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Mutation Studies of Ser7.39 and Ser2.60 in the Human CB₁ Cannabinoid Receptor: Evidence for a Serine-Induced Bend in CB₁ Transmembrane Helix 7

California Pacific Medical Center Research Institute, San Francisco, California (A.K., D.F., M.E.A.); Center for Drug Design, Department of Chemistry and Biochemistry, University of North Carolina Greensboro, Greensboro, North Carolina (D.P.H., R.W., P.H.R.); Center for Drug Discovery, Northeastern University, Boston, Massachusetts (G.T., A.M.); and Department of Chemistry, Guilford College, Greensboro, North Carolina (R.W.)

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 (–)-11-hydroxy-3-(1',1'-dimethylheptyl)hexahydrocannabinol (AM4056) and (–)-11-hydroxydimethylheptyl-⁸-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–1 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.

Address correspondence to: Dr. Mary E. Abood, California Pacific Medical Center Research Institute, San Francisco, CA 94107. E-mail: aboodm{at}cpmcri.org

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				A. Kapur, P. Samaniego, G. A. Thakur, A. Makriyannis, and M. E. Abood Mapping the Structural Requirements in the CB1 Cannabinoid Receptor Transmembrane Helix II for Signal Transduction J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 341 - 348. [Abstract] [Full Text] [PDF]