G(i) protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: Implication for intracellular signalization cross-regulation.

The peripheral cannabinoid receptor (CB2) is a G protein-coupled receptor t hat is both positively and negatively coupled to the mitogen-activated prot ein kinase (MAPK) and cAMP pathways, respectively, through a Bordetella per tussis 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 a ddition to the inhibition of auto-activated activated CB2 in this model, we found that SR 144528 inhibited the MAPK activation induced by G(i)-depende nt receptors such as receptor-tyrosine kinase (insulin, insulin-like growth factor 1) or G protein-coupled receptors (lysophosphatidic acid), but not by G(i)-independent receptors such as the fibroblast growth factor receptor . We showed that this SR 144528 inhibitory effect on G(i)-dependent recepto rs was mediated by a direct G(i) protein inhibition through CB2 receptors. Indeed, we found that through binding to the CB2 receptors, SR 144528 block ed 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 G(i) prot ein level as shown in Western blotting as well as in confocal microscopic e xperiments. This up-regulation occurred with a concomitant loss of SR 14452 8 ability to inhibit the insulin or lysophosphatidic acid-induced MARK acti vation. This inverse agonist-induced modulation of the G(i) strongly sugges ts that the modulated protein is functionally associated with the complex S R 144528/CB2 receptors, and that the G(i) level may account for the heterol ogous desensitization phenomena.