Gi 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 that is both positively and negatively coupled to the mitogen-activated protein kinase (MAPK) and cAMP pathways, respectively, through a Bordetella pertussis 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 addition to the inhibition of autoactivated CB2 in this model, we found that SR 144528 inhibited the MAPK activation induced by Gi-dependent receptors such as receptor-tyrosine kinase (insulin, insulin-like growth factor 1) or G protein-coupled receptors (lysophosphatidic acid), but not by Gi-independent receptors such as the fibroblast growth factor receptor. We showed that this SR 144528 inhibitory effect on Gi-dependent receptors was mediated by a direct Gi protein inhibition through CB2 receptors. Indeed, we found that through binding to the CB2 receptors, SR 144528 blocked 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 Gi protein level as shown in Western blotting as well as in confocal microscopic experiments. This up-regulation occurred with a concomitant loss of SR 144528 ability to inhibit the insulin or lysophosphatidic acid-induced MAPK activation. This inverse agonist-induced modulation of the Gi strongly suggests that the modulated protein is functionally associated with the complex SR 144528/CB2 receptors, and that the Gi level may account for the heterologous desensitization phenomena.
Bouaboula
M
M
Sanofi Recherche, Montpellier cedex 04, France.
Desnoyer
N
N
Carayon
P
P
Combes
T
T
Casellas
P
P
D000818:Animals; D016466:CHO Cells; D001892:Camphanes / Q000494:pharmacology*; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D037342:Pertussis Toxin; D011720:Pyrazoles / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D043882:Receptors, Cannabinoid; D011956:Receptors, Cell Surface / Q000378:metabolism; D011955:Receptors, Drug / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D015854:Up-Regulation; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D001892:Camphanes; D011720:Pyrazoles; D043882:Receptors, Cannabinoid; D011956:Receptors, Cell Surface; D011955:Receptors, Drug; C110630:SR 144528; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D020794:Receptor Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins