ACTIVATION OF THE HUMAN PERIPHERAL CANNABINOID RECEPTOR RESULTS IN INHIBITION OF ADENYLYL-CYCLASE

The human peripheral cannabinoid (CB2) receptor has been cloned by rev erse transcription-polymerase chain reaction from human spleen RNA and expressed, to study both ligand binding characteristics and signal tr ansduction pathways. Receptor binding assays used the aminoalkylindole [H-3]Win 55212-2 and membranes from transiently transfected COS-MG ce lls. Saturation analysis showed that [H-3]Win 55212-2 specific binding to the CB2 receptor was of high affinity, with a K-d of 2.1 +/- 0.2 n M (four experiments), and a high level of expression was attained, wit h a maximal number of saturable binding sites of 24.1 +/- 4.4 pmol/mg of protein (four experiments). The rates of association and dissociati on for [H-3]Win 55212-2 specific binding were both rapid when measured at 30 degrees. [H-3]Win 55212-2 specific binding to the CB2 receptor was moderately enhanced by divalent and monovalent cations but was onl y slightly inhibited by guanosine-5'-O-(3-thio)-triphosphate. Competit ion for [H-3]Win 55212-2 specific binding to the CB2 receptor was ster eoselective, with the following rank order of potency for the more act ive stereoisomers: HU-210 > (-)-CP-55940 approximate to Win 55212-2 >> (-)Delta(9)-THC > anandamide. The signaling pathway of the human CB2 receptor was investigated in a CB2-CHO-K1 stable cell line. CB2 recept or activation by cannabinoid agonists inhibited forskolin-induced cAMP production in a concentration-dependent and stereoselective manner bu t did not increase either cAMP production or Ca2+ mobilization in fura -2/acetoxymethyl ester-loaded CB2-CHO-K1 cells. The CB2 receptor-media ted inhibition of forskolin-induced cAMP production was abolished by p retreatment of the cells with 10 ng/ml pertussis toxin. These results demonstrate that the CB(?)2 receptor is functionally coupled to inhibi tion of adenylyl cyclase activity via a pertussis toxin-sensitive G pr otein.