Evolutionary origins of bitter taste receptors in jawed vertebrates.

Taste is a sense that detects information about nutrients and toxins in foods. Of the five basic taste qualities, bitterness is associated with the detection of potentially harmful substances like plant alkaloids. In bony vertebrates, type 2 taste receptors (T2Rs), which are G-protein-coupled receptors (GPCRs), act as bitter taste receptors1,2. In vertebrates, six GPCR gene families are described as chemosensory receptor genes, encoding taste receptor families (T1Rs and T2Rs) and olfactory receptor families (ORs, V1Rs, V2Rs, and TAARs). These families of receptors have been found in all major jawed vertebrate lineages, except for the T2Rs, which are confined to bony vertebrates3. Therefore, T2Rs are believed to have emerged later than the other chemosensory receptor genes in the bony vertebrate lineage. So far, only the genomes of two cartilaginous fish species have been mined for TAS2R genes, which encode T2Rs4. Here, we identified novel T2Rs in elasmobranchs, namely selachimorphs (sharks) and batoids (rays, skates, and their close relatives) by an exhaustive search covering diverse cartilaginous fishes. Using functional and mRNA expression analyses, we demonstrate that their T2Rs are expressed in the oral taste buds and contribute to the detection of bitter compounds. This finding indicates the early origin of T2Rs in the common ancestor of jawed vertebrates.

Droplet-based microfluidic platform for detecting agonistic peptides that are self-secreted by yeast expressing a G-protein-coupled receptor.

BACKGROUND: Single-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides. RESULTS: The water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists. CONCLUSIONS: We established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells.

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

GPR27 expression correlates with prognosis and tumor progression in gliomas.

Backgrounds: Glioma is a highly aggressive type of brain tumor, and its prognosis is still poor despite recent progress in treatment strategies. G protein-coupled receptor 27 (GPR27) is a member of the G protein-coupled receptor family and has been reported to be involved in various cellular processes, including tumor progression. Nevertheless, the clinical potential and tumor-related role of GPR27 in glioma remain unknown. Here we aimed to explore the function and role of GPR27 in gliomas. Methods: In the current study, we evaluated the expression and clinical significance of GPR27 in gliomas using data from The Cancer Genome Atlas (TCGA) datasets. We also conducted cellular experiments to evaluate the functional role of GPR27 in glioma cell growth. Results: We found that GPR27 expression level was closely associated with disease status of glioma. Of note, GPR27 was negatively correlated with WHO grade, with grade IV samples showing the lowest GPR27 levels, while grade II samples showed the highest levels. Patients with IDH mutation or 1p/19q co-deletion exhibited higher GPR27 levels. In addition, lower GPR27 levels were correlated with higher death possibilities. In cellular experiments, we confirmed that GPR27 inhibited glioma cell growth. Conclusions: Our results indicate that GPR27 may function as a potential prognostic biomarker and therapeutic target in gliomas. Further studies are needed to illustrate the signaling mechanism and clinical implications of GPR27 in gliomas.

Influence of GPRC5A-Regulated ABCB1 Expression on Lung Adenocarcinoma Proliferation.

Objective Aberrant expression of ATP binding cassette subfamily B member 1 (ABCB1) plays a key role in several cancers. However, influence of G protein coupled receptor family C group 5 type A (GPRC5A)-regulated ABCB1 expression on lung adenocarcinoma proliferation remains unclear. Therefore, this study investigated the effect of GPRC5A regulated ABCB1 expression on the proliferation of lung adenocarcinoma. Methods ABCB1 expressions in lung adenocarcinoma cell lines, human lung adenocarcinoma tissues, and tracheal epithelial cells and lung tissues of GPRC5A knockout mice and wild-type mice were analyzed with RT-PCR, Western blot, or immunohistochemical analysis. Cell counting kit-8 assay was performed to analyze the sensitivity of tracheal epithelial cells from GPRC5A knockout mice to chemotherapeutic agents. Subcutaneous tumor formation assay was performed to confirm whether down-regulation of ABCB1 could inhibit the proliferation of lung adenocarcinoma in vivo. To verify the potential regulatory relationship between GPRC5A and ABCB1, immunofluorescence and immunoprecipitation assays were performed. Results ABCB1 expression was up-regulated in lung adenocarcinoma cell lines and human lung adenocarcinoma tissues. ABCB1 expression in the tracheal epithelial cells and lung tissues of GPRC5Adeficient mice was higher than that in the wild type mice. Tracheal epithelial cells of GPRC5A knockout mice were much more sensitive to tariquidar and doxorubicin than those of GPRC5A wild type mice. Accordingly, 28 days after injection of the transplanted cells, the volume and weight of lung tumor in ABCB1knockout cell-transplanted GPRC5A-/-C57BL/6 mice were significantly smaller than those in wild type cell-transplanted mice (P= 0.0043, P= 0.0060). Furthermore, immunofluorescence and immunoprecipitation assays showed that GPRC5A regulated ABCB1 expression by direct binding.Conclusion GPRC5A reduces lung adenocarcinoma proliferation via inhibiting ABCB1 expression. The pathway by which GPRC5A regulates ABCB1 expression needs to be investigated.

Effective in vitro evaluation of the risk of histamine release related to valemetostat tosylate using MRGPRX2-expressing cells.

Mas-related G-protein-coupled receptor X2 (MRGPRX2), expressed on mast cells, is associated with drug-induced pseudo-allergic reactions. Although it is well known that there are differences of sensitivity between species in the pseudo-allergic reactions, no platform for evaluating a human risk of the pseudo-allergic reactions observed in nonclinical studies has been established. Valemetostat tosylate, developed as an anti-cancer drug, induced histamine release in a nonclinical study with dogs. The purpose of the current study was to identify the mechanism and assess the human risk of valemetostat-tosylate-induced histamine release using dog and human MRGPRX2-expressing cells. In an experiment with human or dog MRGPRX2-expressing cells, valemetostat tosylate caused activation of human and dog MRGPRX2. Importantly, the EC50 for dog MRGPRX2 was consistent with the Cmax value at which histamine release was observed in dogs. Furthermore, the EC50 for human MRGPRX2 was ca. 27-fold higher than that for dog MRGPRX2, indicating a species difference in histamine-releasing activity. In a clinical trial, histamine release was not observed in patients receiving valemetostat tosylate. In conclusion, an in vitro assay using human and animal MRGPRX2-expressing cells would be an effective platform to investigate the mechanism and predict the human risk of histamine release observed in nonclinical studies.

Hepatic LGR4 aggravates cholestasis-induced liver injury in mice.

Current therapy for hepatic injury induced by the accumulation of bile acids is limited. Leucine-rich repeat G protein-coupled receptor 4 (LGR4), also known as GPR48, is critical for cytoprotection and cell proliferation. Here, we reported a novel function for the LGR4 in cholestatic liver injury. In the bile duct ligation (BDL)-induced liver injury model, hepatic LGR4 expression was significantly downregulated. Deficiency of LGR4 in hepatocytes (Lgr4LKO) notably decreased BDL-induced liver injury measured by hepatic necrosis, fibrosis, and circulating liver enzymes and total bilirubin. Levels of total bile acids in plasma and liver were markedly reduced in these mice. However, deficiency of LGR4 in macrophages (Lyz2-Lgr4MKO) demonstrated no significant effect on liver injury induced by BDL. Deficiency of LGR4 in hepatocytes significantly attenuated S1PR2 and the phosphorylation of protein kinase B (AKT) induced by BDL. Recombinant Rspo1 and Rspo3 potentiated the taurocholic acid (TCA)-induced upregulation in S1PR2 and phosphorylation of AKT in hepatocytes. Inhibition of S1PR2-AKT signaling by specific AKT or S1PR2 inhibitors blocked the increase of bile acid secretion induced by Rspo1/3 in hepatocytes. Our studies indicate that the R-spondins (Rspos)-LGR4 signaling in hepatocytes aggravates the cholestatic liver injury by potentiating the production of bile acids in a S1PR2-AKT-dependent manner.NEW & NOTEWORTHY Deficiency of LGR4 in hepatocytes alleviates BDL-induced liver injury. LGR4 in macrophages demonstrates no effect on BDL-induced liver injury. Rspos-LGR4 increases bile acid synthesis and transport via potentiating S1PR2-AKT signaling in hepatocytes.

Buyang huanwu decoction promotes remyelination via miR-760-3p/GPR17 axis after intracerebral hemorrhage.

ETHNOPHARMACOLOGICAL RELEVANCE: The repairment of myelin sheaths is crucial for mitigating neurological impairments of intracerebral hemorrhage (ICH). However, the current research on remyelination processes in ICH remains limited. A representative traditional Chinese medicine, Buyang Huanwu decoction (BYHWD), shows a promising therapeutic strategy for ICH treatment. AIM OF THE STUDY: To investigate the pro-remyelination effects of BYHWD on ICH and explore the underlying mechanisms. MATERIALS AND METHODS: The collagenase-induced mice ICH model was created for investigation. BYHWD's protective effects were assessed by behavioral tests and histological staining. Transmission electron microscopy was used for displaying the structure of myelin sheaths. The remyelination and oligodendrocyte differentiation were evaluated by the expressions of myelin proteolipid protein (PLP), myelin basic protein (MBP), MBP/TAU, Olig2/CC1, and PDGFRα/proliferating cell nuclear antigen (PCNA) through RT-qPCR and immunofluorescence. Transcriptomics integrated with disease database analysis and experiments in vivo and in vitro revealed the microRNA-related underlying mechanisms. RESULTS: Here, we reported that BYHWD promoted the neurological function of ICH mice and improved remyelination by increasing PLP, MBP, and TAU, as well as restoring myelin structure. Besides, we showed that BYHWD promoted remyelination by boosting the differentiation of PDGFRα+ oligodendrocyte precursor cells into olig2+/CC1+ oligodendrocytes. Additionally, we demonstrated that the remyelination effects of BYHWD worked by inhibiting G protein-coupled receptor 17 (GPR17). miRNA sequencing integrated with miRNA database prediction screened potential miRNAs targeting GPR17. By applying immunofluorescence, RNA in situ hybridization and dual luciferase reporter gene assay, we confirmed that BYHWD suppressed GPR17 and improved remyelination by increasing miR-760-3p. CONCLUSIONS: BYHWD improves remyelination and neurological function in ICH mice by targeting miR-760-3p to inhibit GPR17. This study may shed light on the orchestration of remyelination mechanisms after ICH, thus providing novel insights for developing innovative prescriptions with brain-protective properties.

Mast cells help organize the Peyer's patch niche for induction of IgA responses.

Peyer's patches (PPs) are lymphoid structures situated adjacent to the intestinal epithelium that support B cell responses that give rise to many intestinal IgA-secreting cells. Induction of isotype switching to IgA in PPs requires interactions between B cells and TGFß-activating conventional dendritic cells type 2 (cDC2s) in the subepithelial dome (SED). However, the mechanisms promoting cDC2 positioning in the SED are unclear. Here, we found that PP cDC2s express GPR35, a receptor that promotes cell migration in response to various metabolites, including 5-hydroxyindoleacetic acid (5-HIAA). In mice lacking GPR35, fewer cDC2s were found in the SED, and frequencies of IgA+ germinal center (GC) B cells were reduced. IgA plasma cells were reduced in both the PPs and lamina propria. These phenotypes were also observed in chimeric mice that lacked GPR35 selectively in cDCs. GPR35 deficiency led to reduced coating of commensal bacteria with IgA and reduced IgA responses to cholera toxin. Mast cells were present in the SED, and mast cell-deficient mice had reduced PP cDC2s and IgA+ cells. Ablation of tryptophan hydroxylase 1 (Tph1) in mast cells to prevent their production of 5-HIAA similarly led to reduced PP cDC2s and IgA responses. Thus, mast cell-guided positioning of GPR35+ cDC2s in the PP SED supports induction of intestinal IgA responses.

Maternal Age at Menarche Genes Determines Fetal Growth Restriction Risk.

We aimed to explore the potential link of maternal age at menarche (mAAM) gene polymorphisms with risk of the fetal growth restriction (FGR). This case (FGR)-control (FGR free) study included 904 women (273 FGR and 631 control) in the third trimester of gestation examined/treated in the Departments of Obstetrics. For single nucleotide polymorphism (SNP) multiplex genotyping, 50 candidate loci of mAAM were chosen. The relationship of mAAM SNPs and FGR was appreciated by regression procedures (logistic/model-based multifactor dimensionality reduction [MB-MDR]) with subsequent in silico assessment of the assumed functionality pithy of FGR-related loci. Three mAAM-appertain loci were FGR-linked to genes such as KISS1 (rs7538038) (effect allele G-odds ratio (OR)allelic = 0.63/pperm = 0.0003; ORadditive = 0.61/pperm = 0.001; ORdominant = 0.56/pperm = 0.001), NKX2-1 (rs999460) (effect allele A-ORallelic = 1.37/pperm = 0.003; ORadditive = 1.45/pperm = 0.002; ORrecessive = 2.41/pperm = 0.0002), GPRC5B (rs12444979) (effect allele T-ORallelic = 1.67/pperm = 0.0003; ORdominant = 1.59/pperm = 0.011; ORadditive = 1.56/pperm = 0.009). The haplotype ACA FSHB gene (rs555621*rs11031010*rs1782507) was FRG-correlated (OR = 0.71/pperm = 0.05). Ten FGR-implicated interworking models were founded for 13 SNPs (pperm ≤ 0.001). The rs999460 NKX2-1 and rs12444979 GPRC5B interplays significantly influenced the FGR risk (these SNPs were present in 50% of models). FGR-related mAAM-appertain 15 polymorphic variants and 350 linked SNPs were functionally momentous in relation to 39 genes participating in the regulation of hormone levels, the ovulation cycle process, male gonad development and vitamin D metabolism. Thus, this study showed, for the first time, that the mAAM-appertain genes determine FGR risk.

Beyond the Nucleus: Plastic Chemicals Activate G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are central mediators of cell signaling and physiological function. Despite their biological significance, GPCRs have not been widely studied in the field of toxicology. Herein, we investigated these receptors as novel targets of plastic chemicals using a high-throughput drug screening assay with 126 human non-olfactory GPCRs. In a first-pass screen, we tested the activity of triphenol phosphate, bisphenol A, and diethyl phthalate, as well as three real-world mixtures of chemicals extracted from plastic food packaging covering all major polymer types. We found 11 GPCR-chemical interactions, of which the chemical mixtures exhibited the most robust activity at adenosine receptor 1 (ADORA1) and melatonin receptor 1 (MTNR1A). We further confirm that polyvinyl chloride and polyurethane products contain ADORA1 or MTNRA1 agonists using a confirmatory secondary screen and pharmacological knockdown experiments. Finally, an analysis of the associated gene ontology terms suggests that ADORA1 and MTNR1A activation may be linked to downstream effects on circadian and metabolic processes. This work highlights that signaling disruption caused by plastic chemicals is broader than that previously believed and demonstrates the relevance of nongenomic pathways, which have, thus far, remained unexplored.

Evaluating conserved domains and motifs of decapod gonadotropin-releasing hormone G protein-coupled receptor superfamily.

G protein-coupled receptors (GPCRs) are an ancient family of signal transducers that are both abundant and consequential in metazoan endocrinology. The evolutionary history and function of the GPCRs of the decapod superfamilies of gonadotropin-releasing hormone (GnRH) are yet to be fully elucidated. As part of which, the use of traditional phylogenetics and the recycling of a diminutive set of mis-annotated databases has proven insufficient. To address this, we have collated and revised eight existing and three novel GPCR repertoires for GnRH of decapod species. We developed a novel bioinformatic workflow that included clustering analysis to capture likely GnRH receptor-like proteins, followed by phylogenetic analysis of the seven transmembrane-spanning domains. A high degree of conservation of the sequences and topology of the domains and motifs allowed the identification of species-specific variation (up to ~70%, especially in the extracellular loops) that is thought to be influential to ligand-binding and function. Given the key functional role of the DRY motif across GPCRs, the classification of receptors based on the variation of this motif can be universally applied to resolve cryptic GPCR families, as was achieved in this work. Our results contribute to the resolution of the evolutionary history of invertebrate GnRH receptors and inform the design of bioassays in their deorphanization and functional annotation.

Exome sequencing identifies ADGRG4 G-protein-coupled receptors gene as a novel cancer biomarker in ovarian cancer patients from North India.

Adhesion G protein-coupled receptor G4 (ADGRG4) is a G protein-coupled receptor (GPCR) that belongs to the adhesion family. Participation of ADGRG4 in cell adhesion and migration, signaling pathway activation, influence on angiogenesis, and modulation of immune responses are some of the possible ways through which it may contribute to oncogenesis. Conducting extensive omics studies poses budgetary challenges to small labs in peripheral areas, primarily due to restricted research funding and resource limitations. Here we propose a low-budget model for biomarker screening. A total of 11 ovarian cancer samples were sent for exome sequencing. Among various genes, ADGRG4 variants were present in all 11 samples and thus were chosen as a potential biomarker in the present population. However, the precise role of ADGRG4 in cancer is not fully understood. The present study aims to look at the association between the ADGRG4 gene variants and their risk of ovarian cancer in the North Indian region of Jammu and Kashmir, India. Overall, 235 individuals (115 cases and 120 healthy controls) were genotyped for the selected biomarker using Sanger sequencing. Logistic regression was used to assess the relationship between the variant and ovarian cancer. A statistically significant association was identified between the ADGRG4 variant rs5930932 polymorphism and the incidence of ovarian cancer among the study population. When corrected for age and BMI, the dominating OR of variant rs5930932 was 1.035 (1.003-1.069) under HWE patients (0.95) and controls (0.18), with a p-value of (0.03). According to the findings of the current investigation, the ADGRG4 gene variant rs5930932 increases the chance of developing ovarian cancer in the studied population.

CMV-encoded GPCRs in infection, disease, and pathogenesis.

G protein coupled receptors (GPCRs) are seven-transmembrane domain proteins that modulate cellular processes in response to external stimuli. These receptors represent the largest family of membrane proteins, and in mammals, their signaling regulates important physiological functions, such as vision, taste, and olfaction. Many organisms, including yeast, slime molds, and viruses encode GPCRs. Cytomegaloviruses (CMVs) are large, betaherpesviruses, that encode viral GPCRs (vGPCRs). Human CMV (HCMV) encodes four vGPCRs, including UL33, UL78, US27, and US28. Each of these vGPCRs, as well as their rodent and primate orthologues, have been investigated for their contributions to viral infection and disease. Herein, we discuss how the CMV vGPCRs function during lytic and latent infection, as well as our understanding of how they impact viral pathogenesis.

Structure-guided engineering of biased-agonism in the human niacin receptor via single amino acid substitution.

The Hydroxycarboxylic acid receptor 2 (HCA2), also known as the niacin receptor or GPR109A, is a prototypical GPCR that plays a central role in the inhibition of lipolytic and atherogenic activities. Its activation also results in vasodilation that is linked to the side-effect of flushing associated with dyslipidemia drugs such as niacin. GPR109A continues to be a target for developing potential therapeutics in dyslipidemia with minimized flushing response. Here, we present cryo-EM structures of the GPR109A in complex with dyslipidemia drugs, niacin or acipimox, non-flushing agonists, MK6892 or GSK256073, and recently approved psoriasis drug, monomethyl fumarate (MMF). These structures elucidate the binding mechanism of agonists, molecular basis of receptor activation, and insights into biased signaling elicited by some of the agonists. The structural framework also allows us to engineer receptor mutants that exhibit G-protein signaling bias, and therefore, our study may help in structure-guided drug discovery efforts targeting this receptor.

Sex-specific epigenetics drive low GPER expression in gastrointestinal smooth muscles in type 2 diabetic mice.

Type 2 diabetes mellitus (T2D) causes gastroparesis, delayed intestinal transit, and constipation, for unknown reasons. Complications are predominant in women than men (particularly pregnant and postmenopausal women), suggesting a female hormone-mediated mechanism. Low G-protein coupled estrogen receptor (GPER) expression from epigenetic modifications may explain it. We explored sexually differentiated GPER expression and gastrointestinal symptoms related to GPER alterations in wild-type (WT) and T2D mice (db/db). We also created smooth muscle-specific GPER knockout (GPER KO) mice to phenotypically explore the effect of GPER deficiency on gastrointestinal motility. GPER mRNA and protein expression, DNA methylation and histone modifications were measured from stomach and colon samples of db/db and WT mice. Changes in gut motility were also evaluated as daily fecal pellet production patterns. We found that WT female tissues have the highest GPER mRNA and protein expressions. The expression is lowest in all db/db. GPER downregulation is associated with promoter hypermethylation and reduced enrichment of H3K4me3 and H3K27ac marks around the GPER promoter. We also observed sex-specific disparities in fecal pellet production patterns of the GPER KO mice compared to WT. We thus, conclude that T2D impairs gut GPER expression, and epigenetic sex-specific mechanisms matter in the downregulation.

Resolvin D2 limits atherosclerosis progression via myeloid cell-GPR18.

Dysregulated inflammation-resolution programs are associated with atherosclerosis progression. Resolvins, in part, mediate inflammation-resolution programs. Indeed, Resolvin D2 (RvD2) activates GPR18, a G-protein-coupled receptor, and limits plaque progression, though the cellular targets of RvD2 remain unknown. Here, we developed a humanized GPR18 floxed ("fl/fl") and a myeloid (Lysozyme M Cre) GPR18 knockout (mKO) mouse. We functionally validated this model by assessing efferocytosis in bone marrow-derived macrophages (BMDMs) and found that RvD2 enhanced efferocytosis in the fl/fl, but not in the mKO BMDMs. To understand the functions of RvD2-GPR18 in atherosclerosis, we performed a bone marrow transfer of fl/fl or mKO bone marrow into Ldlr-/- recipients. For these experiments, we treated each genotype with either Vehicle/PBS or RvD2 (25 ng/mouse, 3 times/week for 3 weeks). Myeloid loss of GPR18 resulted in significantly more necrosis, increased cleaved caspase-3+ cells and decreased percentage of Arginase-1+ -Mac2+ cells without a change in overall Mac2+ plaque macrophages, compared with fl/fl➔Ldlr-/- transplanted mice. RvD2 treatment decreased plaque necrosis, the percent of cleaved caspase-3+ cells and increased the percent of Arginase-1+ -Mac2+ cells in fl/fl➔Ldlr-/- mice, but not in the mKO➔Ldlr-/- transplanted mice. These results suggest that GPR18 plays a causal role in limiting atherosclerosis progression and that RvD2's ability to limit plaque necrosis is in part dependent on myeloid GRP18.

Methylation of GPRC5A promotes liver metastasis and docetaxel resistance through activating mTOR signaling pathway in triple negative breast cancer.

AIMS: This study aims to explore the function and mechanism of G Protein-coupled receptor class C group 5 member A (GPRC5A) in docetaxel-resistance and liver metastasis of breast cancer. METHODS: Single-cell RNA transcriptomic analysis and bioinformatic analysis are used to screen relevant genes in breast cancer metastatic hepatic specimens. MeRIP, dual-luciferase analysis and bioinformation were used to detect m6A modulation. Mass spectrometry (MS), co-inmunoprecipitation (co-IP) and immunofluorescence colocalization were executed to explore the mechanism of GPRC5A in breast cancer cells. RESULT: GPRC5A was upregulated in triple-negative breast cancer (TNBC) and was associated with a poor prognosis. In vitro and in vivo experiments demonstrated that knockdown of GPRC5A alleviated metastasis and resistance to docetaxel in TNBC. Overexpression of GPRC5A had the opposite effects. The m6A methylation of GPRC5A mRNA was modulated by METTL3 and YTHDF1, which facilitates its translation. GPRC5A inhibited the ubiquitination-dependent degradation of LAMTOR1, resulting in the recruitment of mTORC1 to lysosomes and activating the mTORC1/p70s6k signaling pathway. CONCLUSION: METTL3/YTHDF1 axis up-regulates GPRC5A expression by m6A methylation. GPRC5A activates mTORC1/p70s6k signaling pathway by recruiting mTORC1 to lysosomes, consequently promotes docetaxel-resistance and liver metastasis.

Lactate/GPR81 recruits regulatory T cells by modulating CX3CL1 to promote immune resistance in a highly glycolytic gastric cancer.

Lactate plays an important role in shaping immune tolerance in tumor microenvironment (TME) and correlates with poor prognosis in various solid tumors. Overcoming the immune resistance in an acidic TME may improve the anti-tumor immunity. Here, this study elucidated that via G-protein-coupled receptor 81 (GPR81), lactate could modulate immune tolerance in TME by recruiting regulatory T cells (Tregs) in vitro and in vivo. A high concentration of lactate was detected in cell supernatant and tissues of gastric cancer (GC), which was modulated by lactic dehydrogenase A (LDHA). GPR81 was the natural receptor of lactate and was overexpressed in different GC cell lines and samples, which correlated with poor outcomes in GC patients. Lactate/GPR81 signaling could promote the infiltration of Tregs into TME by inducing the expression of chemokine CX3CL1. GPR81 deficiency could decrease the infiltration of Tregs into TME, thereby inhibiting GC progression by weakening the inhibition of CD8+T cell function in a humanized mouse model. In conclusion, targeting the lactate/GPR81 signaling may potentially serve as a critical process to overcome immune resistance in highly glycolytic GC.

G protein-coupled receptor-based thermosensation determines temperature acclimatization of Caenorhabditis elegans.

Animals must sense and acclimatize to environmental temperatures for survival, yet their thermosensing mechanisms other than transient receptor potential (TRP) channels remain poorly understood. We identify a trimeric G protein-coupled receptor (GPCR), SRH-40, which confers thermosensitivity in sensory neurons regulating temperature acclimatization in Caenorhabditis elegans. Systematic knockdown of 1000 GPCRs by RNAi reveals GPCRs involved in temperature acclimatization, among which srh-40 is highly expressed in the ADL sensory neuron, a temperature-responsive chemosensory neuron, where TRP channels act as accessorial thermoreceptors. In vivo Ca2+ imaging demonstrates that an srh-40 mutation reduced the temperature sensitivity of ADL, resulting in supranormal temperature acclimatization. Ectopically expressing SRH-40 in a non-warmth-sensing gustatory neuron confers temperature responses. Moreover, temperature-dependent SRH-40 activation is reconstituted in Drosophila S2R+ cells. Overall, SRH-40 may be involved in thermosensory signaling underlying temperature acclimatization. We propose a dual thermosensing machinery through a GPCR and TRP channels in a single sensory neuron.