Role of a conserved lysine residue in the peripheral cannabinoid receptor (CB2): evidence for subtype specificity.

The human cannabinoid receptors, central cannabinoid receptor (CB1) and peripheral cannabinoid receptor (CB2), share only 44% amino acid identity overall, yet most ligands do not discriminate between receptor subtypes. Site-directed mutagenesis was employed as a means of mapping the ligand recognition site for the human CB2 cannabinoid receptor. A lysine residue in the third transmembrane domain of the CB2 receptor (K109), which is conserved between the CB1 and CB2 receptors, was mutated to alanine or arginine to determine the role of this charged amino acid in receptor function. The analogous mutation in the CB1 receptor (K192A) was found to be crucial for recognition of several cannabinoid compounds excluding (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN 55,212-2). In contrast, in human embryonic kidney (HEK)-293 cells expressing the mutant or wild-type CB2 receptors, we found no significant differences in either the binding profile of several cannabinoid ligands nor in inhibition of cAMP accumulation. We identified a high-affinity site for (-)-3-[2-hydroxyl-4-(1, 1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP-55,940) in the region of helices 3, 6, and 7, with S3.31(112), T3.35(116), and N7.49(295) in the K109A mutant using molecular modeling. The serine residue, unique to the CB2 receptor, was then mutated to glycine in the K109A mutant. This double mutant, K109AS112G, retains the ability to bind aminoalkylindoles but loses affinity for classical cannabinoids, as predicted by the molecular model. Distinct cellular localization of the mutant receptors observed with immunofluorescence also suggests differences in receptor function. In summary, we identified amino acid residues in the CB2 receptor that could lead to subtype specificity.

High-level expression of the human CB2 cannabinoid receptor using a baculovirus system.

A human CB2 recombinant baculovirus (AcNPV-hCB2) was generated by site-specific transposition and employed to express the human CB2 cannabinoid receptor. Northern analysis of total RNA from Spodoptera frugiperda (Sf9) insect cells infected with AcNPV-hCB2 revealed novel expression of a unique 2.3 kb transcript when probed with hCB2 cDNA. This transcript corresponded to the size expected for hCB2 generated from the recombinant virus construct. Western immunoblot analysis of whole cell homogenates of recombinant baculovirus-infected Sf9 cells, using affinity-purified antibody to a human CB2 carboxy terminal domain (anti-hCB2.CV), revealed the presence of novel immunoreactive protein. In addition, when anti-hCB2.CV was employed in immunofluorescence staining, an intense signal was observed within AcNPV-hCB2-infected cells but not within uninfected cells or cells infected with a control beta-galactosidase recombinant baculovirus. The pattern of immunofluorescence at early periods post-infection was in a perinuclear arrangement with a "signet-ring" appearance, suggestive of glycosylation of the expressed recombinant protein. Transmission electron microscopy revealed regions of intranuclear recombinant virus assembly and the presence of numerous intracytoplasmic proteinaceous vesicular inclusions consistent with hyperproduction of hCB2. Scatchard-Rosenthal analysis of [3H]-(-)3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxypro pyl]cyclohexan-1-ol ([3H]CP 55,940) receptor binding indicated a Kd of 2.24 nM and a Bmax equal to 5.24 pmol/mg of protein. The lack of [3H]CP 55,940 displacement with N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamidehydrochloride (SR 141716A), the CB1-selective antagonist, confirmed the identity of the receptor as CB2. These data indicate that AcNPV-hCB2 expresses high levels of the human CB2, which retains properties of the native receptor. Thus, this recombinant virus may prove suitable for hyperproduction of receptor for basic biochemical and biophysical characterization studies.