Activation of the human peripheral cannabinoid receptor results in inhibition of adenylyl cyclase.
The human peripheral cannabinoid (CB2) receptor has been cloned by reverse transcription-polymerase chain reaction from human spleen RNA and expressed, to study both ligand binding characteristics and signal transduction pathways. Receptor binding assays used the aminoalkylindole [3H]Win 55212-2 and membranes from transiently transfected COS-M6 cells. Saturation analysis showed that [3H]Win 55212-2 specific binding to the CB2 receptor was of high affinity, with a Kd of 2.1 +/- 0.2 nM (four experiments), and a high level of expression was attained, with a maximal number of saturable binding sites of 24.1 +/- 4.4 pmol/mg of protein (four experiments). The rates of association and dissociation for [3H]Win 55212-2 specific binding were both rapid when measured at 30 degrees. [3H]Win 55212-2 specific binding to the CB2 receptor was moderately enhanced by divalent and monovalent cations but was only slightly inhibited by guanosine-5'-O-(3-thio)-triphosphate. Competition for [3H]Win 55212-2 specific binding to the CB2 receptor was stereoselective, with the following rank order of potency for the more active stereoisomers: HU-210 > (-)-CP-55940 approximately 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 receptor activation by cannabinoid agonists inhibited forskolin-induced cAMP production in a concentration-dependent and stereoselective manner but did not increase either cAMP production or Ca2+ mobilization in fura-2/acetoxymethyl ester-loaded CB2-CHO-K1 cells. The CB2 receptor-mediated inhibition of forskolin-induced cAMP production was abolished by pretreatment of the cells with 10 ng/ml pertussis toxin. These results demonstrate that the CB2 receptor is functionally coupled to inhibition of adenylyl cyclase activity via a pertussis toxin-sensitive G protein.
Slipetz
D M
DM
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada.
O'Neill
G P
GP
Favreau
L
L
Dufresne
C
C
Gallant
M
M
Gareau
Y
Y
Guay
D
D
Labelle
M
M
Metters
K M
KM
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D048588:Benzoxazines; D001667:Binding, Competitive; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002460:Cell Line; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009025:Morpholines / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D037342:Pertussis Toxin; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D012709:Serum Albumin / Q000494:pharmacology; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D048588:Benzoxazines; D002413:Cations, Divalent; D002414:Cations, Monovalent; D009025:Morpholines; D009281:Naphthalenes; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D012709:Serum Albumin; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002118:Calcium