TY - JOUR T1 - Mutation Studies of Ser7.39 and Ser2.60 in the Human CB<sub>1</sub> Cannabinoid Receptor: Evidence for a Serine-Induced Bend in CB<sub>1</sub> Transmembrane Helix 7 JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1512 LP - 1524 DO - 10.1124/mol.107.034645 VL - 71 IS - 6 AU - Ankur Kapur AU - Dow P. Hurst AU - Daniel Fleischer AU - Rob Whitnell AU - Ganesh A. Thakur AU - Alexandros Makriyannis AU - Patricia H. Reggio AU - Mary E. Abood Y1 - 2007/06/01 UR - http://molpharm.aspetjournals.org/content/71/6/1512.abstract N2 - 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 &gt;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. The American Society for Pharmacology and Experimental Therapeutics ER -