Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cannabinoids, known for their psychoactive effects, also possess immunomodulatory properties. The recent isolation and cloning of the G-protein-coupled peripheral cannabinoid receptor (CB2), mainly expressed in immune tissues, have provided molecular tools to determine how cannabinoid compounds may mediate immunomodulation. We here investigated the CB2 signaling properties using stably transfected Chinese hamster ovary cells expressing human CB2. First, we showed that stimulation by a cannabinoid agonist activated mitogen-activated protein (MAP) kinase in time- and dose-dependent manners. The rank order of potency for MAP kinase activation of cannabinoid agonists correlated well with their binding capacities. Second, we demonstrated that, following MAP kinase activation, cannabinoids induced the expression of the growth-related gene Krox-24, also known as NGFI-A, zif/268, and egr-1. Pertussis toxin completely prevented both MAP kinase activation and Krox-24 induction, even more these responses appeared to be dependent of specific protein kinase C isoforms and independent of inhibition of adenylyl cyclase. A similar coupling of CB2 to a mitogenic pathway and to the regulation of Krox-24 expression was also observed in human promyelocytic cells HL60. Taken together, these findings provide evidence for a functional role of the CB2 receptor in gene induction mediated by the MAP kinase network.
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PMID:Signaling pathway associated with stimulation of CB2 peripheral cannabinoid receptor. Involvement of both mitogen-activated protein kinase and induction of Krox-24 expression. 864 16

Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors. Furthermore it shows no affinity for any of the more than 70 receptors, ion channels or enzymes investigated (IC50 > 10 microM). In vitro, SR 144528 antagonizes the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines permanently expressing the h CB2 receptor (EC50 = 10 nM) but not in cells expressing the h CB1 (no effect at 10 microM). Furthermore, SR 144528 is able to selectively block the mitogen-activated protein kinase activity induced by CP 55,940 in cell lines expressing h CB2 (IC50 = 39 nM) whereas in cells expressing h CB1 an IC50 value of more than 1 microM is found. In addition, SR 144528 is shown to antagonize the stimulating effects of CP 55,940 on human tonsillar B-cell activation evoked by cross-linking of surface Igs (IC50 = 20 nM). In vivo, after oral administration SR 144528 totally displaced the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes (ED50 = 0.35 mg/kg) with a long duration of action. In contrast, after the oral route it does not interact with the cannabinoid receptor expressed in the mouse brain (CB1). It is expected that SR 144528 will provide a powerful tool to investigate the in vivo functions of the cannabinoid system in the immune response.
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PMID:SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor. 945 10

The effect of anandamide, an endogenous ligand for central (CB1) and peripheral (CB2) cannabinoid receptors, was investigated on the growth of the murine IL-6-dependent lymphoid cell line B9 and the murine IL-3-dependent myeloblastic cell line FDC-P1. In conditions of low serum level, anandamide potentiated the growth of both cytokine-dependent cell lines. Comparison with other fatty acid cannabinoid ligands such as (R)-methanandamide, a ligand with improved selectivity for the CB1 receptor, or palmitylethanolamide, an endogenous ligand for the CB2 receptor, showed a very similar effect, suggesting that cell growth enhancement by anandamide or its analogs could be mediated through either receptor subtype. However, several lines of evidence indicated that this growth-promoting effect was cannabinoid receptor-independent. First, the potent synthetic cannabinoid agonist CP 55940, which displays high affinity for both receptors, was inactive in this model. Second, SR 141716A and SR 144528, which are potent and specific antagonists of CB1 and CB2 receptors respectively, were unable, alone or in combination, to block the anandamide-induced effect. Third, inactivation of both receptors by pretreatment of cells with pertussis toxin did not affect the potentiation of cell growth by anandamide. These data demonstrated that neither CB1 nor CB2 receptors were involved in the anandamide-induced effect. Moreover, using CB2-transfected Chinese hamster ovary cells, we demonstrated that after complete blockade of the receptors by the specific antagonist SR 144528, anandamide was still able to strongly stimulate a mitogen-activated protein (MAP) kinase activity, clearly indicating that the endogenous cannabinoid can transduce a mitogenic signal in the absence of available receptors. Finally, arachidonic acid, a structurally related compound and an important lipid messenger without known affinity for cannabinoid receptors, was shown to trigger MAP kinase activity and cell growth enhancement similar to those observed with anandamide. These findings provide clear evidence for a functional role of anandamide in activating a signal transduction pathway leading to cell activation and proliferation via a non-cannabinoid receptor-mediated process.
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PMID:The endogenous cannabinoid anandamide is a lipid messenger activating cell growth via a cannabinoid receptor-independent pathway in hematopoietic cell lines. 956 6

The peripheral cannabinoid receptor (CB2) is a G protein-coupled receptor that is both positively and negatively coupled to the mitogen-activated protein kinase (MAPK) and cAMP pathways, respectively, through a Bordetella pertussis toxin-sensitive G protein. CB2 receptor-transfected Chinese hamster ovary cells exhibit high constitutive activity blocked by the CB2-selective ligand, SR 144528, working as an inverse agonist. We showed here that in addition to the inhibition of autoactivated CB2 in this model, we found that SR 144528 inhibited the MAPK activation induced by Gi-dependent receptors such as receptor-tyrosine kinase (insulin, insulin-like growth factor 1) or G protein-coupled receptors (lysophosphatidic acid), but not by Gi-independent receptors such as the fibroblast growth factor receptor. We showed that this SR 144528 inhibitory effect on Gi-dependent receptors was mediated by a direct Gi protein inhibition through CB2 receptors. Indeed, we found that through binding to the CB2 receptors, SR 144528 blocked the direct activation of the Gi protein by mastoparan analog in Chinese hamster ovary CB2 cell membranes. Furthermore, we described that sustained treatment with SR 144528 induced an up-regulation of the cellular Gi protein level as shown in Western blotting as well as in confocal microscopic experiments. This up-regulation occurred with a concomitant loss of SR 144528 ability to inhibit the insulin or lysophosphatidic acid-induced MAPK activation. This inverse agonist-induced modulation of the Gi strongly suggests that the modulated protein is functionally associated with the complex SR 144528/CB2 receptors, and that the Gi level may account for the heterologous desensitization phenomena.
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PMID:Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: implication for intracellular signalization cross-regulation. 1005 30

Cannabinoids have a long history of consumption for recreational and medical reasons. The primary active constituent of the hemp plant Cannabis sativa is delta9-tetrahydrocannabinol (delta9-THC). In humans, psychoactive cannabinoids produce euphoria, enhancement of sensory perception, tachycardia, antinociception, difficulties in concentration and impairment of memory. The cognitive deficiencies seem to persist after withdrawal. The toxicity of marijuana has been underestimated for a long time, since recent findings revealed delta9-THC-induced cell death with shrinkage of neurons and DNA fragmentation in the hippocampus. The acute effects of cannabinoids as well as the development of tolerance are mediated by G protein-coupled cannabinoid receptors. The CB1 receptor and its splice variant CB1A, are found predominantly in the brain with highest densities in the hippocampus, cerebellum and striatum. The CB2 receptor is found predominantly in the spleen and in haemopoietic cells and has only 44% overall nucleotide sequence identity with the CB1 receptor. The existence of this receptor provided the molecular basis for the immunosuppressive actions of marijuana. The CB1 receptor mediates inhibition of adenylate cyclase, inhibition of N- and P/Q-type calcium channels, stimulation of potassium channels, and activation of mitogen-activated protein kinase. The CB2 receptor mediates inhibition of adenylate cyclase and activation of mitogen-activated protein kinase. The discovery of endogenous cannabinoid receptor ligands, anandamide (N-arachidonylethanolamine) and 2-arachidonylglycerol made the notion of a central cannabinoid neuromodulatory system plausible. Anandamide is released from neurons upon depolarization through a mechanism that requires calcium-dependent cleavage from a phospholipid precursor in neuronal membranes. The release of anandamide is followed by rapid uptake into the plasma and hydrolysis by fatty-acid amidohydrolase. The psychoactive cannabinoids increase the activity of dopaminergic neurons in the ventral tegmental area-mesolimbic pathway. Since these dopaminergic circuits are known to play a pivotal role in mediating the reinforcing (rewarding) effects of the most drugs of abuse, the enhanced dopaminergic drive elicited by the cannabinoids is thought to underlie the reinforcing and abuse properties of marijuana. Thus, cannabinoids share a final common neuronal action with other major drugs of abuse such as morphine, ethanol and nicotine in producing facilitation of the mesolimbic dopamine system.
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PMID:The effects of cannabinoids on the brain. 1036 32

We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-activated protein kinase activity coupled to the spontaneous autoactivated peripheral cannabinoid receptor (CB2) in the Chinese hamster ovary cell line stably transfected with human CB2. In the present report, we studied the effect of SR 144528 on CB2 phosphorylation. The CB2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB2 which can discriminate between phosphorylated and non-phosphorylated CB2 isoforms at serine 352. We first showed that CB2 is constitutively active, phosphorylated, and internalized at the basal level. By blocking autoactivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB2 receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated and maintained in this phosphorylated state for more than 8 h after agonist treatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB2 phosphorylation was reversed by SR 144528, paradoxically leading to a non-phosphorylated CB2 which could then be fully activated by CP-55,940. The process of CP-55,940-induced receptor phosphorylation followed by SR 144528-induced receptor dephosphorylation kept recurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finally, we showed that autophosphorylation and CP-55, 940-induced serine 352 CB2 phosphorylation involve an acidotropic GRK kinase, which does not use Gibetagamma. In contrast, SR 144528-induced CB2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase.
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PMID:Regulation of peripheral cannabinoid receptor CB2 phosphorylation by the inverse agonist SR 144528. Implications for receptor biological responses. 1040 Jun 64

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.
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PMID:Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528. 1046 63

The function of the peripheral cannabinoid receptor (CB2), which is mainly expressed on hematopoietic cells, remains an enigma. In an attempt to decipher its role, we used Affymetrix DNA chips to investigate the gene expression profile of the promyelocytic cells HL-60 transfected with the CB2 receptor and activated with the cannabinoid agonist CP 55,940. Agonist exposure of these cells led to an activation of a mitogen-activated protein kinase cascade and a receptor desensitization, indicating a functional coupling of the transfected receptors. At the genomic level, activation of the CB2 receptors induced an up-regulation of nine genes involved in cytokine synthesis, regulation of transcription, and cell differentiation. A majority of them are under the control of the transcription factor NF-kappaB, whose nuclear translocation was demonstrated. Many features of the transcriptional events, reported here for the first time, appeared to be related to an activation of a cell differentiation program, suggesting that CB2 receptors could play a role in the initialization of cell maturation. Moreover, we showed that CB2-activated wild-type HL-60 cells developed properties usually found in host defense effector cells such as an enhanced release of chemotactic cytokines and an increased motility, characteristic of more mature cells of the granulocytic-monocytic lineage.
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PMID:Genomic and functional changes induced by the activation of the peripheral cannabinoid receptor CB2 in the promyelocytic cells HL-60. Possible involvement of the CB2 receptor in cell differentiation. 1082 43

1. The effects of cannabinoid (CB) receptor stimulation on membrane currents in single cells from the Syrian hamster vas deferens cell line DDT1MF-2 were investigated using the whole cell patch clamp technique. 2. The CB receptor agonist CP55,940 evoked a concentration-dependent transient outward current. The selective CB1 receptor ligand SR141716 (1 microM), but not the selective CB2 receptor ligand SR144528 (1 microM), inhibited the outward current. Pertussis toxin (100 ng ml-1 for 20 h) completely abolished the outward current. 3. Western blotting with an antibody against the rat (r)CB1 receptor showed a band characteristic for the CB1 receptor around 63 kDa in DDT1MF-2 cells. 4. The reversal potential for the outward current measured using a voltage ramp protocol was -84 +/- 5 mV. The current was inhibited by the Ca2+-dependent K+ channel blockers iberiotoxin (10 nM) and charybdotoxin (10 nM). 5. Removal of Ca2+ from the bathing solution, or the addition of 0.1 mM Cd2+ completely abolished the outward current evoked by 10 microM CP55,940. 6. The sarcoplasmic Ca2+ pump inhibitor thapsigargin reduced the outward current evoked by 10 microM CP55,940 in a concentration-dependent manner. 7. The mitogen-activating protein kinase (MAP kinase) inhibitor PD98059, but not the phospholipase C inhibitor U73122, inhibited the outward current evoked by 10 microM CP55,940. 8. The adenylyl cyclase inhibitor SQ22,536 (100 microM) and 8-Br-cyclic AMP (10 microM) significantly reduced the outward current evoked by 10 microM CP55,940. 9. Our data suggest that CB1 receptor stimulation in DDT1MF-2 cells leads to activation of a large conductance Ca2+-dependent K+ channel through a Gi/Go protein-mediated rise in [Ca2+]i, for which both inhibition of adenylyl cyclase and activation of MAP kinase are required. In addition, the cannabinoid-induced increase in [Ca2+]i is likely to arise from capacitive Ca2+ entry.
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PMID:Signal transduction of cannabinoid CB1 receptors in a smooth muscle cell line. 1117 94

2-Arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand, was shown to induce rapid phosphorylation of p42/44 mitogen-activated protein kinase (MAP kinase) in HL-60 cells. We confirmed that the enzyme activity of p42/44 MAP kinase in HL-60 cells was augmented markedly when the cells were stimulated with 2-AG. The addition of SR144528, a cannabinoid CB2 receptor-specific antagonist, to the cells prior to the addition of 2-AG abolished the response induced by 2-AG, indicating that the CB2 receptor is involved in the response. G protein G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells nullified the response induced by 2-AG. CP55940 and anandamide also induced the activation of p42/44 MAP kinase, although the activation by anandamide was less pronounced than that by 2-AG or CP55940. These results suggest that 2-AG may play an important physiological role in this type of cell through the activation of the p42/44 MAP kinase cascade.
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PMID:Activation by 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, of p42/44 mitogen-activated protein kinase in HL-60 cells. 1132 86


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