Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor.

We have cloned the peripheral cannabinoid receptor, mCB2, from a mouse splenocyte cDNA library. The 3.7 kb sequence contains an open reading frame encoding a protein of 347 residues sharing 82% overall identity with the only other known peripheral receptor, human CB2 (hCB2) and shorter than hCB2 by 13 amino acids at the carboxyl terminus. Binding experiments with membranes from COS-3 cells transiently expressing mCB2 showed that the synthetic cannabinoid WIN 55212-2 had a 6-fold lower affinity for mCB2 than for hCB2, whereas both receptors showed similar affinities for the agonists CP 55,940, delta(9)-THC and anandamide and almost no affinity for the central receptor- (CB1) specific antagonist SR 141716A. Both hCB2 and mCB2 mediate agonist-stimulated inhibition of forskolin-induced cAMP production in CHO cell lines permanently expressing the receptors. SR 141716A failed to antagonize this activity in either cell line, confirming its specificity for CB1.

Modulation of CB1 cannabinoid receptor functions after a long-term exposure to agonist or inverse agonist in the Chinese hamster ovary cell expression system.

We have investigated the adaptive changes of the human central cannabinoid receptor (CB1) stably expressed in Chinese hamster ovary cells (CHO-CB1), after agonist (CP 55,940) or selective CB1 inverse agonist (SR 141716) treatment. CB1 receptor density and affinity constant as measured by binding assays with both tritiated ligands remained essentially unchanged after varying period exposure of CHO-CB1 cells (from 30 min to 72 hr) to saturating concentrations of CP 55,940 or SR 141716. However, using a C-myc-tagged version of the CB1 receptor, FACS analysis and confocal microscopy studies on CB1 expression indicated that the agonist promoted a disappearance of cell surface receptor although inverse agonist increased its cell surface density. Taken together these results suggest that 1) agonist induces internalization of the receptor into a cellular compartment that would be still accessible to both the hydrophobic ligands CP 55,940 or SR 141716; 2) inverse-agonist promotes externalization of the receptor from an intracellular preexisting pool to the cell surface. In parallel, we also investigated the associated effects of CP 55,940 and SR 141716 on CB1 receptor-coupled second messengers. We showed that preexposure of cells to CP 55,940 induced a rapid desensitization of the CB1 to the agonist response. The ability of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase and to activate the mitogen-activated protein kinase activity was dramatically reduced. By striking contrast, SR 141716 pretreatment of CHO-CB1 cells not only had no significant effect on the potency of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase but also induced a significant enhancement of the CP 55,940 ability to stimulate the mitogen-activated protein kinase activity. These results suggest that the modulation of the number of cell surface receptor could lead to functional desensitization or sensitization of the CB1 receptors.

Characterization of two cloned human CB1 cannabinoid receptor isoforms.

We have investigated the pharmacology of two central human cannabinoid receptor isoforms, designated CB1 and CB1A, stably expressed in Chinese hamster ovary cell lines, designated as CHO-CB1 and CHO-CB1A, respectively. In direct binding assays on isolated membranes the agonist [3H]CP 55,940 bound in a saturable and highly specific manner to both cannabinoid receptor isoforms. Competition binding experiments performed with other commonly used receptor agonists showed the following rank order of potency: CP 55,940 > tetrahydrocannabinol > WIN 55212-2 > anandamide. Except for the endogenous ligand anandamide (CB1, Ki = 359.6 nM vs. CB1A, Ki = 298 nM), these agonists bound to CB1A (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 7.24,345 and 26.7 nM, respectively) with about 3-fold less affinity than to CB1 (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 2.26, 93 and 7.1 nM, respectively). The cannabinoid receptor antagonist SR 141716A also bound to CB1A (Ki = 43.3 nM) with slightly less affinity than to CB1 (Ki = 4.9 nM). Cannabinoid receptor-linked second messenger system studies performed in the CHO-CB1 and CHO-CB1A cells showed that both receptors mediated their action through the agonist-induced inhibition of forskolin-stimulated cAMP accumulation. This activity was totally blocked by pretreatment with PTX. Additionally, both isoforms activated mitogen-activated protein kinase. The selective antagonist SR 141716A was able to selectively block these responses in both cell lines, to an extent that reflected its binding characteristics. Our results show that the amino-truncated and -modified CB1 isoform CB1A exhibits all the properties of CB1 to a slightly attenuated extent.

SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor.

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.