Cannabidiol skews biased agonism at cannabinoid CB1 and CB2 receptors with smaller effect in CB1-CB2 heteroreceptor complexes

Navarro, Gemma; Reyes‐Resina, Irene; Rivas‐Santisteban, Rafael; Medina, Verónica Sánchez de; Morales, Paula; Casano, S.; Ferreiro‐Vera, Carlos; Lillo, Alejandro; Aguinaga, David; Jagerovic, Nadine; Nadal, Xavier; Franco, Rafael

doi:10.1016/j.bcp.2018.08.046

Cited by 76 publications

(57 citation statements)

References 47 publications

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“…Anti‐MYC antibodies were here used as positive control (Figure B, right panel). The number of PLA signals was comparable to those reported for other GPCR heterodimeric complexes . The lower staining observed for the MT 1 /5‐HT 2C dimer compared to MT 2 /5‐HT 2C might be explained by the higher BRET 50 value (lower propensity to form dimers) of the former, as previously reported .…”

Section: Resultssupporting

confidence: 87%

“…The number of PLA signals was comparable to those reported for other GPCR heterodimeric complexes. 38,39 The lower staining observed for the MT 1 /5-HT 2C dimer compared to MT 2 /5-HT 2C might be explained by the higher BRET 50 value (lower propensity to form dimers) of the former, as previously reported. 37 Collectively, mAB-A06 and mAB-A84 are able to detect heterodimeric complexes involving mMT 1 or mMT 2 , respectively.…”

Section: Resultssupporting

confidence: 61%

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Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Cecon

Ivanova

Luka

et al. 2019

Journal of Pineal Research

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Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT1 and MT2, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT1 and MT2. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT1 and MT2 by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT1 or MT2. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT2 expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.

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Section: Resultssupporting

confidence: 87%

Section: Resultssupporting

confidence: 61%

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Cecon

Ivanova

Luka

et al. 2019

Journal of Pineal Research

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“…As well, adenylyl cyclase isoforms can differ in their Gα sensitivities and expression levels can vary between immune cell types 55 . Furthermore, G protein coupling "switches" have been reported for receptors in heterodimers 57,58 . The particular profile and stoichiometries of interaction partners can give rise to differential signaling "units" between cell types reviewed in 59 .…”

Section: Discussionmentioning

confidence: 99%

Cannabinoid Receptor 2 (CB₂) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes

Saroz

Kho

Glass

et al. 2019

Preprint

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Cannabinoid receptor 2 (CB2) is a promising therapeutic target for immunological modulation.There is, however, a deficit of knowledge regarding CB2 signaling and function in human primary immunocompetent cells. We applied an experimental paradigm which closely models the in situ state of human primary leukocytes (PBMC; peripheral blood mononuclear cells) to characterize activation of a number of signaling pathways in response to a CB2-selective ligand (HU308). We observed a "lag" phase of unchanged cAMP concentration prior to development of classically-expected Gαi-mediated inhibition of cAMP synthesis. Application of G protein inhibitors revealed that this apparent lag was a result of counteraction of Gαi effects by concurrent Gαs activation. Monitoring downstream signaling events, activation of p38 was mediated by Gαi whereas ERK1/2 and Akt phosphorylation were mediated by Gαi-coupled βγ.Activation of CREB integrated multiple components; Gαs and βγ mediated ~85% of the response, while ~15% was attributed to Gαi. Responses to HU308 had an important functional outcome -secretion of interleukins 6 (IL-6) and 10 (IL-10). IL-2, IL-4, IL-12, IL-13, IL-17A, MIP-1α, and TNF-α were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB2. Additionally, signaling and functional effects were completely blocked by a CB2selective inverse agonist, giving additional evidence for CB2 involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB2 ligands as immunomodulatory therapeutics.Keywords: cannabinoid receptor 2 (CB2), G protein-coupled receptor (GPCR), signaling, leukocytes, interleukin 6, interleukin 10 Significance statementCannabinoid receptor 2 (CB2) is a G protein-coupled receptor which plays a complex role in immunomodulation and is a promising target in a range of disorders with immune system involvement. However, to date the majority of the studies in this field have been performed on cell lines, rodent models, or stimulated primary cells. Here we provide a detailed account of CB2-mediated signaling in primary human immune cells under conditions which closely mimic their in vivo state. We reveal a complex signaling system involving an unprecedented CB2 signaling pathway and leading to immunomodulatory functional outcomes. This work provides not only a critical foundation impacting CB2-targeted drug discovery, but reveals important wider considerations for GPCR signaling studies and model validity.

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“…Arrestin recruitment was determined as previously described (Navarro et al, , 2018b(Navarro et al, , 2018c Wildbad, GE) that allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify protein-Rluc expression, luminescence readings were also performed 10 min after adding 5 μM coelenterazine h.…”

Section: ß-Arrestin 2 Recruitment Assaysmentioning

confidence: 99%

“…More recently, we have reported that microglia from a transgenic AD model has a similar phenotype as that found in primary microglial cultures (from wild type mice) activated by a combination of lipopolysaccharide and interferon γ (IFN‐γ). Interestingly, one of the common features was an increased expression of the CB 2 R (Navarro et al, a). It should be noted that transgenic AD models do not display significant neuronal loss and that the cognitive symptoms appear late in the life of the animal.…”

Section: Introductionmentioning

confidence: 99%

Potentiation of cannabinoid signaling in microglia by adenosine A_2A receptor antagonists

Franco

Reyes‐Resina

Aguinaga

et al. 2019

Glia

Self Cite

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Neuroprotective M2‐skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein‐coupled receptors (GPCRs) are potential targets to achieve such microglial polarization. A common feature of adenosine A2A (A2AR) and cannabinoid CB2 (CB2R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB2R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A2AR antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A2A‐CB2 receptor heteromer (A2A‐CB2Het). Particularly relevant is the upregulation of A2A‐CB2Het expression in samples from the APPSw,Ind AD transgenic mice model. The most relevant finding, confirmed in both heterologous cells and in primary cultures of microglia, was that blockade of A2A receptors results in increased CB2R‐mediated signaling. This heteromer‐specific feature suggests that A2AR antagonists would potentiate, via microglia, the neuroprotective action of endocannabinoids with implications for AD therapy.

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Cannabidiol skews biased agonism at cannabinoid CB1 and CB2 receptors with smaller effect in CB1-CB2 heteroreceptor complexes

Cited by 76 publications

References 47 publications

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Cannabinoid Receptor 2 (CB₂) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes

Potentiation of cannabinoid signaling in microglia by adenosine A_2A receptor antagonists

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Cannabidiol skews biased agonism at cannabinoid CB1 and CB2 receptors with smaller effect in CB1-CB2 heteroreceptor complexes

Cited by 76 publications

References 47 publications

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Cannabinoid Receptor 2 (CB2) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes

Potentiation of cannabinoid signaling in microglia by adenosine A2A receptor antagonists

Contact Info

Product

Resources

About

Cannabinoid Receptor 2 (CB₂) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes

Potentiation of cannabinoid signaling in microglia by adenosine A_2A receptor antagonists