The Difference between the CB1 and CB2Cannabinoid Receptors at Position 5.46 Is Crucial for the Selectivity of WIN55212-2 for CB2

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It has been reported that WIN55212-2, a prototypic aminoalkylindole, has higher affinity for CB2 than for CB1. To explain the selectivity of WIN55212-2 for CB2, molecular modeling studies were performed to probe the interacting sites between WIN55212-2 and cannabinoid receptors. In TMH5 the position 5.46 is a Phe in CB2 versus a Val in CB1. Docking of WIN55212-2 into the models of CB1 and CB2 predicts that F5.46 will result in a greater aromatic stacking of CB2 with WIN55212-2. Using site-directed mutagenesis, this hypothesis was tested by exchanging the amino acids at position 5.46 between CB1 and CB2. Two mutations, including a Phe to Val mutation at the position 5.46 in CB2 (CB2F5.46V), and a corresponding Val to Phe mutation at the position 5.46 in CB1 (CB1V5.46F), were made. The mutant receptors were transfected into 293 cells, and stable cell lines expressing similar numbers of receptors as wild-type receptors were chosen for additional ligand binding and cAMP accumulation studies. In ligand- binding assays, the CB2F5.46V mutation decreased the affinity of WIN55212-2 for CB2 by 14-fold. In contrast, the CB1V5.46F mutation increased the affinity of WIN55212-2 for CB1 by 12-fold. However, these mutations did not change the affinity of HU-210, CP-55940, and anandamide for CB1 and CB2. In cAMP accumulation assays, the changes in EC50 values of WIN55212-2 were consistent with the changes in its binding affinity caused by the mutations. These results strongly support the hypothesis that the selectivity of WIN55212-2 for CB2 over CB1 is attributable to the change from Val in CB1 at position 5.46 to Phe in CB2.