Tag Archives: G-Protein coupled receptors

Quantitation of Plasma Membrane (G Protein-Coupled) Receptor Trafficking in Cultured Cells.

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“Measuring the functional behavior of G protein-coupled receptors (GPCRs) has been a major focus of academic and pharmaceutical research for many decades. These efforts have led to the development of many assays to measure the downstream effects of ligand binding on receptor activity. In this chapter, we describe an internalization/recycling assay that can be used to track changes in receptor number at the plasma membrane. Used in concert with other assays, this antibody-based technique can provide important information on GPCR activation by receptor-specific ligands.”

http://www.ncbi.nlm.nih.gov/pubmed/27245911

Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors.

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“Cannabinoid CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) that recruit β-arrestins upon activation by (partial) agonists. β-Arrestin recruitment is induced by phosphorylation of their C-terminal tails, and is associated with the termination of GPCR signaling; yet, it may also activate cellular signaling pathways independent of G-proteins. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1 and CB2 receptors, by using the PathHunter(®) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter(®) assay makes use of β-galactosidase complementation, and has a chemiluminescent readout. We used this assay to characterize a set of reference ligands (both agonists and antagonists) on human CB1 and CB2 receptors.”

http://www.ncbi.nlm.nih.gov/pubmed/27245896

The Cyclic AMP Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

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“The cyclic AMP assay is a functional assay that is commonly used to determine the pharmacological behavior (agonists, antagonists, inverse agonists) of G-protein-coupled receptor (GPCR) ligands. Here, we describe the cyclic AMP assay that is carried out with commercially available non-radioligand ready-to-use kits and Chinese hamster ovarian (CHO) cells stably transfected with the human cannabinoid CB2 receptor.”

http://www.ncbi.nlm.nih.gov/pubmed/27245894

A Functional Assay for GPR55: Envision Protocol.

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“AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.”

http://www.ncbi.nlm.nih.gov/pubmed/27245893

Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

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“Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse.”

http://www.ncbi.nlm.nih.gov/pubmed/27186994

Functional selectivity of CB2 cannabinoid receptor ligands at a canonical and non-canonical pathway.

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“The CB2 cannabinoid receptor remains a tantalizing, but unrealized therapeutic target. CB2 receptor ligands belong to varied structural classes and display extreme functional selectivity. Here we have screened diverse CB2 receptor ligands at canonical (inhibition of adenylyl cyclase) and non-canonical (arrestin recruitment) pathways. The non-classical cannabinoid, CP55940 was the most potent agonist for both pathways, while the classical cannabinoid ligand JWH133 was the most efficacious agonist amongst all the ligands profiled in cyclase assays. In the cyclase assay, other classical cannabinoids showed little (Δ9THC, KM233) to no efficacy (L759633 and L759656). Most aminoalkylindoles including WIN55212-2 were moderate efficacy agonists. The cannabilactone AM1710 was equi-efficacious to CP55940 to inhibit adenylyl cyclase, albeit with lower potency. In the arrestin recruitment assays, all classical cannabinoid ligands failed to recruit arrestins, indicating a bias towards G protein coupling for this class of compound. All aminoalkylindoles tested, except for WIN55212-2 and UR144, failed to recruit arrestin. WIN55212-2 was a low efficacy agonist for arrestin recruitment, while UR144 was arrestin biased with no significant inhibition of cyclase. Endocannabinoids were G protein biased with no arrestin recruitment. The diarylpyrazole antagonist, SR144528 was an inverse agonist in cyclase and arrestin recruitment assays while the aminoalkylindole AM630 and carboxamide JTE907 were inverse agonists in cyclase but low efficacy agonists in arrestin recruitment assays. Thus CB2 receptor ligands display strong and varied functional selectivity at both pathways. Therefore extreme care must be exercised when using these compounds to infer the role of CB2 receptors in vivo.”

http://www.ncbi.nlm.nih.gov/pubmed/27194477

Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent

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“Breast cancer is the leading cause of cancer-related deaths among women aged 34–50 worldwide, and is the most commonly diagnosed metastasizing tumor in women of all ages. Despite advances in understanding breast cancer as a disease, there remains a critical need for novel disease-modifying therapeutics.

Nonspecific cannabinoids, cannabinoid receptor 2 (CB2)-selective, as well as cannabinoid receptor 1 (CB1)-selective compounds have yielded similar antitumor results in several tumor models. The lack of neuronal expression of CB2 receptors precludes CB2 selective compounds from inducing the psychotropic effects that typically accompany CB1 activation.

 Our group and others have shown that CB2 agonists displaying selectivity for the CB2 receptor can decrease tumor cell viability and significantly attenuate cancer-induced bone pain without displaying psychoactive or addictive properties.

…antitumor effects of cannabinoids have been demonstrated in a variety of tumor models…

The antiproliferative effects of a CB2 agonist along with our previous work demonstrating significant efficacy in inhibiting bone cancer pain and slowing bone loss in a murine model of advanced breast cancer strongly suggest that CB2 agonists should be investigated in humans as adjunct therapy for advanced stages of breast cancer.

 Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems.
The results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor.
Several groups have shown that both nonselective cannabinoid and CB2-specific compounds decrease breast cancer viability in vitro and in vivo: Δ9-tetrahydrocannabinol and CB2-selective agonist, JWH-133, have been demonstrated to exert considerable antitumoral effects…”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4847606/

The role of cannabinoid receptors and the endocannabinoid system in mantle cell lymphoma and other non-Hodgkin lymphomas.

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“The initiating oncogenic event in mantle cell lymphoma (MCL) is the translocation of cyclin D1, t(11;14)(q13;q32). However, other genetic aberrations are necessary for an overt lymphoma to arise. Like other B cell lymphomas, MCL at some points during the oncogenesis is dependent on interactions with other cells and factors in the microenvironment.

The G protein coupled receptors cannabinoid receptors 1 and 2 (CB1 and CB2) are expressed at low levels on non-malignant lymphocytes and at higher levels in MCL and other lymphoma subtypes.

In this review we give an overview of what is known on the role of the cannabinoid receptors and their ligands in lymphoma as compared to non-malignant T and B lymphocytes.

In MCL cannabinoids mainly reduce cell proliferation and induce cell death.

Importantly, our recent findings demonstrate that cannabinoids may induce either apoptosis or another type of programmed cell death, cytoplasmic vacuolation/paraptosis in MCL.”

http://www.ncbi.nlm.nih.gov/pubmed/22024769

Stimulated CB1 Cannabinoid Receptor Inducing Ischemic Tolerance and Protecting Neuron from Cerebral Ischemia.

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“Anandamide system is mainly made up of cannabinoid receptors, their endogenous ligands and some related enzymes. Activation of the system mediates various molecular events, thereafter leading to vasodilation, bradycardia and anti-inflammation.

The stimulated cannabinoid receptors may take part in protection of endothelial cells from injury and therefore can be potential targets in therapy for some diseases, especially cardio or cerebral vascular disturbances.

Cerebral ischemia is a deadly disease that modern people have to face and will probably face for a long period of time. Ischemic tolerance has the protective effect of brain as an endogenous event in cerebral ischemia, in which variety of inducers such as transient cerebral ischemia, hypoxia, hypothermia and drug agents are involved.

Most of cannabinoid 1 receptors (CB1Rs), a member in G protein-coupled receptor family, exist in central nervous systems.

Mechanism of neuroprotection mediated by the receptor is considered through facilitating neurotransmitter release and regulating other molecular events. In this review, advance of the neuroprotection against cerebral ischemia and the mechanism of the action are overviewed.”

http://www.ncbi.nlm.nih.gov/pubmed/27142423

“Cerebral ischemia or brain ischemia, is a condition that occurs when there isn’t enough blood flow to the brain to meet metabolic demand. This leads to limited oxygen supply or cerebral hypoxia and leads to the death of brain tissue, cerebral infarction, or ischemic stroke. It is a sub-type of stroke along with subarachnoid hemorrhage and intracerebral hemorrhage. There are two kinds of ischemia: focal ischemia: confined to a specific region of the brain; global ischemia: encompasses wide areas of brain tissue.”  http://www.columbianeurosurgery.org/conditions/cerebral-ischemia/

Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent

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“Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems.

The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands.

The results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor.”

https://www.dovepress.com/modulation-of-breast-cancer-cell-viability-by-a-cannabinoid-receptor-2-peer-reviewed-article-BCTT