PT - JOURNAL ARTICLE AU - Qing Tao AU - Sean D. McAllister AU - John Andreassi AU - Katharine W. Nowell AU - Guy A. Cabral AU - Dow P. Hurst AU - Kevin Bachtel AU - Marie C. Ekman AU - Patricia H. Reggio AU - Mary E. Abood TI - Role of a Conserved Lysine Residue in the Peripheral Cannabinoid Receptor (CB<sub>2</sub>): Evidence for Subtype Specificity DP - 1999 Mar 01 TA - Molecular Pharmacology PG - 605--613 VI - 55 IP - 3 4099 - http://molpharm.aspetjournals.org/content/55/3/605.short 4100 - http://molpharm.aspetjournals.org/content/55/3/605.full SO - Mol Pharmacol1999 Mar 01; 55 AB - 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. The American Society for Pharmacology and Experimental Therapeutics