Expression of the murine CB2 cannabinoid receptor using a recombinant Semliki Forest virus.
A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB(2) RNA, was employed for the high-level expression of the murine CB(2) (mCB(2)) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate-polyacrylamide gel electrophoresis (urea-SDS-PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40kDa were produced by the construct. The 40kDa product, but not the 26kDa product, was glycosylated as determined by 2-deoxy-D-glucose incorporation and peptide-N-glycosidase F digestion analysis. Assessment of [3H]CP55940 ([3H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB(2) RNA indicated a K(d) of 0.35+/-0.04nM and a B(max) of 24.4+/-2.7pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB(2) receptors, was observed. The CB(2) receptor-specific antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [3H]CP55940, while the CB(1) receptor-specific antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB(2) RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB(2) receptor. However, the presence of both 26 and 40kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB(2) RNA expression system.
Olson
John M
JM
Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095-1737, USA.
Kennedy
Suzanne J
SJ
Cabral
Guy A
GA
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D015152:Blotting, Northern; D002186:Cannabinoids / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003847:Deoxyglucose / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012313:RNA / Q000032:analysis; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012672:Semliki forest virus / Q000235:genetics*; D014162:Transfection
D000998:Antiviral Agents; D002186:Cannabinoids; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D011994:Recombinant Proteins; D012313:RNA; D003847:Deoxyglucose; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase