Characterisation of inverse agonism of the orphan-G protein-coupled receptor GPR52 by cannabinoid ligands Cannabidiol and O-1918.
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ABSTRACT:
SUBMITTER: Stott LA
PROVIDER: S-EPMC8219759 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:G protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors and transmit critical signals from the extracellular to the intracellular spaces. Transcriptomic data of resected breast tumors shows that low mRNA expression of the orphan GPCR GPR52 correlates with reduced overall survival in breast cancer patients, leading to the hypothesis that loss of GPR52 supports breast cancer progression. CRISPR-Cas9 was used to knockout GPR52 in human triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line, MCF10A. Loss of GPR52 was found to be associated with increased cell-cell interaction in 2D cultures, altered 3D spheroid morphology, and increased propensity to organize and invade collectively in Matrigel. Furthermore, GPR52 loss was associated with features of EMT in MDA-MB-468 cells. To determine the in vivo impact of GPR52 loss, MDA-MB-468 cells were injected into zebrafish and loss of GPR52 was associated with a greater total cancer area compared to control cells. RNA-sequencing and proteomic analyses of GPR52-null breast cancer cells reveal an increased cAMP signaling signature. Consistently, we found that treatment of wild-type (WT) cells with forskolin, which stimulates production of cAMP, induces some phenotypic changes associated with GPR52 loss, and inhibition of cAMP production rescued some of the GPR52 KO phenotypes. Overall, our results reveal GPR52 loss as a potential mechanism by which breast cancer progression may occur and support the investigation of GPR52 agonism as a therapeutic option in breast cancer.
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Project description:The cannabinoid receptor 1 (CB(1)) and CB(2) cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a beta-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing beta-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CbetaII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of beta-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CbetaII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy.
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Project description:Pharmacological blockade of the cannabinoid type 1 receptor, a G protein-coupled receptor expressed in the central nervous system and various peripheral tissues, reverses diet-induced obesity and dyslipidemia through the reduction of food intake and altered nutrient partitioning. This strategy is being explored for a number of therapeutic applications; however, its potency for the treatment of atherosclerotic cardiovascular disease via improvements in lipid metabolism remains unclear. Therefore, here, we aimed to investigate whether inhibition of the endocannabinoid system can attenuate atherosclerosis development through improvement of dyslipidemia. Lean, dyslipidemic female APOE∗3-Leiden.CETP transgenic mice were fed a Western-type diet supplemented with or without the cannabinoid type 1 receptor inverse agonist rimonabant (20 mg·kg body weight-1 day-1) for up to 20 weeks. Plasma lipids and bile acids were determined, and atherosclerotic lesions were scored in the aortic valve region. Rimonabant lowered plasma levels of triglyceride (TG) (-56%) and non-HDL-C (-19%) and increased HDL-C (+57%). These effects were explained by decreased VLDL-TG production (-52%) and accelerated VLDL-TG turnover accompanied by pronounced browning of white adipose tissue. In addition, rimonabant attenuated reverse cholesterol transport (-30%), increased plasma bile acid levels (+160%), and increased hepatic cholesterol accumulation (+88%). Importantly, rimonabant markedly lowered atherosclerotic lesion size (-64%), which coincided with decreased lesion severity (28% vs. 56% severe lesions) and which strongly correlated with non-HDL-C exposure (R2 = 0.60). Taken together, inhibition of the endocannabinoid system potently reverses dyslipidemia and prevents atherogenesis, even in the absence of obesity.
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Project description:Prolonged status epilepticus (SE) in humans causes high mortality and brain inflammation-associated neuronal injury and morbidity in survivors. Currently, the only effective treatment is to terminate the seizures swiftly to prevent brain damage. However, reliance on acute therapies alone would be imprudent due to the required short response time. Follow-on therapies that can be delivered well after the SE onset are in an urgent need. Cannabinoid receptor type 2 (CB2), a G protein-coupled receptor that can be expressed by activated brain microglia, has emerged as an appealing anti-inflammatory target for brain conditions. In the current study, we reported that the CB2 inverse agonism by our current lead compound SMM-189 largely prevented the rat primary microglia-mediated inflammation and showed moderate neuroprotection against N-methyl-D-aspartic acid (NMDA) receptor-mediated excitotoxicity in rat primary hippocampal cultures containing both neurons and glia. Using a classical mouse model of epilepsy, in which SE was induced by systemic administration of kainate (30 mg/kg, i.p.) and proceeded for 1 h, we demonstrated that SE downregulated the CB1 but slightly upregulated CB2 receptor in the hippocampus. Transient treatment with SMM-189 (6 mg/kg, i.p., b.i.d.) after the SE was interrupted by diazepam (10 mg/kg, i.p.) prevented the seizure-induced cytokine surge in the brain, neuronal death, and behavioral impairments 24 h after SE. Our results suggest that CB2 inverse agonism might provide an adjunctive anti-inflammatory therapy that can be delivered hours after SE onset, together with NMDA receptor blockers and first-line anti-convulsants, to reduce brain injury and functional deficits following prolonged seizures.
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Project description:Background and purposeCannabinoid CB(1) receptor antagonists/inverse agonists, potentiate toxin-induced nausea and vomiting in animal models. Here, we sought to determine if this potentiated nausea was mediated by inverse agonism or neutral antagonism of the CB(1) receptor, and if the potentiated nausea would be produced by intracerebroventricular (icv) administration of an inverse agonist.Experimental approachThe conditioned gaping model of nausea in rats was used to compare the CB(1) receptor antagonist/inverse agonist, AM251, and the CB(1) receptor neutral antagonists, AM6527 (centrally and peripherally active) and AM6545 (peripherally active), in potentiating conditioned gaping produced by lithium chloride (LiCl) solution. The effect of icv (lateral ventricle and 4th ventricle) administration of AM251 on LiCl-induced gaping in this model was also evaluated.Key resultsAt a dose that did not produce conditioned gaping on its own, systemically administered AM251 (1.25 mg.kg(-1)) potentiated LiCl-induced conditioned gaping and reduced sucrose palatability; however, even doses as high as 8 mg.kg(-1) of AM6545 and AM6527 neither potentiated LiCl-induced conditioned gaping nor reduced sucrose palatability. Infusions of AM251 into the lateral ventricles (1.25, 12.5 and 125 microg) or the 4th ventricle (2.5, 12.5 and 125 microg) did not potentiate LiCl-induced conditioned gaping reactions, but all doses attenuated saccharin palatability during the subsequent test.Conclusions and implicationsInverse agonism, but not neutral antagonism, of CB(1) receptors potentiated toxin-induced nausea. This effect may be peripherally mediated or may be mediated centrally by action on CB(1) receptors, located distal to the cerebral ventricles.
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Project description:O-1602 and O-1918 are atypical cannabinoid ligands for GPR55 and GPR18, which may be novel pharmaceuticals for the treatment of obesity by targeting energy homeostasis regulation in skeletal muscle. This study aimed to determine the effect of O-1602 or O-1918 on markers of oxidative capacity and fatty acid metabolism in the skeletal muscle. Diet-induced obese (DIO) male Sprague Dawley rats were administered a daily intraperitoneal injection of O-1602, O-1918 or vehicle for 6 weeks. C2C12 myotubes were treated with O-1602 or O-1918 and human primary myotubes were treated with O-1918. GPR18 mRNA was expressed in the skeletal muscle of DIO rats and was up-regulated in red gastrocnemius when compared with white gastrocnemius. O-1602 had no effect on mRNA expression on selected markers for oxidative capacity, fatty acid metabolism or adiponectin signalling in gastrocnemius from DIO rats or in C2C12 myotubes, while APPL2 mRNA was up-regulated in white gastrocnemius in DIO rats treated with O-1918. In C2C12 myotubes treated with O-1918, PGC1?, NFATc1 and PDK4 mRNA were up-regulated. There were no effects of O-1918 on mRNA expression in human primary myotubes derived from obese and obese T2DM individuals. In conclusion, O-1602 does not alter mRNA expression of key pathways important for skeletal muscle energy homeostasis in obesity. In contrast, O-1918 appears to alter markers of oxidative capacity and fatty acid metabolism in C2C12 myotubes only. GPR18 is expressed in DIO rat skeletal muscle and future work could focus on selectively modulating GPR18 in a tissue-specific manner, which may be beneficial for obesity-targeted therapies.
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