- and kappa-opioid receptors (MOP and KOP, respectively) are structurally related G-protein-coupled receptors (GPCRs), but they are anatomically differently distributed and functionally distinct, often mediating opposite responses, with MOP typically promoting euphoria and reward, while KOP is associated with dysphoria and aversive states. While the actions of NTX on MOP are extensively characterized, the interactions with KOP are not. Here, we used sensitive fluorescence-based methods with single-molecule sensitivity to study in live cells the influence of alcohol (ethanol, EtOH) on KOP and the interaction between KOP and NTX. Our data show that alcohol, at relevant concentrations (10–40 mM), alters KOP interactions with the lipid environment in the plasma membrane. The counteracting effects of NTX are exerted by both its canonical action on KOP and its hitherto unrevealed effects on the lateral dynamics and organization of lipids in the plasma membrane. The KOP-specific antagonist LY2444296, in clinical trial for major depressive disorder (MDD), blocks KOP but does not show the full action profile of NTX. The therapeutic effect of NTX treatment in AUD may in part be due to direct actions on KOP and in part due to its effect on the surrounding lipid environment.

The cell adhesion molecule CD44 acts as a modulator of 5-HT7 receptor functions

BackgroundHomo- and heteromerization of G protein-coupled receptors (GPCRs) plays an important role in the regulation of receptor functions. Recently, we demonstrated an interaction between the serotonin receptor 7 (5-HT7R), a class A GPCR, and the cell adhesion molecule CD44. However, the functional consequences of this interaction on 5-HT7R-mediated signaling remained enigmatic.MethodsUsing a quantitative FRET (Förster resonance energy transfer) approach, we determined the affinities for the formation of homo- and heteromeric complexes of 5-HT7R and CD44. The impact of heteromerization on 5-HT7R-mediated cAMP signaling was assessed using a cAMP responsive luciferase assay and a FRET-based cAMP biosensor under basal conditions as well as upon pharmacological modulation of the 5-HT7R and/or CD44 with specific ligands. We also investigated receptor-mediated G protein activation using BRET (bioluminescence resonance energy transfer)-based biosensors in both, homo- and heteromeric conditions. Finally, we analyzed expression profiles for 5-HT7R and CD44 in the brain during development.ResultsWe found that homo- and heteromerization of the 5-HT7R and CD44 occur at similar extent. Functionally, heteromerization increased 5-HT7R-mediated cAMP production under basal conditions. In contrast, agonist-mediated cAMP production was decreased in the presence of CD44. Mechanistically, this might be explained by increased Gαs and decreased GαoB activation by 5-HT7R/CD44 heteromers. Unexpectedly, treatment of the heteromeric complex with the CD44 ligand hyaluronic acid boosted constitutive 5-HT7R-mediated cAMP signaling and receptor-mediated transcription, suggesting the existence of a transactivation mechanism.ConclusionsInteraction with the hyaluronan receptor CD44 modulates both the constitutive activity of 5-HT7R as well as its agonist-mediated signaling. Heteromerization also results in the transactivation of 5-HT7R-mediated signaling via CD44 ligand.