Pharmacological comparison of a recombinant CB1 cannabinoid receptor with its G(alpha 16) fusion product

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB(1) agonist-mediated signal transduction via an engineered G(alpha 16) system is different than that of the G(i/o) coupling normally preferred by the CB(1) receptor, we transfected the human recombinant CB(1) receptor (hCB(1)) or a fusion protein comprising the hCB(1) receptor and G(alpha 16) (hCB(1)-G(alpha 16)) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB(1)-G(alpha 16) fusion protein was found to be similar to that for hCB(1): HU 210 > CP 55,940 > or = SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [(35)S]GTP gamma S binding or inhibited forskolin-stimulated cAMP, presumably by coupling to G(i/o), in cells expressing hCB(1) but not hCB(1)-G(alpha 16). However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCB(1)-G(alpha 16) but not hCB(1). These data demonstrate that ligand affinities for the hCB(1) receptor are not affected by fusion to the G(alpha 16) subunit. Furthermore, there is essentially no difference in the function of the hCB(1) receptor when coupled to G(i/o) or G (alpha 16).     

A neutral CB1 receptor antagonist reduces weight gain in rat

Cannabinoid (CB)1 receptor inverse agonists inhibit food intake in animals and humans but also potentiate emesis. It is not clear whether these effects result from inverse agonist properties or from the blockade of endogenous cannabinoid signaling. Here, we examine the effect of a neutral CB1 antagonist, AM4113, on food intake, weight gain, and emesis. Neutral antagonist and binding properties were confirmed in HEK-293 cells transfected with human CB1 or CB2 receptors. AM4113 had no effect on forskolin-stimulated cAMP production at concentrations up to 630 nM. The Ki value of AM4113 (0.80 +/- 0.44 nM) in competitive binding assays with the CB1/2 agonist [3H]CP55,940 was 100-fold more selective for CB1 over CB2 receptors. We determined that AM4113 antagonized CB1 receptors in brain by blocking hypothermia induced by CP55,940. AM4113 (0-20 mg/kg) significantly reduced food intake and weight gain in rat. Compared with AM251, higher doses of AM4113 were needed to produce similar effects on food intake and body weight. Unlike AM251 (5 mg/kg), a highly anorectic dose of AM4113 (10 mg/kg) did not significantly potentiate vomiting induced by the emetic morphine-6-glucoronide. We show that a centrally active neutral CB1 receptor antagonist shares the appetite suppressant and weight loss effects of inverse agonists. If these compounds display similar properties in humans, they could be developed into a new class of antiobesity agents.     

UHRF1 depletion causes a G2/M arrest, activation of DNA damage response and apoptosis

The ECS (endocannabinoid system) plays an important role in the onset of obesity and metabolic disorders, implicating central and peripheral mechanisms predominantly via CB1 (cannabinoid type 1) receptors. CB1 receptor antagonist/inverse agonist treatment improves cardiometabolic risk factors and insulin resistance. However, the relative contribution of peripheral organs to the net beneficial metabolic effects remains unclear. In the present study, we have identified the presence of the endocannabinoid signalling machinery in skeletal muscle and also investigated the impact of an HFD (high-fat diet) on lipid-metabolism-related genes and endocannabinoid-related proteins. Finally, we tested whether administration of the CB1 inverse agonist AM251 restored the alterations induced by the HFD. Rats were fed on either an STD (standard/low-fat diet) or an HFD for 10 weeks and then treated with AM251 (3 mg/kg of body weight per day) for 14 days. The accumulated caloric intake was progressively higher in rats fed on the HFD than the STD, resulting in a divergence in body weight gain. AM251 treatment reduced accumulated food/caloric intake and body weight gain, being more marked in rats fed on the HFD. CB2 (cannabinoid type 2) receptor and PPARα (peroxisome-proliferator-activated receptor α) gene expression was decreased in HFD-fed rats, whereas MAGL (monoglyceride lipase) gene expression was up-regulated. These data suggest an altered endocannabinoid signalling as a result of the HFD. AM251 treatment reduced CB2 receptor, PPARγ and AdipoR1 (adiponectin receptor 1) gene expression in STD-fed rats, but only partially normalized the CB2 receptor in HFD-fed rats. Protein levels corroborated gene expression results, but also showed a decrease in DAGL (diacylglycerol) β and DAGLα after AM251 treatment in STD- and HFD-fed rats respectively. In conclusion, the results of the present study indicate a diet-sensitive ECS in skeletal muscle, suggesting that blockade of CB1 receptors could work towards restoration of the metabolic adaption imposed by diet.     

Role of intracellular loops of cannabinoid CB(1) receptor in functional interaction with G(alpha16)

The cannabinoid CB(1) but not the CB(2) receptor was demonstrated to couple via G(alpha16) to activate phospholipase C after co-expression in COS7 cells. Chimeric CB(1)/CB(2) receptors were used as a model to study receptor-G(alpha16) interaction. Sequences of the second and third intracellular loops and the carboxy-terminus were substituted from the CB(1) into the CB(2) receptor. Only the triple mutant with all three regions replaced activated phospholipase C to a similar extent as the CB(1) receptor, suggesting that all three intracellular regions are required for interacting with G(alpha16). Several sub-domains within the third intracellular loop were identified for receptor-G(alpha16) interaction.     

Combined stimulation of glucagon-like peptide-1 receptor and inhibition of cannabinoid CB1 receptor act synergistically to reduce food intake and body weight in the rat

Pharmacological activation of the glucagon-like peptide-1 (GLP-1) receptor and inhibition of the cannabinoid CB1 receptor were found to reduce food intake and body weight in humans and animals. Since earlier studies revealed that endocannabinoids may interact with other neurotransmitters to affect feeding behavior, we have examined whether a stable GLP-1 agonist, exendin-4 and a CB1 receptor antagonist, AM 251, may reciprocally enhance their inhibitory effects on food consumption in the rat. Additionally, we have tested whether the blockade of the GLP-1 receptor by exendin (9-39) modifies AM 251-dependent effects on energy balance. In a dose-response study, male Wistar rats were injected intraperitoneally with either 1.5-6.0 μg/kg exendin-4, 0.5-2 mg/kg AM 251, 80-320 μg/kg exendin (9-39) or their vehicle and the daily food and water intake as well as body weight changes were monitored two days before and two days after the injection. Exendin-4 at a dose of 3.0 and 6.0 μg/kg and AM 251 at a dose 2 mg/kg decreased significantly 24-hour food intake and body weight. Therefore, in the next study, the effects of lower doses of exendin-4 (1.5 μg/kg) and AM 251 (1.0 mg/kg) administered alone or together on food consumption were compared. As opposed to being injected alone, the co-administration of the two resulted in a marked decrease in both daily food intake and body weight. Exendin (9-39) did not modify the suppressory effect of the highest AM 251 dose on food consumption. Apparently, the effect of AM 251 on the appetite is not mediated by GLP-1. The concomitant stimulation of GLP-1 receptor and blockade of CB1 receptor, however, may act synergistically to inhibit appetite in the rat.     

Determination of endocannabinoid receptor antagonist SR141716 (rimonabant) in plasma by liquid chromatograph tandem mass spectrometry

SR141716 (rimonabant) is an endocannabinoid receptor antagonist. Endocannabinoids are a class of chemicals that affect neurotransmission via G-protein coupled CB1 (brain) and CB2 (peripheral tissue) receptors. Numerous animal studies have shown that SR141716 binds with the CB1 receptor in the brain, resulting in several biological consequences including reduced alcohol intake and reward as well as reduced food consumption. In this work, an analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for the quantitative measurement of SR141716 in both human and rat plasma to support the investigation of this compound. A suitable internal standard (AM251) has been chosen and the experimental conditions have been optimized for the separation and detection of singly charged positive ions of SR141716 and the internal standard. A protein precipitation protocol has been developed for extraction of SR141716 and the internal standard from plasma samples. Quantitation was achieved using multiple-reaction-monitoring (MRM) mode for SR141716 (m/z 463-->m/z 363) and the internal standard (m/z 555-->m/z 455) and calibration curve over the concentration range of 5.00-1000 ng/ml was plotted using the peak-area ratio versus the concentration of SR141716 with a LOD and LLOQ of 1.09 and 3.62 ng/ml, respectively. The method developed has been used to analyze SR141716 in rat plasma samples from an animal study.     

Inverse agonism and neutral antagonism at cannabinoid CB1 receptors

There are at least two types of cannabinoid receptor, CB1 and CB2, both G protein coupled. CB1 receptors are expressed predominantly at nerve terminals and mediate inhibition of transmitter release whereas CB2 receptors are found mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous cannabinoid receptor agonists also exist and these "endocannabinoids" together with their receptors constitute the "endocannabinoid system". These discoveries were followed by the development of a number of CB1- and CB2-selective antagonists that in some CB1 or CB2 receptor-containing systems also produce "inverse cannabimimetic effects", effects opposite in direction from those produced by cannabinoid receptor agonists. This review focuses on the CB1-selective antagonists, SR141716A, AM251, AM281 and LY320135, and discusses possible mechanisms by which these ligands produce their inverse effects: (1) competitive surmountable antagonism at CB1 receptors of endogenously released endocannabinoids, (2) inverse agonism resulting from negative, possibly allosteric, modulation of the constitutive activity of CB1 receptors in which CB1 receptors are shifted from a constitutively active "on" state to one or more constitutively inactive "off" states and (3) CB1 receptor-independent mechanisms, for example antagonism of endogenously released adenosine at A1 receptors. Recently developed neutral competitive CB1 receptor antagonists, which are expected to produce inverse effects through antagonism of endogenously released endocannabinoids but not by modulating CB1 receptor constitutive activity, are also discussed. So too are possible clinical consequences of the production of inverse cannabimimetic effects, there being convincing evidence that released endocannabinoids can have "autoprotective" roles.     

GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.     

Autocrine and paracrine regulation of lymphocyte CB2 receptor expression by TGF-beta.

The marijuana-derived cannabinoid Delta(9)-tetrahydrocannabinol (THC) has been shown to be immunosuppressive. We report that THC induces the immunosuppressive cytokine TGF-beta by human peripheral blood lymphocytes (PBL). The ability of THC to stimulate TGF-beta production was blocked by the CB2 receptor specific antagonist SR144528 but not by the CB1 specific antagonist AM251. Furthermore, our data suggest that TGF-beta actively regulates lymphocyte CB2 receptor expression in an autocrine and paracrine manner. Whereas the addition of recombinant TGF-beta to PBL cultures downregulated CB2 receptor expression, anti-TGF-beta antibody treatment increased CB2 receptor expression. We conclude that one mechanism by which THC contributes to immune suppression is by stimulating an enhanced production of lymphocyte TGF-beta.     

Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through G(alpha)o/i-triggered proteasomal degradation of Rap1GAPII

The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth.     

CB1 receptor-G protein association. Subtype selectivity is determined by distinct intracellular domains.

The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C-terminal domain (CB(1)401-417). Guanosine 5'-O-(3-thio)triphosphate binding to G proteins can be stimulated by both peptide CB(1)401-417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol. 53, 504-510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry 38, 3447-3455]. In Chaps-solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB(1) receptor-G alpha(i) association and attenuated the CB(1)401-417-induced inhibition of adenylate cyclase. CB(1)401-417 significantly reduced the CB(1) receptor association with G alpha(i3), but not with G alpha(i1) or G alpha(i2). In contrast, third intracellular loop peptides significantly reduced the CB(1) receptor association with G alpha(i1) and G alpha(i2), but not G alpha(i3). These interactions are specific for the CB(1) receptor because a peptide corresponding to the juxtamembrane C-terminal domain of the CB(2) receptor failed to compete for the association of the CB1 receptor with any of the Gi alpha subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB(1) receptor direct the interaction with specific G protein subtypes.     

Constitutively active alpha subunits of G(q/11) and G(12/13) families inhibit activation of the pro-survival Akt signaling cascade

Accumulating evidence indicates that G protein signaling plays an active role in the regulation of cell survival. Our previous study demonstrated the regulatory effects of G(i/o) proteins in nerve growth factor-induced activation of pro-survival Akt kinase. In the present study we explored the role of various members of the G(s), G(q/11) and G(12/13) subfamilies in the regulation of Akt in cultured mammalian cells. In human embryonic kidney 293 cells transiently expressing constitutively active mutants of G alpha11, G alpha14, G alpha16, G alpha12, or G alpha13 (G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, respectively), basal phosphorylation of Akt was attenuated, as revealed by western blotting analysis using a phosphospecific anti-Akt immunoglobulin. In contrast, basal Akt phosphorylation was unaffected by the overexpression of a constitutively active G alpha(s) mutant (G alpha(s)QL). Additional experiments showed that G alpha11QL, G alpha14QL, G alpha16QL, G alpha12QL and G alpha13QL, but not G alpha(s)QL, attenuated phosphorylation of the Akt-regulated translation regulator tuberin. Moreover, they were able to inhibit the epidermal growth factor-induced Akt activation and tuberin phosphorylation. The inhibitory mechanism of Gq family members was independent of phospholipase Cbeta activation and calcium signaling because G alpha11QL, G alpha14QL and G alpha16QL remained capable of inhibiting epidermal growth factor-induced Akt activation in cells pretreated with U73122 and the intracellular calcium chelator, BAPTA/AM. Finally, overexpression of the dominant negative mutant of RhoA blocked G alpha12QL- and G alpha13QL-mediated inhibition, suggesting that activated G alpha12 and G alpha13 inhibit Akt signaling via RhoA. Collectively, this study demonstrated the inhibitory effect of activated G alpha11, G alpha14, G alpha16, G alpha12 and G alpha13 on pro-survival Akt signaling.     

Stimulation of increases in intracellular calcium and prostaglandin E2 generation in Chinese hamster ovary cells expressing receptor-Galpha16 fusion proteins

We examined whether fusion proteins of G protein-coupled receptors with the alpha subunit of G(16) (Galpha(16)) could activate downstream signals. We expressed fusion proteins of G(i)-coupled receptors, i.e. CX(3)C chemokine receptor 1 (CX(3)CR1) and M(2) receptor, in Chinese hamster ovary cells. An agonist for CX(3)CR1 induced greater increases in intracellular Ca(2+) and prostaglandin E(2) generation in cells expressing CX(3)CR1-Galpha(16) fusion protein than in cells expressing CX(3)CR1 alone or both CX(3)CR1 and Galpha(16) separately. Similarly, agonist-induced prostaglandin E(2) generation was greater in cells expressing M(2)-Galpha(16) fusion protein than ones expressing M(2) alone or both M(2) and Galpha(16) separately. In cells expressing fusion proteins with Galpha(16) of G(q)-coupled receptors, i.e. urotensin II receptor and M(1) receptor, the relevant agonists induced similar increases in intracellular Ca(2+) and prostaglandin E(2) generation as in ones expressing the receptor alone. In cells expressing urotensin II receptor-Galpha(16) fusion protein, prostaglandin E(2) generation exhibited a lower EC(50) value than the intracellular Ca(2+) increase. These results indicate that agonist-stimulated receptor-Galpha(16) fusion proteins are coupled to downstream signaling pathways, and suggest that receptor-Galpha(16) fusion proteins may be useful for screening for ligands of orphan G protein-coupled receptors and G(i)-coupled receptors.     

The G alpha(o/i)-coupled cannabinoid receptor-mediated neurite outgrowth involves Rap regulation of Src and Stat3

The study of the signaling pathways regulating neurite outgrowth in culture is important because of their potential role in neuronal differentiation in vivo. We have previously shown that the G alpha(o/i)-coupled CB1 cannabinoid receptor (CB1R) activates Rap1 to induce neurite outgrowth. G alpha(o/i) also activates the Src-Stat3 pathway. Here, we studied the relationship between the G alpha(o/i)-Rap1 and Src-Stat3 pathways and the role of these signaling pathways in CB1R-mediated neurite outgrowth in Neuro-2A cells. The CB1 agonist HU-210 induced pertussis toxin-sensitive Src and Stat3 phosphorylation. Dominant negative (DN) mutants of Src and Stat3 blocked CB1R-induced neurite outgrowth. Constitutively active Rap 1B and Ral-activated Src and CB1R-induced Src phosphorylation was inhibited by Rap1-DN and Ral-DN, indicating that both Rap1 and Ral mediate downstream signaling from G alpha(o/i) for Src activation. Rap1-activated Ral and Ral-DN blocked Rap-induced Src phosphorylation. G alpha(o)-induced Stat3 activation was blocked by Ral-DN, whereas v-Src-induced Stat3 activation was not inhibited by Ral-DN, indicating that the CB1R, through G alpha(o), mediates the sequential activation of Rap1 to Ral to Src to Stat3 in Neuro-2A cells. Downstream of Src, the CB1R also activated Rac1 and JNK, which enhanced CBR1-mediated Stat3 activation. Rac-DN blocked CB1R-induced activation of JNK. Pharmacological inhibition of JNK blocked Src and CB1R activation of Stat3, indicating that Rac and JNK are also involved in CB1R-mediated neurite outgrowth. Overall, this study demonstrated that G alpha(o/i)-coupled CB1R triggers neurite outgrowth in Neuro-2A through the activation of a signaling network containing two pathways that bifurcate at Src and converge at Stat3.     

Effect of lipid rafts on Cb2 receptor signaling and 2-arachidonoyl-glycerol metabolism in human immune cells

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5'-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (approximately 55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD in DAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to approximately 50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells.     

The effect of protein kinase C activation on G(z)-mediated regulation of type 2 and 6 adenylyl cyclases

Three serine-to-alanine mutants of the alpha subunit of the heterotrimeric G protein G(z) (alpha(z)) were examined for their signaling properties in the presence of phorbol ester treatment. All three alpha(z) mutants resembled wild-type alpha(z) in their abilities to inhibit alpha(s)-stimulated type 6 adenylyl cyclase (AC6) and phorbol ester treatment reduced their magnitudes of inhibition. Depending on the permissive condition, the betagamma-mediated stimulation of type 2 adenylyl cyclase (AC2) was differentially regulated by alpha(z) and the three mutants. Mutation of Ser(27) but not Ser(16) of alpha(z) affected the efficient release of betagamma subunits upon receptor activation and abolished the stimulation of phosphorylated but not alpha(s)-stimulated AC2.