The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

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The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to simply exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Here we reveal a more complex modulatory role of GHS-R1b. Differential co-expression of GHS-R1a and GHS-R1b, both in HEK-293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHSR1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively. We found a preferential G i/o coupling of the GHSR1a-GHS-R1b complex in HEK-293T cells and, unexpectedly, a preferential G s/olf coupling in both striatal and hippocampal neurons in culture. A dopamine D 1 receptor (D1R) antagonist blocked ghrelin-induced cAMP accumulation in striatal but not hippocampal neurons, indicating the involvement of D1R in the striatal GHS-R1a-G s/olf coupling. Experiments in HEK-293T cells demonstrated that D1R co-expression promotes a switch in GHS-R1a-G protein coupling from G i/o to G s/olf , but only upon co-expression of GHS-R1b. Furthermore, resonance energy transfer experiments showed that D1R interacts with GHS-R1a, but only in the presence of GHS-R1b. Therefore, GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling but also determines the ability of GHS-R1a to form oligomeric complexes with other receptors, promoting profound qualitative changes in ghrelin-induced signaling.Ghrelin is an orexigenic hormone, an internal signal for the animal to engage in food-directed behavior (1, 2). It is produced by stomach oxyntic cells, which provide plasma levels that fluctuate diurnally with a peak in the day and trough at night. Notably, oxyntic cells qualify as food-entrained oscillators, and ghrelin plasma levels increase during anticipated mealtimes and decrease after meals (1). These and other less well characterized central neuronal functions of ghrelin depend on its ability to cross the blood-brain barrier by still unclear mechanisms and reaching ghrelin receptors localized in specific brain areas, such as the hypothalamus, hippocampus, amygdala, mesencephalic dopaminergic regions, and striatum (2-4).Ghrelin acts on the class A G protein-coupled receptor known as growth hormone secretagogue (GHS) 5 receptor or GHS-R1a. Cells expressing GHS-R1a also express GHS-R1b, a truncated variant of GHS-R1a lacking transmembrane domains 6 and 7. Ghrelin does not bind and therefore does not signal through GHS-R1b (5), and the role of this truncated "receptor" on ghrelin-mediated signaling is just beginning to be understood. Evidence has been provided for the ability of GHS-R1a to homodimerize and heterodimerize with GHS-R1b, which might allow GHS-R1b to produce a dominant negative effect on GHSR1a signaling. Two different mechanisms have b...