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 per ipheral cannabinoid receptor (CB2), share only 44% amino acid identity over all, yet most ligands do not discriminate between receptor subtypes. Site-d irected mutagenesis was employed as a means of mapping the ligand recogniti on site for the human CB2 cannabinoid receptor. A lysine residue in the thi rd transmembrane domain of the CB2 receptor (K109), which is conserved betw een the CB1 and CB2 receptors, was mutated to alanine or arginine to determ ine the role of this charged amino acid in receptor function. The analogous mutation in the CB1 receptor (K192A) was found to be crucial for recogniti on of several cannabinoid compounds excluding (R)-(+)-[2,3-dihydro-5-methyl -3-[(4-morpholinyl)methyl]pyrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthale nyl)methanone (WIN 55,212-2). In contrast, in human embryonic kidney (HEK)2 93 cells expressing the mutant or wild-type CB2 receptors, we found no sign ificant differences in either the binding profile of several cannabinoid li gands nor in inhibition of cAMP accumulation. We identified a high-affinity site for (-)-3-[2-hydroxyl-4-1,1 -dimethylheptyl)phenyl]-4-[3-hydroxyl pro pyl] 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 th e ability to bind aminoalkylindoles but loses affinity for classical cannab inoids, as predicted by the molecular model. Distinct cellular localization of the mutant receptors observed with immunofluorescence also suggests dif ferences in receptor function. In summary, we identified amino acid residue s in the CB2 receptor that could lead to subtype specificity.