Role Of G Protein-coupled Receptors Research Articles

GPR160 regulates the self-renewal and pluripotency of mouse embryonic stem cells via JAK1/STAT3 signal pathway

G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and regulate various physiological and pathological processes. Despite extensive studies, the roles of GPCRs in mouse embryonic stem cells (mESCs) represent a significant data gap. Here, we show that GPR160, a class A member of GPCRs, is dramatically downregulated concurrent with mESC differentiation into embryoid bodies in vitro. Knockdown of GPR160 leads to downregulation of the expression of pluripotency-associated transcription factors and upregulation of the expression of lineage markers, accompanying with the arrest of the mESC cell cycle in the G0/G1 phase. RNA-seq analysis shows that GPR160 participates in the JAK/STAT signaling pathway crucial for maintaining ESC stemness, and the knockdown of GPR160 results in the downregulation of STAT3 phosphorylation level, which in turn is partially rescued by colivelin, a STAT3 activator. Constant with these observations, GPR160 physically interacts with JAK1, and cooperates with leukemia inhibitory factor receptor (LIFR) and gp130 to activate the STAT3 pathway. In summary, our results suggest that GPR160 regulates mESC self-renewal and pluripotency by interacting with the JAK1-LIFR-gp130 complex to mediate the JAK1/STAT3 signaling pathway.

Genome-wide identification of the opsin protein in Leptosphaeria maculans and comparison with other fungi (pathogens of Brassica napus).

The largest family of transmembrane receptors are G-protein-coupled receptors (GPCRs). These receptors respond to perceived environmental signals and infect their host plants. Family A of the GPCR includes opsin. However, there is little known about the roles of GPCRs in phytopathogenic fungi. We studied opsin in Leptosphaeria maculans, an important pathogen of oilseed rape (Brassica napus) that causes blackleg disease, and compared it with six other fungal pathogens of oilseed rape. A phylogenetic tree analysis of 31 isoforms of the opsin protein showed six major groups and six subgroups. All three opsin isoforms of L. maculans are grouped in the same clade in the phylogenetic tree. Physicochemical analysis revealed that all studied opsin proteins are stable and hydrophobic. Subcellular localization revealed that most isoforms were localized in the endoplasmic reticulum membrane except for several isoforms in Verticillium species, which were localized in the mitochondrial membrane. Most isoforms comprise two conserved domains. One conserved motif was observed across all isoforms, consisting of the BACTERIAL_OPSIN_1 domain, which has been hypothesized to have an identical sensory function. Most studied isoforms showed seven transmembrane helices, except for one isoform of V. longisporum and four isoforms of Fusarium oxysporum. Tertiary structure prediction displayed a conformational change in four isoforms of F. oxysporum that presumed differences in binding to other proteins and sensing signals, thereby resulting in various pathogenicity strategies. Protein-protein interactions and binding site analyses demonstrated a variety of numbers of ligands and pockets across all isoforms, ranging between 0 and 13 ligands and 4 and 10 pockets. According to the phylogenetic analysis in this study and considerable physiochemically and structurally differences of opsin proteins among all studied fungi hypothesized that this protein acts in the pathogenicity, growth, sporulation, and mating of these fungi differently.

Role of GPER in the expression and secretion of pro-inflammatory cytokines and calcium signaling in bone marrow-derived macrophages

Macrophages play an important role in mediating inflammation in numerous disease states by secreting pro-inflammatory cytokines including IL-1β and IL-18. Macrophages are driven into the M1-proinflammatory phenotype upon detection of pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs). As macrophages are polarized into the M1 phenotype, they upregulate the expression of pro-inflammatory cytokines and components of the NLRP3 inflammasome. An additional activating stimulus is required to induce the assembly of the NLRP3 inflammasome, which facilitates the activation of pro-caspase 1 and the subsequent conversion of the pro-forms of both IL-18 and IL-1β into their mature forms for their secretion through gasdermin D membrane pores. A canonical activator of the NLRP3 inflammasome is extracellular ATP. ATP activation of the P2X7 receptor induces potassium efflux and calcium influx, both of which are necessary for NLRP3 inflammasome activation. Previous studies suggest that the G protein-coupled receptor (GPER) agonist G-1 reduced inflammation in animal disease models. However, the GPER-dependent effect of G-1 on pro-inflammatory cytokine expression and NLRP3 inflammasome activation in macrophages is unknown. In the current study, we utilized bone marrow-derived macrophages (BMDMs) from either WT or GPER-/- mice to test the hypothesis that G-1 reduces inflammatory cytokine production and NLRP3 inflammasome expression in a GPER-dependent manner. We found that the treatment of WT BMDMs with 100 nM G-1 for 24 hrs inhibited LPS-induced NLRP3, pro-IL-1β, IL-6, CCL5 and expression, and IL-1β secretion. Importantly, G-1 had no effect on GPER-/- BMDMs. To begin to determine if GPER modulates NLRP3 inflammasome activation, we characterized ATP-induced Ca2+ signals in both WT and GPER BMDMs with and without LPS pretreatment. No differences were observed between unprimed WT and GPER-/- BMDMs with respect to basal Ca2+ level, ATP-induced intracellular Ca2+ release, and subsequent bulk Ca2+ signal upon reestablishment of extracellular Ca2+. However, priming with LPS significantly increased ATP-induced intracellular Ca2+ release in both WT and GPER-/- BMDMs. Compared to WT cells, LPS-primed GPER-/- BMDMs exhibit significantly higher basal Ca2+ yet significantly lower ATP-induced Ca2+ release. Consistently, 15-minute treatment with 100 nM G-1 agonist strongly reduces ATP-induced Ca2+ release in WT BMDMs but had no effect in GPER-/- BMDMs. These results suggest a role for GPER in mediating Ca2+ uptake into IP3-sensitive intracellular compartments in BMDMs during LPS priming. In keeping with the known role for mobilization of intracellular Ca2+ in ATP-induced secretion of pro-inflammatory cytokines, preliminary data suggests that short-term G-1 pretreatment reduced IL-1β secretion induced by ATP in LPS-primed BMDMs. Overall, these data indicate a role for GPER in the priming and activation of the NLRP3 inflammasome. This research was supported by Des Moines University IOER Research & Grant Award #03-22-10. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2868: A chemogenetic approach reveals a GPCR-Gαs-PKA signaling axis promoting T cell dysfunction and cancer immunotherapy failure

Abstract Recent advances in immune checkpoint blockade (ICB) inhibiting programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein (CTLA-4) have revolutionized the standard of care for cancer treatment. However, the limited response rates to ICB across multiple cancer types suggest that new approaches and targets are clearly needed to fully elucidate the underlying biology of dysfunctional and exhausted CD8 T cells in cancer in order to achieve durable responses (cure). G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, primarily due to their druggability and relevance to most physiological processes and disease conditions. However, the role of GPCRs in cancer and particularly in cancer immunology has been largely underexplored. We have applied a new computational pipeline to cross-integrate hundreds of thousands of CD8 T cells from multiple single cell RNA-seq datasets from 13 distinct cancer types. Using this approach, we identified many GPCRs that are overrepresented in exhausted CD8 T cells. A new computational strategy enabled us to predict the G protein coupling specificity of most GPCRs. This led to the discovery that Gαs-coupled GPCRs are significantly enriched in exhausted CD8 T cells. These include EP2, EP4, A2AR, β1AR, and β2AR, and we found that all of these receptors promote CD8 T cell dysfunction by inhibiting cytotoxicity and cytokine secretion. However, these receptors are also expressed in other immune cells within the tumor microenvironment. To address the direct impact of these receptors on T cell function we used a novel synthetic biology approach, involving the development of a chemogenetic CD8-restricted Gαs-DREADD (Designer Receptor Exclusively Activated by A Designer Drug) transgenic mouse model in which activation of Gαs signaling is temporally and spatially controlled. Utilizing this Gαs-DREADD model, we discovered that the Gαs-signaling axis represents a previously uncharacterized signaling axis that dampens the anti-tumor CD8 T cell activity and leads to ICB immunotherapy failure. Our central hypothesis is that secretion of GPCR ligands in the tumor microenvironment and their actions on CD8 T cells lead to the activation of the Gαs signaling cascade leading to T cell dysfunction, by decreasing cytotoxic and migratory activity that abolished the effectiveness of ICB. Our findings reveal that Gαs-coupled GPCRs may represent new targetable immune checkpoints that can be combined with ICB as part of novel multimodal precision approaches to enhance the response to immunotherapies. Citation Format: Bryan S. Yung, Victoria H. Wu, Farhoud Faraji, Robert Saddawi-Konefka, Zhiyong Wang, Alexander T. Wenzel, Miranda Song, Meghana S. Pagadala, Lauren M. Clubb, Joshua Chiou, Sanju Sinha, Marin Matic, Francesco Raimondi, Thomas Hoang, Rebecca Berdeaux, Dario A. Vignali, Ramiro Iglesias-Bartolome, Hannah Carter, Eytan Ruppin, Jill P. Mesirov, J. Silvio Gutkind. A chemogenetic approach reveals a GPCR-Gαs-PKA signaling axis promoting T cell dysfunction and cancer immunotherapy failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2868.

Antimalarial and Plasmodium falciparum serpentine receptor 12 targeting effect of FDA approved purinergic receptor antagonist

Intraerythrocytic stages of Plasmodium falciparum responsible for all clinical manifestations of malaria are regulated by array of signalling cascades that represent attractive targets for antimalarial therapy. G-protein coupled receptors (GPCRs) are druggable targets in the treatment of various pathological conditions, however, there is limited understanding about the role of GPCRs in malaria pathogenesis. In Plasmodium, serpentine receptors (PfSR1, PfSR10, PfSR12 and PfSR25) with GPCR-like membrane topology have been reported with the finite knowledge about their potential as antimalarial targets. We analyzed the localization of these receptors in malaria parasite by immunofluorescence assays. All four receptors were expressed in blood stages with PfSR12 expressing more in late intraerythrocytic stages. Further, we evaluated the druggability of PfSR12 using FDA-approved P2Y purinergic receptor antagonist, Prasugrel and its active metabolite R138727, which is proposed to be specific towards PfSR12. Interestingly, biophysical analysis indicated strong binding between PfSR12 and R138727 as compared to the prodrug Prasugrel. This binding interaction was further confirmed by thermal shift assay. Treatment of parasite with Prasugrel and R138727 resulted in growth inhibition of P. falciparum indicating an important role of purinergic signalling and PfSR12 in parasite survival. Next, progression studies indicated the inhibitory effect of Prasugrel begins in late erythrocyte stages corroborating with PfSR12 expression at these stages. Furthermore, Prasugrel also blocked in vivo growth of malaria parasite in a mouse experimental model. This study indicates the presence of P2Y type of purinergic signalling in growth and development of malaria parasite and suggests PfSR12, putative purinergic receptor druggability through Prasugrel. Communicated by Ramaswamy H. Sarma

Targeting GPCRs and Their Signaling as a Therapeutic Option in Melanoma.

Simple SummarySixteen G-protein-coupled receptors (GPCRs) have been involved in melanogenesis or melanomagenesis. Here, we review these GPCRs, their associated signaling, and therapies.G-protein-coupled receptors (GPCRs) serve prominent roles in melanocyte lineage physiology, with an impact at all stages of development, as well as on mature melanocyte functions. GPCR ligands are present in the skin and regulate melanocyte homeostasis, including pigmentation. The role of GPCRs in the regulation of pigmentation and, consequently, protection against external aggression, such as ultraviolet radiation, has long been established. However, evidence of new functions of GPCRs directly in melanomagenesis has been highlighted in recent years. GPCRs are coupled, through their intracellular domains, to heterotrimeric G-proteins, which induce cellular signaling through various pathways. Such signaling modulates numerous essential cellular processes that occur during melanomagenesis, including proliferation and migration. GPCR-associated signaling in melanoma can be activated by the binding of paracrine factors to their receptors or directly by activating mutations. In this review, we present melanoma-associated alterations of GPCRs and their downstream signaling and discuss the various preclinical models used to evaluate new therapeutic approaches against GPCR activity in melanoma. Recent striking advances in our understanding of the structure, function, and regulation of GPCRs will undoubtedly broaden melanoma treatment options in the future.

P088 Metabolomic analysis reveals differences among UC and non-IBD human colonic resections: role of GPCRs

Abstract Background Metabolomics is a recent technique that has bounced into Inflammatory Bowel Diseases (IBD) due to its capacity to elucidate specific metabolites involved in the pathology and changes in the metabolomic profile have been detected in urine, blood or feces from UC patients. G-protein coupled receptors (GPCRs) have been recently identified as promising pharmacological targets. We aim to characterize the metabolomic profile and metabolite-sensing GPCRs expression in colonic resections from UC patients. Methods Colonic resections from UC (n=18) and non-IBD (n=20) patients were obtained. Metabolomic analysis was performed using Nuclear Magnetic Resonance (NMR) to study polar metabolites and Gas-Chromatography or Liquid-Chromatography Mass-Spectrometry to study non-polar metabolites. Results are expressed as μg of metabolite per gram of tissue. Gene expression of GPCRs was analyzed by qPCR. Data were expressed as fold induction vs control (mean±SEM) and compared by a t-test. A p-value<0.05 was considered statistically significant. Pearson correlation matrix were performed with R or mixOmics library in R. Results Metabolomic analysis revealed in colon of UC patients significant increased levels of propionic acid (7.7±1.1), undecanoic acid (0.3±0.02) decanoic acid (4.5±0.9), myristic acid (64.5±8.7), α-linoleic acid (23.1±4.0), aspartic acid (33.9±3.4), phenylalanine (8.8±1.6), glutamic acid, (136.3±10.8), b-hydroxybutyric (8.3±0.9) acid and lactic acid (297.5±34.7) vs non-IBD patients. Gene expression of GPR43 (22.7±9.5), GPR41 (4.7±1.7), GPR109a (19.1±5.3), GPR109b (13.6±4.6), GPR91 (13.2±5.7), GPR84 (6.7±2.0), GPR40 (6.6±1.8), GPR65 (3.0±0.5) and GPR68 (4.6±1.0) were significantly increased in UC patients compared with controls. In contrast, GPR120 (0.9±0.3), GPR119 (0.6±0.2) and GPR35 (0.5±0.1) were significantly reduced in UC patients. N-oleylethanolamide positively correlates with most of GPCRs, especially GPR68, GPR84 and GPR91 while acetic and succinic acids negatively correlate with GPR4 (Fig.1A). In UC patients, medium-chain fatty acids (FA) and most long-chain FA positively correlate with GPR84, GPR142, GPR43, GPR109a, GPR109b, GPR91 and GPR132 (Fig. 1C). Fig1. Graphs show the correlations between data relative to mRNA expression of GPCRs (expressed as ΔCt) vs metabolites. (A) Data from controls and UC. (B) Only controls. (C) Only UC. Conclusion Increased levels of several metabolites and GPCRs are detected in the colon of UC patients and their correlations suggest potential candidates to be involved in UC pathophysiology.

An Insight into GPCR and G-Proteins as Cancer Drivers

G-protein-coupled receptors (GPCRs) are the largest family of cell surface signaling receptors known to play a crucial role in various physiological functions, including tumor growth and metastasis. Various molecules such as hormones, lipids, peptides, and neurotransmitters activate GPCRs that enable the coupling of these receptors to highly specialized transducer proteins, called G-proteins, and initiate multiple signaling pathways. Integration of these intricate networks of signaling cascades leads to numerous biochemical responses involved in diverse pathophysiological activities, including cancer development. While several studies indicate the role of GPCRs in controlling various aspects of cancer progression such as tumor growth, invasion, migration, survival, and metastasis through its aberrant overexpression, mutations, or increased release of agonists, the explicit mechanisms of the involvement of GPCRs in cancer progression is still puzzling. This review provides an insight into the various responses mediated by GPCRs in the development of cancers, the molecular mechanisms involved and the novel pharmacological approaches currently preferred for the treatment of cancer. Thus, these findings extend the knowledge of GPCRs in cancer cells and help in the identification of therapeutics for cancer patients.

G Protein-Coupled Receptors in Osteoarthritis: A Novel Perspective on Pathogenesis and Treatment.

G protein-coupled receptors (GPCRs) are transmembrane receptor proteins that trigger numerous intracellular signaling pathways in response to the extracellular stimuli. The GPCRs superfamily contains enormous structural and functional diversity and mediates extensive biological processes. Until now, critical roles have been established in many diseases, including osteoarthritis (OA). Existing studies have shown that GPCRs play an important role in some OA-related pathogenesis, such as cartilage matrix degradation, synovitis, subchondral bone remodeling, and osteophyte formation. However, current pharmacological treatments are mostly symptomatic and there is a paucity of disease-modifying OA drugs so far. Targeting GPCRs is capable of inhibiting cartilage matrix degradation and synovitis and up-regulating cartilage matrix synthesis, providing a new therapeutic strategy for OA. In this review, we have comprehensively summarized the structures, biofunctions, and the novel roles of GPCRs in the pathogenesis and treatment of OA, which is expected to lay the foundation for the development of novel therapeutics against OA. Even though targeting GPCRs may ameliorate OA progression, many GPCRs-related therapeutic strategies are still in the pre-clinical stage and require further investigation.

G protein-coupled receptors as potential targets for nonalcoholic fatty liver disease treatment.

Nonalcoholic fatty liver disease (NAFLD) is a broad-spectrum disease, ranging from simple hepatic steatosis to nonalcoholic steatohepatitis, which can progress to cirrhosis and liver cancer. Abnormal hepatic lipid accumulation is the major manifestation of this disease, and lipotoxicity promotes NAFLD progression. In addition, intermediate metabolites such as succinate can stimulate the activation of hepatic stellate cells to produce extracellular matrix proteins, resulting in progression of NAFLD to fibrosis and even cirrhosis. G protein-coupled receptors (GPCRs) have been shown to play essential roles in metabolic disorders, such as NAFLD and obesity, through their function as receptors for bile acids and free fatty acids. In addition, GPCRs link gut microbiota-mediated connections in a variety of diseases, such as intestinal diseases, hepatic steatosis, diabetes, and cardiovascular diseases. The latest findings show that gut microbiota-derived acetate contributes to liver lipogenesis by converting dietary fructose into hepatic acetyl-CoA and fatty acids. GPCR agonists, including peptides and natural products like docosahexaenoic acid, have been applied to investigate their role in liver diseases. Therapies such as probiotics and GPCR agonists may be applied to modulate GPCR function to ameliorate liver metabolism syndrome. This review summarizes the current findings regarding the role of GPCRs in the development and progression of NAFLD and describes some preclinical and clinical studies of GPCR-mediated treatment. Overall, understanding GPCR-mediated signaling in liver disease may provide new therapeutic options for NAFLD.

Hypoxia-induced GPR4 suppresses trophoblast cell migration and proliferation through the MAPK signaling pathway

Pre-eclampsia (PE), a common pregnancy-systemic syndrome, is characterized by proteinuria and hypertension and is the leading cause of maternal and fetal mortality. Thus, we aim to investigate the role of G-Protein Coupled Receptor 4 (GPR4) in PE and the underlying molecular mechanism. In this study, GSE66273 microarray data were obtained from the Gene Expression Omnibus(GEO) database of the National Center for Biotechnology Information, and Gene set enrichment analysis (GSEA) was performed by GSEA software. qRT-PCR and Immunohistochemistry (IHC) or western blotting were used to assay for the expression of GPR4 in PE placentas and HTR8/SVneo cells. The influence of acidosis and hypoxia environments on the expression of GPR4 was explored using western blotting. Cell proliferation and migration of HTR8/SVneo cells were measured using EdU and MTT assays and migration assay, respectively. Moreover, expressions of MEK1/2, p-MEK1/2, ERK1/2, and p-ERK1/2 in HTR8/SVneo cells were assayed by western blotting. Our data demonstrated that the expression of GPR4 was up regulated in PE placentas. Increase in acidic pH and hypoxic levels increased the expression of GPR4 in HTR8/SVneo cells. GPR4 inhibited cell proliferation and migration in the HTR8/SVneo cells. GPR4 silencing enhanced the phosphorylation of p-MEK1/2and p-ERK1/2 in HTR8/SVneo cells. Additionally, we found that pathway inhibitor partially reversed the effects of GPR4 on proliferation and migration of HTR8/SVneo cells. In conclusions, these results show that GPR4 suppressed cell proliferation and migration by inhibiting MAPK signaling pathway in PE.

Identification of G protein-coupled receptors required for vitellogenesis and egg development in an insect with panoistic ovary.

G protein-coupled receptors (GPCRs), a superfamily of integral transmembrane proteins regulate a variety of physiological processes in insects. Juvenile hormone (JH) is known to stimulate Vitellogenin (Vg) synthesis in the fat body, secretion into the hemolymph and uptake by developing oocytes. However, the role of GPCRs in JH-dependent insect vitellogenesis and oocyte maturation remains elusive. In the present study, we performed transcriptomic analysis and RNA interference (RNAi) screening in vitellogenic females of the migratory locust Locusta migratoria. Of 22 GPCRs identified in ovarian transcriptome, LGR4, OR-A1, OR-A2, Mthl1, Mthl5 and Smo were most abundant in the ovary. By comparison, mAChR-C expressed at higher levels in the fat body, whereas Oct/TyrR, OARβ, AdoR and ADGRA3 were at higher expression levels in the brain. Our RNAi screening demonstrated that knockdown of six GPCRs resulted in defective phenotypes of Vg accumulation in developing oocytes, accompanied by blocked ovarian development and impaired oocyte maturation. While LGR4 and Oct/TyrR appeared to control Vg synthesis in the fat body, OR-A1, OR-A2, mAChR-C and CirlL regulated Vg transportation and uptake. The findings provide fundamental evidence for deciphering the regulatory mechanisms of GPCRs in JH-stimulated insect reproduction.

2111-P: Role of G-Protein Coupled Receptor 31 (GPR31) in ß-Cell Health and Disease

Islet β-cell inflammation, dysfunction, and death are characteristic of diabetes. 12/15-Lipoxygenase (12/15-LOX) produces 12-HETE and is found activated under conditions of insulin resistance and systemic inflammation. 12-HETE acts as an inflammatory mediator that promotes β-cell dysfunction and death through oxidative stress. Since 12-HETE has been shown to bind the G-protein coupled receptor 31 (GPR31), we hypothesized that this binding activates pathways leading to β-cell dysfunction. To test our hypothesis, we screened primary islets and β-cell lines for the presence of GPR31 and studied islets and glucose homeostasis in Gpr31b-/- mice. We observed that GPR31 is present in human and mouse islets, and the mouse β cell-derived cell line (βTC3). Consistent with a role in inflammation, GPR31 expression was increased upon stimulation with a cocktail of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β). We also created a germline knockout of the gene encoding GPR31 (Gpr31b) in mice. Similar to mice with deletion of the gene for 12/15-LOX (Alox15-/-), Gpr31b-/- mice were viable and healthy, with body weight and glucose tolerance indistinguishable from control littermates. To mimic conditions of insulin resistance in vitro, we treated isolated islets Gpr31-/- and control littermates with 10 µM S961, an insulin receptor inhibitor. Treated control islets showed an expected decrease in β-cell identity genes (Ins1/2, Pdx1), genes encoding antioxidant enzymes (Gpx1), and an increase in genes associated with ER stress (Chop, sXbp1). In contrast, Gpr31b-/- islets expression of these genes was resistant to S961. Compared to control littermates, Gpr31-/- mice were resistant to glucose intolerance induced by the β-cell inflammatory toxin streptozotocin. Together these data support the hypothesis that the major product of 12/15-LOX, 12-HETE, acts via GPR31 in promoting β-cell dysfunction in the setting of insulin resistance and inflammation. Disclosure M. Hernandez-Perez: None. I. Haider: None. R.M. Anderson: None. S.A. Tersey: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. Funding JDRF (3-PDF-2019-750-A-N); National Institutes of Health (T32DK064466, R01DK105588, (P30DK097512)

New insights from The Cancer Genome Atlas: Implications for GPCR expression in cell compartments in solid tumors

Assessment of the transcriptome of tumors via high‐throughout methods such as RNA‐sequencing (RNA‐seq) has yielded insights that enable understanding of tumor biology and facilitate drug discovery efforts. A challenge with using such “omics” methods is the presence of technical variation between groups of samples (often termed ‘batch effects’) which arise from differences in sample collection, handling and processing, especially with large, multi‐center, consortium‐based efforts such as The Cancer Genome Atlas (TCGA). Approaches to correct for technical artifacts should seek to minimize variance introduced by known technical factors, while preserving the biological variability within the dataset. We present a workflow for assessing the magnitude of identifiable technical artifacts within RNA‐seq data in TCGA and to identify the most relevant sources of variation are most relevant and steps to correct for such artifacts using bioinformatics tools. We show that certain TCGA datasets are highly impacted by technical variation and that correction for these artifacts can alter downstream analyses such as association studies and survival analysis. Correction for these effects using batch correction tools removes the influence of technical artifacts, while preserving biological variation and the overall structure and integrity of the data. We use these corrected data to establish an updated list of significant genes that impact survival in a range of solid tumor types in TCGA . In addition, we use methods to identify networks of co‐expressed and co‐regulated genes and identify genes that are sensitive to molecular features of tumors, such as the presence of specific driver mutations.Using these “tidied‐up” datasets, we analyzed the expression of GPCRs (G protein‐coupled receptors) in tumors to identify the cellular compartments within the tumor microenvironment that express particular GPCRs. Due to their widespread utility as pharmacological targets and numerous recent results re‐emphasizing a role for GPCRs in cancer, such analyses provide a valuable tool for drug discovery. We document a number of GPCRs that are widely expressed in immune cells in a range of solid tumors and that whose functions may impact the phenotype of immune cells, including in cancer. Such widely expressed immune‐associated GPCRs are examples of novel putative targets in the rapidly growing field of immuno‐oncology. These analyses for GPCRs can be expanded to other classes of targets as well, providing a general tool for using large databases of gene expression to identify and evaluate genes that are likely to be expressed in cell types within tumors in vivo, thereby avoiding pitfalls that arise when working with isolated cells or single‐cell based approaches.Support or Funding InformationASPET David Lehr Award

Role of G-Protein Coupled Receptors in Chemotaxis of Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) are a recently identified family of immune cells mostly present at barrier surfaces. They play an important role in the induction, regulation, and resolution of inflammatory responses. Environmental signals play an important role in development and function of ILCs. G-protein coupled receptors (GPCRs) sense and mediate cellular responses to the environmental signals. ILCs express several G-protein coupled receptors, which play a critical role in migration of these cells to appropriate sites. Here, we describe a method to test the migration of ILCs toward 7α,25-hydroxycholesterol, which is mediated by cell surface-expressed GPR183. A similar strategy can be employed to test the role of other GPCRs in mediating the migration of ILCs toward other chemotactic ligands.

Identifying miRNA Biomarkers and Predicted Targets Associated with Venous Thromboembolism in Colorectal Cancer Patients

Introduction Venous thromboembolism (VTE) is the second leading cause of mortality among cancer patients and is potentially preventable through the use of anticoagulation therapy. Risk models exist to guide use of prophylactic therapy but have low positive predictive value. New biomarkers are needed, particularly in intermediate-risk cancer types. Extracellular small RNAs, such as miRNAs, are promising biomarkers that have been implicated in tumor-dependent modification of platelets, a key component of thrombus formation. In this pilot study, we explore miRNAs as potential biomarkers for VTE risk in patients with colorectal cancer. Methods We conducted a case-control study utilizing specimens from a population enrolled in a prospective colorectal cancer biorepository at the Cleveland Clinic. Cases were defined as patients who developed VTE, including deep vein thrombosis and pulmonary embolism, within 6 months after cancer diagnosis and had their blood drawn prior to VTE. Cases were matched to controls (who did not have VTE and had a minimum of 6 months survival after cancer diagnosis) on a 1:2 ratio based on age, sex, cancer stage at diagnosis, and cancer treatment received prior to blood collection for a total of 21 patients. Total RNA from plasma specimens were sequenced on a Ion Proton platform (Thermo Fisher). Sequencing data were analyzed using the limma-voom R package. As this study was meant to be exploratory, miRNA were determined to be differentially expressed at a corrected Benjamini-Hochberg false detection rate (FDR) < 0.2. Target genes of differentially expressed miRNAs were predicted using mirDB and target gene pathways constructed with PANTHER. Results The study population had a median age of 65 (IQR 51-72). Of these patients, 85.7% were male; 42.9% had stage I/II cancer, 42.9% stage III cancer, 14.3% stage IV cancer; 71.4% had received no treatment prior to blood collection, 14.3% received chemo/chemoradiation therapy, and 14.3% received surgery. A total of 2426 unique miRNAs (median 1524, IQR 1371-1665) were expressed in the study group. Of these, 9 miRNAs were significantly differentially expressed (FDR < 0.2) and downregulated in cases compared to controls: hsa-miR-4451, 942-3p, 8063, 3132, 3118, 105-5p, 891a-5p, 200a-5p, and 6832-3p. From these miRNAs, 609 target genes were predicted and classified into 75 pathways, including angiogenesis, G-protein coupled receptors (GPCRs), inflammation mediated by chemo/cytokines, and integrin signaling. Target genes within these notable pathways included EPHA3, PDGFA, PTK2/FAK1, and IL15. Conclusions We identified 9 significantly downregulated miRNAs in the blood of colorectal cancer patients who developed VTE compared to controls in this pilot study. These data suggest that colorectal cancer patients may express unique miRNA profiles prior to VTE development which may be useful as biomarkers in future predictive models. In addition, this study identified potential new mechanistic targets for understanding cancer-associated thrombosis. While the role of GPCRs, integrins, and inflammation in platelet activation and function is widely known, this study also identified factors within the angiogenesis pathway that have been linked to increased platelet aggregation and tissue factor activation. Thus, downregulation of inhibitory miRNAs may cause disinhibition of pathways important for platelet and vascular function and other prothrombotic factors. Disclosures Khorana: Janssen: Consultancy; Bayer: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy. McCrae:Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Family C G-Protein-Coupled Receptors in Alzheimer's Disease and Therapeutic Implications.

Alzheimer’s disease (AD), particularly its sporadic or late-onset form (SAD/LOAD), is the most prevalent (96–98% of cases) neurodegenerative dementia in aged people. AD’s neuropathology hallmarks are intrabrain accumulation of amyloid-β peptides (Aβs) and of hyperphosphorylated Tau (p-Tau) proteins, diffuse neuroinflammation, and progressive death of neurons and oligodendrocytes. Mounting evidences suggest that family C G-protein-coupled receptors (GPCRs), which include γ-aminobutyric acid B receptors (GABABRs), metabotropic glutamate receptors (mGluR1-8), and the calcium-sensing receptor (CaSR), are involved in many neurotransmitter systems that dysfunction in AD. This review updates the available knowledge about the roles of GPCRs, particularly but not exclusively those expressed by brain astrocytes, in SAD/LOAD onset and progression, taking stock of their respective mechanisms of action and of their potential as anti-AD therapeutic targets. In particular, GABABRs prevent Aβs synthesis and neuronal hyperexcitability and group I mGluRs play important pathogenetic roles in transgenic AD-model animals. Moreover, the specific binding of Aβs to the CaSRs of human cortical astrocytes and neurons cultured in vitro engenders a pathological signaling that crucially promotes the surplus synthesis and release of Aβs and hyperphosphorylated Tau proteins, and also of nitric oxide, vascular endothelial growth factor-A, and proinflammatory agents. Concurrently, Aβs•CaSR signaling hinders the release of soluble (s)APP-α peptide, a neurotrophic agent and GABABR1a agonist. Altogether these effects progressively kill human cortical neurons in vitro and likely also in vivo. Several CaSR’s negative allosteric modulators suppress all the noxious effects elicited by Aβs•CaSR signaling in human cortical astrocytes and neurons thus safeguarding neurons’ viability in vitro and raising hopes about their potential therapeutic benefits in AD patients. Further basic and clinical investigations on these hot topics are needed taking always heed that activation of the several brain family C GPCRs may elicit divergent upshots according to the models studied.

Emerging Roles for G-protein Coupled Receptors in Development and Activation of Macrophages.

Macrophages have emerged as a key component of the innate immune system that emigrates to peripheral tissues during gestation and in the adult organism. Their complex pathway to maturity, their unique plasticity and their various roles as effector and regulatory cells during an immune response have been the focus of intense research. A class of surface molecules, the G-Protein coupled receptors (GPCRs) play important roles in many immune processes. They have drawn attention in regard to these functions and the potential for therapeutic targets that can modulate the response of immune cells in pathologies such as diabetes, atherosclerosis, and chronic inflammatory diseases. Of the more than 800 GPCRs identified, ~100 are currently targeted with drugs which have had their activity investigated in vivo. Macrophages express a number of GPCRs which have central roles during cell differentiation and in the regulation of their functions. While some macrophage GPCRs such as chemokine receptors have been studied in great detail, the roles of other receptors of this large family are still not well understood. This review summarizes new insights into macrophage biology, differences of human, and mouse macrophages and gives details of some of the GPCRs expressed by this cell type.

Expression of G protein‐coupled receptors in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and has an overall 5‐year survival rate ~7%. PDAC has poor response to chemotherapy and radiotherapy; thus new treatments for PDAC are an important, unmet medical need. G protein‐coupled receptor (GPCR) regulate many biological functions, including in cancer cells, but GPCR expression and the role of GPCRs in PDAC cells is unclear. We generated two primary PDAC cell lines (79E and 34E) and an immortalized normal pancreatic ductal epithelial cell line (HPDE6). Hypothesizing that GPCRs may regulate PDAC cells, we used Taqman GPCR array to assess GPCR expression in four PDAC cell lines, 79E, 34E, Miapaca, and AsPC. We identified 68 commonly expressed GPCRs in all four PDAC cell lines, among which 22 GPCRs were significantly up‐regulated (at least 2‐fold) in PDAC cells compared to normal control cell HPDE6; 13 GPCRs were significantly down‐regulated and 3 GPCRs were uniquely expressed in PDACs. We further tested the 13 most up‐regulated GPCRs using real time qPCR and confirmed that LPHN1, CHRM4, FZD4, HRH1 and CD97 have much higher expression in PDACs compared to HPED6, suggesting that these GPCRs might contribute to the malignant phenotype and perhaps could be novel treatment targets for pancreatic cancer, in particular for PDAC.Support or Funding InformationThis work is supported by NIH/NCI Research Grants R21 CA189477, R01 CA155620, and Padres Pedal the Cause PTC2017.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heterotrimeric G protein signaling in plant

Heterotrimeric guanine nucleotide-binding protein (G protein) signaling is an evolutionarily conserved mechanism in diverse eukaryotic organisms. In the past half century, five Nobel Prizes are awarded to researchers working in the G protein field, these seminal studies make the G protein pathway become the best understood signaling transmit system in the world. In animals, heterotrimeric G proteins contains α , β and γ subunits, perceives extracellular stimuli through G protein-coupled receptors (GPCR), and transmit signals to ion channels, enzymes and other effectors to affect numerous cellular behaviors. In recent years, much has been learned about the diversity of signal transduction through plant G proteins thanks to the identification and mutation of genes in Arabidopsis and rice. Plant cells have most of the core elements found in animal G signaling, such as α , β and γ subunits. However, plant G proteins transmit signals by atypical mechanisms. The plant G α subunit spontaneously releases GDP and forms a stable GTP bound state in vitro , and thus are self-activating. The self-activating characteristic also means that plant G protein paradigm do not need and therefore do not have typical animal GPCR. Indeed, the current evidence supports that no typical GPCR exists in plant. In most plants, the role of GPCR is performed by GTPase-accelerating protein (GAP), such as Arabidopsis regulator of G protein signaling 1 (AtRGS1). Interestingly, there is no ortholog of AtRGS1 in rice genome. Recently, an important advance has been proved that up-regulation of COLD1 is positively correlated with chilling tolerance in rice. COLD1 has nine-transmembrane domains, and localize to plasma membrane (PM) and endoplasmic reticulum (ER). The COLD1 physically interacts with RGA1 and acts as RGS protein that senses low temperature signals and accelerates G-protein GTPase activity. These results indicate that non-GPCR proteins in plant can topologically and functionally resemble animal GPCRs. In plants, the repertoire of the heterotrimeric G protein is much simpler than that in mammalian, but G γ subunits exhibit an extraordinary level of structural diversity and show significant differences from their animal counterparts. The non-canonical G γ subunits in plant kingdom have a large cysteine domain on C terminal, can be four times larger than the average mammalian G γ subunits size. The non-canonical G γ subunits present an inhibitory effects of N terminal on C terminal. Recent studies have shown that the manipulation of two non-canonical G γ subunits, GS3 (grain size 3) and DEP1 (dense and erect panicle 1), represents new strategy to simultaneously increase grain yield and nitrogen use efficiency in rice breeding. In this review, we focus on the recent insights of G protein network derived from the research about the model plant rice and Arabidopsis , the difference of G protein signal between plants and animals, and the potential of G protein to improve the crops productivity.