Two proteins with seven transmembrane-spanning domains typical of guanosine-nucleotide-bindingprotein-coupled receptors have been identified as cannabinoid receptors ; the central cannabinoid receptor, CB1, and the peripheral cannabinoid receptor, CB2, initially described in rat brain and spleen, respectively. Here, we report the distribution patterns for both CB1 and CB2 transcripts in human immune cells and in several human tissues, as analysed using a highly sensitive and quantitative PCR-based method. CB1 was mainly expressed in the central nervous system and, to a lower extent, in several peripheral tissues such as adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. In contrast, the CB2 gene, which is not expressed in the brain, was particularly abundant in immune tissues, with an expression level 10-100-fold higher than that of CB1. Although CB2 mRNA was also detected in some other peripheral tissues, its level remained very low. In spleen and tonsils, the CB2 mRNA content was equivalent to that of CB1 mRNA in the central nervous system. Among the main human blood cell subpopulations, the distribution pattern of the CB2 mRNA displayed important variations. The rank order of CB2 mRNA levels in these cells was B-cells > natural killer cells S monocytes > polymorphonuclear neutrophil cells > T8 cells > T4 cells. The same rank order was also established in human cell lines belonging to the inyeloid, monocytic and lymphoid lineages. The prevailing expression of the CB2 gene in immune tissues was confirmed by Northern-blot analysis. In addition, the expression of the CB2 protein was demonstrated by an immunohistological analysis performed on tonsil sections using specific anti-(human CB2) IgG; this experiment showed that CB2 expression was restricted to Blymphocyte-enriched areas of the mantle of secondary lymphoid follicles. These results suggest that (a) CB1 and CB2 can be considered as tissue-selective antigens of the central nervous system and immune system, respectively, and (b) cannabinoids may exert specific receptor-mediated actions on the immune system through the CB2 receptor.Keywords: cannabinoid ; cannabinoid receptors (CB1; CB2) ; human immune system ; B cells; natural killer cells.A-9-Tetrahydrocannabinol, the major active component of cannabis, as well as other cannabinoids, are known to exert a wide range of physiological effects such as drowsiness, alterations in cognition and memory, analgesia, orexigenic effects, anti-emetic effects, a decrease in intra-ocular pressure, anti-inflammatory effects and immunosuppression [l]. Many studies have been conducted to decipher the cannabinoid system. First attributed to non-specific cell membrane disruption, the major cannabinoid effects are now thought to be mediated through specific cannabinoid receptors. A guanosine-nucleotide-bindingprotein-coupled receptor of 472 amino-acid residues, CB1, was initially characterized in rat brain [Z] and further cloned both in rat [3] and human [4]. As the expression of the c...

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 (CB 2 ) in the Chinese hamster ovary cell line stably transfected with human CB 2 . In the present report, we studied the effect of SR 144528 on CB 2 phosphorylation. The CB 2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB 2 which can discriminate between phosphorylated and non-phosphorylated CB 2 isoforms at serine 352. We first showed that CB 2 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 CB 2 receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB 2 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 CB 2 phosphorylation was reversed by SR 144528, paradoxically leading to a nonphosphorylated CB 2 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 CB 2 phosphorylation involve an acidotropic GRK kinase, which does not use G i ␤␥. In contrast, SR 144528-induced CB 2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase. Several studies revealed that receptor activation can occur spontaneously in the absence of an agonist. This discovery led to a reclassification of antagonists as neutral antagonists or inverse agonists. Neutral antagonists block agonist action without any effect on constitutive activity (16 -19), whereas agonists block agonist action but also suppress constitutive activity.We recently demonstrated the agonist-independent activity of CB 1 and CB 2 receptors expressed in mammalian cells following transfection (20,21). We also showed that the CB 1 antagonist SR141716 and the CB 2 antagonist SR 144528 not only block the actions of cannabinoid agonists but they also suppress the constitutive activity of these receptors, indicating that these molecules act as inverse agonists. Furthermore, we revealed for the first time a novel property of these inverse agonists. We demonstrated that they also switch off the activation induced by other unrelated G i -dependent receptors such as insulin or insulin-like growth factor-1 receptors...

Several putative functions have been attributed to the peripheral benzodiazepine receptor (PBR), but its precise physiologic role has not been elucidated. In the present study, we investigated PBR function by quantifying this receptor in leukocyte subsets from healthy donors and in leukemic blasts from lymphoid and myeloid lineages. Using a monoclonal antibody (MoAb) directed against the human PBR and a quantitative flow cytometric assay, we found that phagocytic cells from healthy donors displayed a higher level of PBRs than lymphocytes or natural killer (NK) cells. Among the lymphoid lineage, thymocytes and IgD-negative B cells expressed the lowest levels. However, because of the wide heterogeneity of PBR levels among 42 acute or chronic lymphoid and myeloid leukemias, it was not possible to assign PBR expression to a stage of maturation or a cell lineage. Although the PBR displayed a mitochondrial subcellular localization, its expression was not correlated with the mitochondrial content, suggesting a modulation of PBR density at the level of the mitochondria. This modulation was confirmed when we studied in detail the PBR expression during T-cell development by both flow cytometry and confocal microscopy. We found that the PBR was expressed with a bimodal profile during T-cell development, identical to the one observed with the proto-oncogene, Bcl- 2. The high similarity in the expression of both the PBR and the Bcl-2 proto-oncogene in T-cell and B-cell subsets, their common mitochondrial localization, and the observation of high quantities of PBR in phagocytic cells, which are known to produce high levels of radical oxygen species, suggested that PBRs may participate in an antioxidant pathway. Indeed, a strong correlation was established between the ability of hematopoietic cell lines to resist H202 cytotoxicity and their level of PBR expression. Demonstration of the role of PBR in the protection against H202 was obtained by transfecting JURKAT cells with the human PBR cDNA. Transfected cells exhibited increased resistance to H202 compared with wild-type cells, suggesting that PBR may prevent mitochondria from radical damages and thereby modulate apoptosis in the hematopoietic system.

The P2Y 11 receptor is an ATP receptor positively coupled to the cAMP and phosphoinositide pathways. Ssf1 is a Saccharomyces cerevisiae nuclear protein, which plays an important role in mating. The gene encoding the human orthologue of SSF1 is adjacent to the P2Y 11 gene on chromosome 19. During the screening of placenta cDNA libraries, we isolated a chimeric clone resulting from the intergenic splicing between the P2Y 11 and SSF1 genes. The fusion protein was stably expressed in CHO-K1 cells where it generated a cAMP response to ATP qualitatively indistinguishable from that of the P2Y 11 receptor. According to both Western blotting and cAMP response, the expression of the fusion protein in the transfected cells was clearly lower than that of the P2Y 11 receptor. Both P2Y 11 and SSF1 probes detected a 5.6-kb messenger RNA with a similar pattern of intensity in each of 11 human tissues. The ubiquitous presence of chimeric transcripts and their up-regulation during granulocytic differentiation indicate that the transgenic splicing between the P2Y 11 and the SSF1 genes is a common and regulated phenomenon. There are very few examples of intergenic splicing in mammalian cells, and this is the first case involving a G-protein-coupled receptor.

Two subtypes of G-protein–coupled cannabinoid receptors have been identified to date: the CB1 central receptor subtype, which is mainly expressed in the brain, and the CB2 peripheral receptor subtype, which appears particularly abundant in the immune system. We investigated the expression of CB2 receptors in leukocytes using anti-CB2 receptor immunopurified polyclonal antibodies. We showed that peripheral blood and tonsillar B cells were the leukocyte subsets expressing the highest amount of CB2 receptor proteins. Dual-color confocal microscopy performed on tonsillar tissues showed a marked expression of CB2 receptors in mantle zones of secondary follicles, whereas germinal centers (GC) were weakly stained, suggesting a modulation of this receptor during the differentiation stages from virgin B lymphocytes to memory B cells. Indeed, we showed a clear downregulation of CB2 receptor expression during B-cell differentiation both at transcript and protein levels. The lowest expression was observed in GC proliferating centroblasts. Furthermore, we investigated the effect of the cannabinoid agonist CP55,940 on the CD40-mediated proliferation of both virgin and GC B-cell subsets. We found that CP55,940 enhanced the proliferation of both subsets and that this enhancement was blocked by the CB2 receptor antagonist SR 144528 but not by the CB1 receptor antagonist SR 141716. Finally, we observed that CB2 receptors were dramatically upregulated in both B-cell subsets during the first 24 hours of CD40-mediated activation. These data strongly support an involvement of CB2 receptors during B-cell differentiation.