The third transmembrane helix of the cannabinoid receptor plays a role in the selectivity of aminoalkylindoles for CB2, peripheral cannabinoid receptor

Two subtypes of the human cannabinoid receptor have been identified. The CB 1 receptor is primarily distributed in the central nervous system, whereas the CB2 receptor is associated with peripheral tissue, including the spleen . These two subtypes are also distinguished by their ligand-binding profile s. The goal of this study was to identify critical residues in transmembran e region III (TM3) of the receptors that contribute to subtype specificity in ligand binding. For this purpose, a chimeric cannabinoid receptor [CB1/2 (TM3)] was generated in which the TM3 of CB1 was replaced with the correspo nding region of CB2. These receptors were stably expressed in Chinese hamst er ovary cells for evaluation. The binding affinities of CB1/2(TM3) and the wild-type CB1 receptor to several prototype ligands were similar with one notable exception: the chimeric receptor exhibited a 4-fold enhancement in binding affinity to WIN 55,212-2 (K-d = 4.8 nM) relative to that observed w ith CB1 (K-d = 21.7 nM). Two additional aminoalkylindoles, JWH 015 and JWH 018, also bound the chimeric receptor (K-i = 1.0 mu M and 1.4 nM, respectiv ely) with higher affinity compared with the wild-type CB1 (K-i = 5.2 mu M a nd 9.8 nM, respectively). Furthermore, the increase in binding affinities o f the aminoalkylindoles were reflected in the EC50 values for the ligand-in duced inhibition of intracellular cAMP levels mediated by the chimeric rece ptor. This pattern mirrors the selectivity of WIN 55,212-2 binding to CB2 c ompared with CB1. Site-specific mutagenesis of the most notable amino acid changes in the chimeric receptor, Gly195 to Ser and Ala198 to Met, revealed that the enhancement in WIN 55,212-2 binding is contributed to by the Ser but not by the Met residue. The data indicate that the amino acid differenc es in TM3 between CB1 and CB2 play a critical role in subtype selectivity f or this class of compounds.