Abstract

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 CB₂receptor (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.