Expression of the murine CB2 cannabinoid receptor using a recombinant Semliki Forest virus

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB2 RNA, was employed for the high-level expression of the murine CB2 (mCB2) 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 40 kDa were produced by the construct. The 40 kDa product, but not the 26 kDa product, was glycosylated as determined by 2-deoxy-d-glucose incorporation and peptide-N-glycosidase F digestion analysis. Assessment of [<F>3H</F>]CP55940 ([<F>3H</F>]-(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB2 RNA indicated a Kd of <F>0.35±0.04</F> nM and a Bmax of <F>24.4±2.7</F> pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB2 receptors, was observed. The CB2 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 [<F>3H</F>]CP55940, while the CB1 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-mCB2 RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB2 receptor. However, the presence of both 26 and 40 kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB2 RNA expression system. [Copyright &y& Elsevier]