Multiple G Protein-coupled Receptors Research Articles

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

The nematode-trapping fungus Arthrobotrys oligospora detects prey pheromones via G protein-coupled receptors.

The ability to sense prey-derived cues is essential for predatory lifestyles. Under low-nutrient conditions, Arthrobotrys oligospora and other nematode-trapping fungi develop dedicated structures for nematode capture when exposed to nematode-derived cues, including a conserved family of pheromones, the ascarosides. A. oligospora senses ascarosides via conserved MAPK and cAMP-PKA pathways; however, the upstream receptors remain unknown. Here, using genomic, transcriptomic and functional analyses, we identified two families of G protein-coupled receptors (GPCRs) involved in sensing distinct nematode-derived cues. GPCRs homologous to yeast glucose receptors are required for ascaroside sensing, whereas Pth11-like GPCRs contribute to ascaroside-independent nematode sensing. Both GPCR classes activate conserved cAMP-PKA signalling to trigger trap development. This work demonstrates that predatory fungi use multiple GPCRs to sense several distinct nematode-derived cues for prey recognition and to enable a switch to a predatory lifestyle. Identification of these receptors reveals the molecular mechanisms of cross-kingdom communication via conserved pheromones also sensed by plants and animals.

Naodesheng decoction regulating vascular function via G-protein-coupled receptors: network analysis and experimental investigations.

Introduction: Ischemic stroke (IS) is a detrimental neurological disease with limited treatment options. Recanalization of blocked blood vessels and restoring blood supply to ischemic brain tissue are crucial for post-stroke rehabilitation. The decoction Naodesheng (NDS) composed of five Chinese botanical drugs, including Panax notoginseng (Burk.) F. H. Chen, Ligusticum chuanxiong Hort., Carthamus tinctorius L., Pueraria lobata (Willd.) Ohwi, and Crataegus pinnatifida Bge., is a blood-activating and stasis-removing herbal medicine commonly used for the clinical treatment of cerebrovascular diseases in China. However, the material basis of NDS on the effects of blood circulation improvement and vascular tone regulation remains unclear. Methods: A database comprising 777 chemical metabolites of NDS was constructed. Then, the interactions between various herbal metabolites of NDS and five vascular tone modulation G-protein-coupled receptors (GPCRs), including 5-HT1AR, 5-HT1BR, β2-AR, AT1R, and ETBR, were assessed by molecular docking. Using network analysis and vasomotor experiment of the cerebral basilar artery, the potential material basis underlying the vascular regulatory effects of NDS was further explored. Results: The Naodesheng Effective Component Group (NECG) was found to induce relaxation of rat basilar artery rings precontracted using Endothelin-1 (ET-1) and KCl in vitro in a dose-dependent manner. Several metabolites of NDS, including C. tinctorius, C. pinnatifida, and P. notoginseng, were found to be the main plant resources of metabolites with high docking scores. Furthermore, several metabolites in NDS, including formononetin-7-glucoside, hydroxybenzoyl-coumaric anhydride, methoxymecambridine, puerarol, and pyrethrin II, were found to target multiple vascular GPCRs. Metabolites with moderate-to-high binding energy were verified to have good rat basilar artery-relaxing effects, and the maximum artery relaxation effects of all three metabolites, namely, isorhamnetin, kaempferol, and daidzein, were found to exceed 90%. Moreover, metabolites of NDS were found to exert a synergistic effect by interacting with vascular GPCR targets, and these metabolites may contribute to the cerebrovascular regulatory function of NDS. Discussion: The study reports that various metabolites of NDS contribute to its vascular tone regulating effects and demonstrates the multi-component and multi-target characteristics of NDS. Among them, metabolites with moderate-to-high binding scores in NDS may play an important role in regulating vascular function.

Inhibition of a novel Dickkopf-1-LDL receptor-related proteins 5 and 6 axis prevents diabetic cardiomyopathy in mice.

Anti-hypertensive agents are one of the most frequently used drugs worldwide. However, no blood pressure-lowering strategy is superior to placebo with respect to survival in diabetic hypertensive patients. Previous findings show that Wnt co-receptors LDL receptor-related proteins 5 and 6 (LRP5/6) can directly bind to several G protein-coupled receptors (GPCRs). Because angiotensin II type 1 receptor (AT1R) is the most important GPCR in regulating hypertension, this study examines the possible mechanistic association between LRP5/6 and their binding protein Dickkopf-1 (DKK1) and activation of the AT1R and further hypothesizes that the LRP5/6-GPCR interaction may affect hypertension and potentiate cardiac impairment in the setting of diabetes. The roles of serum DKK1 and DKK1-LRP5/6 signalling in diabetic injuries were investigated in human and diabetic mice. Blood pressure up-regulation positively correlated with serum DKK1 elevations in humans. Notably, LRP5/6 physically and functionally interacted with AT1R. The loss of membrane LRP5/6 caused by injection of a recombinant DKK1 protein or conditional LRP5/6 deletions resulted in AT1R activation and hypertension, as well as β-arrestin1 activation and cardiac impairment, possibly because of multiple GPCR alterations. Importantly, unlike commonly used anti-hypertensive agents, administration of the anti-DKK1 neutralizing antibody effectively prevented diabetic cardiac impairment in mice. These findings establish a novel DKK1-LRP5/6-GPCR pathway in inducing diabetic injuries and may resolve the long-standing conundrum as to why elevated blood DKK1 has deleterious effects. Thus, monitoring and therapeutic elimination of blood DKK1 may be a promising strategy to attenuate diabetic injuries.

Unraveling the G protein-coupled receptor superfamily in aphids: Contractions and duplications linked to phloem feeding

The G Protein-Coupled Receptor (GPCR) superfamily is the largest and most diverse transmembrane receptor family, playing crucial roles in regulating various physiological processes. As one of the most destructive pests, aphids have been subject to previous studies, which revealed fewer GPCR superfamily members in Acyrthosiphon pisum and Aphis gossypii and the loss of multiple neuropeptide GPCRs. To elucidate the contraction patterns and evolutionary features of the aphid GPCR superfamily, we identified 97, 105, and 95 GPCR genes in Rhopalosiphum maidis, A. pisum, and A. gossypii, respectively. Comparative analysis and phylogenetic investigations with other hemipteran insects revealed a contracted GPCR superfamily in aphids. This contraction mainly occurred in biogenic amine receptors, GABA-B-R, and fz families, and several neuropeptide receptors such as ACPR, CrzR, and PTHR were completely lost. This phenomenon may be related to the parasitic nature of aphids. Additionally, several GPCRs associated with aphid feeding and water balance underwent duplication, including Lkr, NPFR, CCHa1-R, and DH-R, Type A LGRs, but the SK/CCKLR that inhibits feeding was completely lost, indicating changes in feeding genes that underpin the aphid's prolonged phloem feeding behavior. Furthermore, we observed fine-tuning in opsins, with reduced long-wavelength opsins and additional duplications of short-wavelength opsin, likely associated with daytime activity. Lastly, we found variations in the number of mthl genes in aphids. In conclusion, our investigation sheds light on the GPCR superfamily in aphids, revealing its association with diet lifestyle and laying the foundation for understanding and developing control strategies for the aphid GPCR superfamily.

Towards the convergent therapeutic potential of GPCRs in autism spectrum disorders.

Autism spectrum disorders (ASD) are diagnosed in 1/100 children worldwide, based on two core symptoms, deficits in social interaction and communication and stereotyped behaviours. G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors that transduce extracellular signals to convergent intracellular signalling and downstream cellular responses that are commonly dysregulated in ASD. Despite hundreds of GPCRs being expressed in the brain, only 23 are genetically associated with ASD according to the Simons Foundation Autism Research Initiative (SFARI) gene database: oxytocin OTR, vasopressin V1A , V1B , metabotropic glutamate mGlu5 , mGlu7 , GABAB2 , dopamine D1 , D2 , D3 , serotoninergic 5-HT1B , β2 -adrenoceptor, cholinergic M3 , adenosine A2A , A3 , angiotensin AT2 , cannabinoid CB1 , chemokine CX3 CR1, orphan GPR37, GPR85 and olfactory OR1C1, OR2M4, OR2T10, OR52M1. Here, we review the therapeutic potential of these 23 GPCRs, 5-HT2A and 5-HT7 for ASD. For each GPCR, we discuss its genetic association, genetic and pharmacological manipulation in animal models, pharmacopeia for core symptoms of ASD and rank them based on these factors. Among these GPCRs, we highlight that D2 , 5-HT2A , CB1 , OTR and V1A are the most promising targets for ASD. We discuss that the dysregulation of GPCRs and their signalling is a convergent pathological mechanism of ASD. Their therapeutic potential has only begun as multiple GPCRs could mitigate ASD.

Single transmembrane GPCR modulating proteins: neither single nor simple.

The discovery of G-protein coupled receptor (GPCR) accessory proteins has fundamentally redefined the pharmacological concept of GPCR signaling, demonstrating a more complex molecular basis for receptor specificity on the plasma membrane and impressionable downstream intracellular cascades. GPCR accessory proteins not only contribute to the proper folding and trafficking of receptors, but also exhibit selectable receptor preferences. The melanocortin receptor accessory proteins (MRAP1 and MRAP2) as well as receptor activity-modifying proteins (RAMPs) are two well-known single transmembrane partners for the regulation of the melanocortin receptors (MC1R-MC5R) and the glucagon receptor (GCGR), respectively. Especially, MRAP family participates in the pathological control of multiple endocrine disorders and RAMPs contribute to the endogenous regulation of glucose homeostasis. However, the precise mechanisms by which the MRAP and RAMP proteins regulate receptor signaling at the atomic resolution remain unknown. Recent progress made in the determination of RAMP2-bound GCGR complexes published on Cell (Krishna Kumar et al., 2023) unraveled the importance of RAMP2 for the promotion of extracellular receptor dynamics leading to cytoplasmic surface inactivation. Moreover, the new findings on Cell Research (Luo et al., 2023) of the adrenocorticotropic hormone (ACTH)-bound MC2R-Gs-MRAP1 complex disclosed the essential role of MRAP1 for MC2R activation and specificity of ligand recognition. In this article, we reviewed a series of key findings of MRAP proteins in the last decade, and the recent structural investigation of MRAP-MC2R and RAMP-GCGR functional complex and the expanded identification of new GPCR partners of MRAP proteins. In-depth understanding of GPCR modulation by single transmembrane accessory proteins will provide valuable insights for the therapeutic drug development to treat multiple GPCR associated human disorders.

Role of G-protein coupled receptors in cardiovascular diseases.

Cardiovascular diseases (CVDs) are the leading cause of death globally, with CVDs accounting for nearly 30% of deaths worldwide each year. G-protein-coupled receptors (GPCRs) are the most prominent family of receptors on the cell surface, and play an essential regulating cellular physiology and pathology. Some GPCR antagonists, such as β-blockers, are standard therapy for the treatment of CVDs. In addition, nearly one-third of the drugs used to treat CVDs target GPCRs. All the evidence demonstrates the crucial role of GPCRs in CVDs. Over the past decades, studies on the structure and function of GPCRs have identified many targets for the treatment of CVDs. In this review, we summarize and discuss the role of GPCRs in the function of the cardiovascular system from both vascular and heart perspectives, then analyze the complex ways in which multiple GPCRs exert regulatory functions in vascular and heart diseases. We hope to provide new ideas for the treatment of CVDs and the development of novel drugs.

Multiple Subthreshold GPCR Signals Combined by the G-Proteins Gαq and Gαs Activate the Caenorhabditis elegans Egg-Laying Muscles.

Individual neurons or muscle cells express many G-protein-coupled receptors (GPCRs) for neurotransmitters and neuropeptides, yet it remains unclear how cells integrate multiple GPCR signals that all must activate the same few G-proteins. We analyzed this issue in the Caenorhabditis elegans egg-laying system, where multiple GPCRs on muscle cells promote contraction and egg laying. We genetically manipulated individual GPCRs and G-proteins specifically in these muscle cells within intact animals and then measured egg laying and muscle calcium activity. Two serotonin GPCRs on the muscle cells, Gαq-coupled SER-1 and Gαs-coupled SER-7, together promote egg laying in response to serotonin. We found that signals produced by either SER-1/Gαq or SER-7/Gαs alone have little effect, but these two subthreshold signals combine to activate egg laying. We then transgenically expressed natural or designer GPCRs in the muscle cells and found that their subthreshold signals can also combine to induce muscle activity. However, artificially inducing strong signaling through just one of these GPCRs can be sufficient to induce egg laying. Knocking down Gαq and Gαs in the egg-laying muscle cells induced egg-laying defects that were stronger than those of a SER-1/SER-7 double knockout, indicating that additional endogenous GPCRs also activate the muscle cells. These results show that in the egg-laying muscles multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they combine to produce sufficient levels of Gαq and Gαs signaling to promote muscle activity and egg laying.SIGNIFICANCE STATEMENT How can neurons and other cells gather multiple independent pieces of information from the soup of chemical signals in their environment and compute an appropriate response? Most cells express >20 GPCRs that each receive one signal and transmit that information through three main types of G-proteins. We analyzed how this machinery generates responses by studying the egg-laying system of C. elegans, where serotonin and multiple other signals act through GPCRs on the egg-laying muscles to promote muscle activity and egg laying. We found that individual GPCRs within an intact animal each generate effects too weak to activate egg laying. However, combined signaling from multiple GPCR types reaches a threshold capable of activating the muscle cells.

Targeting G protein-coupled receptors to regulate energy homeostasis.

G protein-coupled receptors (GPCRs) have a critical role in energy homeostasis, contributing to food intake, energy expenditure and glycaemic control. Dysregulation of energy expenditure can lead to metabolic syndrome (abdominal obesity, elevated plasma triglyceride, LDL cholesterol and glucose, and high blood pressure), which is associated with increased risk of developing obesity, diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular complications. As the prevalence of these chronic diseases continue to rise worldwide, there is an increased need to understand the molecular mechanisms by which energy expenditure is regulated to facilitate the development of effective therapeutic strategies to treat and prevent these conditions. In recent years, drugs targeting GPCRs have been the focus of efforts to improve treatments for type-2 diabetes and obesity, with GLP-1R agonists a particular success. In this review, we focus on nine GPCRs with roles in energy homeostasis that are current and emerging targets to treat obesity and diabetes. We discuss findings from pre-clinical models and clinical trials of drugs targeting these receptors and challenges that must be overcome before these drugs can be routinely used in clinics. We also describe new insights into how these receptors signal, including how accessory proteins, biased signalling and complex spatial signalling could provide unique opportunities to develop more efficacious therapies with fewer side effects. Finally, we describe how combined therapies, in which multiple GPCRs are targeted, may improve clinical outcomes and reduce off-target effects.

Combinatorial depletions of G-protein coupled receptor kinases in immune cells identify pleiotropic and cell type-specific functions

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts, which eventually blocks signal propagation and often induces receptor internalization. However, there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2, GRK3, GRK5, GRK6) simultaneously, but we have very limited knowledge about their interplay in primary immune cells. In particular, we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type, and (b) one specific GPCR between several immune cell subsets. To address this issue, we generated mouse models of single, combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils, T cells, B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes, many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands, whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types, and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly, we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis, which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together, our findings demonstrate the complexity of GRK functions in immune cells, which go beyond GPCR desensitization in specific leukocyte types. Furthermore, they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset.

The Many Faces of G Protein-Coupled Receptor 143, an Atypical Intracellular Receptor.

GPCRs transform extracellular stimuli into a physiological response by activating an intracellular signaling cascade initiated via binding to G proteins. Orphan G protein-coupled receptors (GPCRs) hold the potential to pave the way for development of new, innovative therapeutic strategies. In this review we will introduce G protein-coupled receptor 143 (GPR143), an enigmatic receptor in terms of classification within the GPCR superfamily and localization. GPR143 has not been assigned to any of the GPCR families due to the lack of common structural motifs. Hence we will describe the most important motifs of classes A and B and compare them to the protein sequence of GPR143. While a precise function for the receptor has yet to be determined, the protein is expressed abundantly in pigment producing cells. Many GPR143 mutations cause X-linked Ocular Albinism Type 1 (OA1, Nettleship-Falls OA), which results in hypopigmentation of the eyes and loss of visual acuity due to disrupted visual system development and function. In pigment cells of the skin, loss of functional GPR143 results in abnormally large melanosomes (organelles in which pigment is produced). Studies have shown that the receptor is localized internally, including at the melanosomal membrane, where it may function to regulate melanosome size and/or facilitate protein trafficking to the melanosome through the endolysosomal system. Numerous additional roles have been proposed for GPR143 in determining cancer predisposition, regulation of blood pressure, development of macular degeneration and signaling in the brain, which we will briefly describe as well as potential ligands that have been identified. Furthermore, GPR143 is a promiscuous receptor that has been shown to interact with multiple other melanosomal proteins and GPCRs, which strongly suggests that this orphan receptor is likely involved in many different physiological actions.

MD Simulations Revealing Special Activation Mechanism of Cannabinoid Receptor 1.

Cannabinoid receptor 1 (CB1) is a G protein-coupled receptor (GPCR) that is gaining much interest for its regulating role in the central nervous system and its value as a drug target. Structures of CB1 in inactive and active states have revealed conformational change details that are not common in other GPCRs. Here, we performed molecular dynamics simulations of CB1 in different ligand binding states and with mutations to reveal its activation mechanism. The conformational change of the “twin toggle switch” residues F2003.36 and W3566.48 that correlates with ligand efficacy is identified as a key barrier step in CB1 activation. Similar conformational change of residues 3.36/6.48 is also observed in melanocortin receptor 4, showing this “twin toggle switch” residue pair is crucial for the activation of multiple GPCR members.

Blocking Gi/o-Coupled Signaling Eradicates Cancer Stem Cells and Sensitizes Breast Tumors to HER2-Targeted Therapies to Inhibit Tumor Relapse.

Simple SummaryCancer stem cells (CSCs) are associated with therapeutic resistance and tumor relapse but effective approaches for eliminating CSCs are still lacking. The aim of this study was to assess the role of G protein-coupled receptors (GPCRs) in regulating CSCs in breast cancer. We showed that a subgroup of GPCRs that coupled to Gi/o proteins (Gi/o-GPCRs) was required for maintaining the tumor-forming capability of CSCs in HER2+ breast cancer. Targeting Gi/o-GPCRs or their downstream PI3K/AKT and Src pathways was able to enhance HER2-targeted elimination of CSCs and therapeutic efficacy. These findings suggest that targeting Gi/o-GPCR signaling is an effective strategy for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor recurrence.Cancer stem cells (CSCs) are a small subpopulation of cells within tumors that are resistant to anti-tumor therapies, making them a likely origin of tumor relapse after treatment. In many cancers including breast cancer, CSC function is regulated by G protein-coupled receptors (GPCRs), making GPCR signaling an attractive target for new therapies designed to eradicate CSCs. Yet, CSCs overexpress multiple GPCRs that are redundant in maintaining CSC function, so it is unclear how to target all the various GPCRs to prevent relapse. Here, in a model of HER2+ breast cancer (i.e., transgenic MMTV-Neu mice), we were able to block the tumorsphere- and tumor-forming capability of CSCs by targeting GPCRs coupled to Gi/o proteins (Gi/o-GPCRs). Similarly, in HER2+ breast cancer cells, blocking signaling downstream of Gi/o-GPCRs in the PI3K/AKT and Src pathways also enhanced HER2-targeted elimination of CSCs. In a proof-of-concept study, when CSCs were selectively ablated (via a suicide gene construct), loss of CSCs from HER2+ breast cancer cell populations mimicked the effect of targeting Gi/o-GPCR signaling, suppressing their capacity for tumor initiation and progression and enhancing HER2-targeted therapy. Thus, targeting Gi/o-GPCR signaling in HER2+ breast cancer is a promising approach for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor reemergence.

Recurrent high-impact mutations at cognate structural positions in class A G protein-coupled receptors expressed in tumors

G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated somatic cancer mutations across class A GPCRs and found a nonrandom distribution of positions with variant amino acid residues. Individual cancer types were enriched for highly impactful, recurrent mutations at selected cognate positions of known functional motifs. We also discovered that no single receptor drives this pattern, but rather multiple receptors contain amino acid substitutions at a few cognate positions. Phenotypic characterization suggests these mutations induce perturbation of G protein activation and/or β-arrestin recruitment. These data suggest that recurrent impactful oncogenic mutations perturb different GPCRs to subvert signaling and promote tumor growth or survival. The possibility that multiple different GPCRs could moonlight as drivers or enablers of a given cancer through mutations located at cognate positions across GPCR paralogs opens a window into cancer mechanisms and potential approaches to therapeutics.

Role of G Protein-Coupled Receptors in Hepatic Stellate Cells and Approaches to Anti-Fibrotic Treatment of Non-Alcoholic Fatty Liver Disease.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is globally increasing. Gaining control over disease-related events in non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD, is currently an unmet medical need. Hepatic fibrosis is a critical prognostic factor in NAFLD/NASH. Therefore, a better understanding of the pathophysiology of hepatic fibrosis and the development of related therapies are of great importance. G protein-coupled receptors (GPCRs) are cell surface receptors that mediate the function of a great variety of extracellular ligands. GPCRs represent major drug targets, as indicated by the fact that about 40% of all drugs currently used in clinical practice mediate their therapeutic effects by acting on GPCRs. Like many other organs, various GPCRs play a role in regulating liver function. It is predicted that more than 50 GPCRs are expressed in the liver. However, our knowledge of how GPCRs regulate liver metabolism and fibrosis in the different cell types of the liver is very limited. In particular, a better understanding of the role of GPCRs in hepatic stellate cells (HSCs), the primary cells that regulate liver fibrosis, may lead to the development of drugs that can improve hepatic fibrosis in NAFLD/NASH. In this review, we describe the functions of multiple GPCRs expressed in HSCs, their roles in liver fibrogenesis, and finally speculate on the development of novel treatments for NAFLD/NASH.

Structure determination of GPCRs: cryo-EM compared with X-ray crystallography.

G protein-coupled receptors (GPCRs) are the largest single family of cell surface receptors encoded by the human genome and they play pivotal roles in co-ordinating cellular systems throughout the human body, making them ideal drug targets. Structural biology has played a key role in defining how receptors are activated and signal through G proteins and β-arrestins. The application of structure-based drug design (SBDD) is now yielding novel compounds targeting GPCRs. There is thus significant interest from both academia and the pharmaceutical industry in the structural biology of GPCRs as currently only about one quarter of human non-odorant receptors have had their structure determined. Initially, all the structures were determined by X-ray crystallography, but recent advances in electron cryo-microscopy (cryo-EM) now make GPCRs tractable targets for single-particle cryo-EM with comparable resolution to X-ray crystallography. So far this year, 78% of the 99 GPCR structures deposited in the PDB (Jan–Jul 2021) were determined by cryo-EM. Cryo-EM has also opened up new possibilities in GPCR structural biology, such as determining structures of GPCRs embedded in a lipid nanodisc and multiple GPCR conformations from a single preparation. However, X-ray crystallography still has a number of advantages, particularly in the speed of determining many structures of the same receptor bound to different ligands, an essential prerequisite for effective SBDD. We will discuss the relative merits of cryo-EM and X-ray crystallography for the structure determination of GPCRs and the future potential of both techniques.

Neuronal Gα subunits required for the control of response to polystyrene nanoparticles in the range of μg/L in C. elegans

The aim of this study was to identify Gα proteins mediating function of neuronal G protein-coupled receptors (GPCRs) in controlling the response to polystyrene nanoparticles (PS-NPs). Caenorhabditis elegans was used as an animal model, and both gene expression and functional analysis were performed to identify the Gα proteins in controlling PS-NPs toxicity. In nematodes, exposure to PS-NPs (1–100 μg/L) significantly altered transcriptional expressions of some neuronal Gα genes, including gpa-5, gpa-10, gpa-11, gpa-15 gsa-1, egl-30, and goa-1. Among these 7 Gα genes, only neuronal RNAi knockdown of gsa-1, gpa-10, and goa-1 affected toxicity of PS-NPs in inducing ROS production and in decreasing locomotion behavior. Some neuronal GPCRs (such as GTR-1, DCAR-1, DOP-2, NPR-8, NPR-12, NPR-9, and DAF-37) functioned upstream of GOA-1, some neuronal GPCRs (such as DCAR-1, DOP-2, NPR-9, NPR-8, and DAF-37) functioned upstream of GSA-1, and some neuronal GPCRs (such as DOP-2, NPR-8, DAF-37, and DCAR-1) functioned upstream of GPA-10 to regulate the toxicity of PS-NPs. Moreover, GOA-1 acted upstream of MPK-1/ERK MAPK, JNK-1/JNK MAPK, DBL-1/TGF-β, and DAF-7/ TGF-β, GSA-1 functioned upstream of MPK-1/ERK MAPK, JNK-1/JNK MAPK, and DBL-1/TGF-β, and GPA-10 functioned upstream of GLB-1/Globin and DBL-1/TGF-β to control the PS-NPs toxicity. Therefore, neuronal Gα proteins of GOA-1, GSA-1, and GPA-10 functioned to transduce signals of multiple GPCRs to different downstream signaling pathways during the control of PS-NPs toxicity in nematodes. Our results provide clues for understanding the important function of GPCRs-Gα signaling cascade in the neurons in controlling response to nanoplastics in organisms.

Discovery of ciliary G protein-coupled receptors regulating pancreatic islet insulin and glucagon secretion.

Multiple G protein-coupled receptors (GPCRs) are expressed in pancreatic islet cells, but the majority have unknown functions. We observed specific GPCRs localized to primary cilia, a prominent signaling organelle, in pancreatic α and β cells. Loss of cilia disrupts β-cell endocrine function, but the molecular drivers are unknown. Using functional expression, we identified multiple GPCRs localized to cilia in mouse and human islet α and β cells, including FFAR4, PTGER4, ADRB2, KISS1R, and P2RY14. Free fatty acid receptor 4 (FFAR4) and prostaglandin E receptor 4 (PTGER4) agonists stimulate ciliary cAMP signaling and promote glucagon and insulin secretion by α- and β-cell lines and by mouse and human islets. Transport of GPCRs to primary cilia requires TULP3, whose knockdown in primary human and mouse islets relocalized ciliary FFAR4 and PTGER4 and impaired regulated glucagon or insulin secretion, without affecting ciliary structure. Our findings provide index evidence that regulated hormone secretion by islet α and β cells is controlled by ciliary GPCRs providing new targets for diabetes.

Differential methylation of G-protein coupled receptor signaling genes in gastrointestinal neuroendocrine tumors

Neuroendocrine tumors (NETs) of the small intestine undergo large chromosomal and methylation changes. The objective of this study was to identify methylation differences in NETs and consider how the differentially methylated genes may impact patient survival. Genome-wide methylation and chromosomal copy number variation (CNV) of NETs from the small intestine and appendix were measured. Tumors were divided into three molecular subtypes according to CNV results: chromosome 18 loss (18LOH), Multiple CNV, and No CNV. Comparison of 18LOH tumors with MultiCNV and NoCNV tumors identified 901 differentially methylated genes. Genes from the G-protein coupled receptor (GPCR) pathways are statistically overrepresented in the differentially methylated genes. One of the highlighted genes from the GPCR pathway is somatostatin (SST), a clinical target for NETs. Patient survival based on low versus high methylation in all samples identified four significant genes (p < 0.05) OR2S2, SMILR, RNU6-653P, and AC010543.1. Within the 18LOH molecular subtype tumors, survival differences were identified in high versus low methylation of 24 genes. The most significant is TRHR (p < 0.01), a GPCR with multiple FDA-approved drugs. By separating NETs into different molecular subtypes based on chromosomal changes, we find that multiple GPCRs and their ligands appear to be regulated through methylation and correlated with survival. These results suggest opportunities for better treatment strategies for NETs based on molecular features.