Abstract

Ligands of structurally diverse natures are able to bind at the CB₁ cannabinoid receptor, suggesting the existence of multiple binding sites on the receptor. Modeling studies have implicated Ser2.60(173) and Ser7.39(383) as possible interaction site(s) for CB₁ agonists. To test the importance of these residues for receptor recognition, recombinant human CB₁ receptors, stably expressed in human embryonic kidney 293 cells, were used to investigate the consequences of mutating Ser2.60 (to S2.60A) or Ser7.39 (to S7.39A) in radioligand binding and guanosine 5′-3-O-(thio)triphosphate functional assays. The S7.39A mutant resulted in a total ablation of [³H](–)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP55,940) high-affinity binding. However, [³H](R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55,212-2) binding properties at S7.39A were comparable with those of the wild-type (WT) receptor. The binding affinity of (–)-11β-hydroxy-3-(1′,1′-dimethylheptyl)hexahydrocannabinol (AM4056) and (–)-11-hydroxydimethylheptyl-Δ⁸-tetrahydrocannabinol (HU210) were drastically reduced (50- to 100-fold) at the S7.39A mutant. Likewise, the EC₅₀ for HU210 and AM4056-mediated activation of the S7.39A receptor was increased by >200-fold. In contrast, the binding affinity and potency of WIN55,212-2, CP55,940, HU210, and AM4056 were unaltered at the S2.60A mutant compared with WT human CB₁ receptors. These results clearly suggest that Ser7.39, but not Ser2.60, plays a crucial role in mediating ligand specific interactions for CP55,940, HU210, and AM4056 at the human CB₁ receptor. Our modeling studies predict that Ser7.39 in a g–χ1 conformation may induce a helix bend in TMH7 that provides docking space for CP55,940 binding; the S7.39A mutation may alter this binding space, precluding CP55,940 binding.