Mutation Studies of Ser7.39 and Ser2.60 in the Human CB 1 Cannabinoid Receptor: Evidence for a Serine-Induced Bend in CB 1 Transmembrane Helix 7 Ankur Kapur, Dow P. Hurst, Daniel Fleischer, Rob Whitnell, Ganesh A. Thakur, Alexandros Makriyannis, Patricia H. Reggio, and Mary E. Abood 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.) Received January 30, 2007; accepted March 23, 2007 ABSTRACT Ligands of structurally diverse natures are able to bind at the CB 1 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 CB 1 agonists. To test the importance of these residues for receptor recognition, recombinant human CB 1 receptors, sta- bly 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 [ 3 H](-)-3-[2-hy- droxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cy- clohexan-1-ol (CP55,940) high-affinity binding. However, [ 3 H](R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]- pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)meth- anone (WIN55,212-2) binding properties at S7.39A were com- parable with those of the wild-type (WT) receptor. The binding affinity of (-)-11-hydroxy-3-(1',1'-dimethylheptyl)hexahydro- cannabinol (AM4056) and (-)-11-hydroxydimethylheptyl- 8 - tetrahydrocannabinol (HU210) were drastically reduced (50- to 100-fold) at the S7.39A mutant. Likewise, the EC 50 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 CB 1 receptors. These results clearly suggest that Ser7.39, but not Ser2.60, plays a crucial role in mediating ligand specific inter- actions for CP55,940, HU210, and AM4056 at the human CB 1 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. The CB 1 cannabinoid receptor is a member of the G-protein coupled receptor (GPCR) family 1A, which includes the CB 2 receptor and the prototype rhodopsin (Howlett et al., 2002; Reggio, 2005). The human CB 1 and CB 2 receptors share only 44% amino acid overall homology, with a higher homology (68%) within the seven transmembrane domains (Munro et al., 1993). Both the CB 1 and CB 2 receptors share common signal transduction pathways, such as negative modulation of adenylyl cyclase activity (Felder et al., 1995) and also share certain common structural features with rhodopsin, including an extracellularly oriented N terminus, an intra- cellular carboxyl terminus, and hydrophobic transmembrane helices (TMHs). Although neither CB 1 nor CB 2 proteins have been crystal- lized, the crystal structure of rhodopsin (Palczewski et al., 2000) serves as a valuable template to model the putative CB 1 ligand binding domains. Ligands of structural diverse This study was supported by National Institutes of Health grants DA09978 and DA05274 (to M.E.A.), DA00489 and DA039434 (to P.H.R.), and DA09158 (to A.M. and M.E.A.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.034645. ABBREVIATIONS: GPCR, G-protein coupled receptor; TMH, transmembrane helix; CP55,940, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)- phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; AAI, aminoalkylindole; WIN55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyr- rolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth- yl-1H-pyrazole-3-carboxamide; HU-210, (-)-11-hydroxydimethylheptyl- 8 -tetrahydrocannabinol; AM841, (-)-7'-isothiocyanato-11-hydroxy- 1',1'-dimethylheptylhexahydrocannabinol; AM4056, (-)-11-hydroxy-3-(1',1'-dimethylheptyl)hexahydrocannabinol; GTPS, guanosine 5'-3-O- (thio)triphosphate; (+)-7-OH-CBD-DMH, 7-OH-cannabidiol, 1,1-dimethylheptyl; CB, cannabinoid receptor; WT, wild type; HEK, human embryonic kidney; CM, conformational memories; Rho, rhodopsin; MD, molecular dynamics; EC, extracellular; IC, intracellular; h, human; SAH, southern aliphatic hydroxyl; NAH, northern aliphatic hydroxyl; HU-243, 3-dimethylheptyl-11-hydroxyhexahydrocannabinol. 0026-895X/07/7106-1512–1524$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 6 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 34645/3213164 Mol Pharmacol 71:1512–1524, 2007 Printed in U.S.A. 1512 at ASPET Journals on October 4, 2017 molpharm.aspetjournals.org Downloaded from