TY - JOUR T1 - Mapping the Structural Requirements in the CB<sub>1</sub> Cannabinoid Receptor Transmembrane Helix II for Signal Transduction JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 341 LP - 348 DO - 10.1124/jpet.107.133256 VL - 325 IS - 1 AU - Ankur Kapur AU - Patrick Samaniego AU - Ganesh A. Thakur AU - Alexandros Makriyannis AU - Mary E. Abood Y1 - 2008/04/01 UR - http://jpet.aspetjournals.org/content/325/1/341.abstract N2 - Amino acid residues in the transmembrane domains of the CB1 receptor are important for ligand recognition and signal transduction. We used site-directed mutagenesis to identify the role of two novel and adjacent residues in the transmembrane helix II domain, Ile2.62 and Asp2.63. We investigated the role of the conserved, negatively charged aspartate at position 2.63 in cannabinoid receptor (CB1) function by substituting it with asparagine (D2.63N) and glutamate (D2.63E). In addition, the effect of the mutant I2.62T alone and in combination with D2.63N (double mutant) on the affinity and potency of structurally diverse ligands was investigated. Recombinant human CB1 receptors, stably expressed in human embryonic kidney 293 cells, were assayed for ligand affinity and agonist-stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding. The charge-conserved mutant D2.63E behaved similar to wild type. The charge-neutralization mutation D2.63N attenuated the potency of (–)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP,55940), (R)-(–)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2), (–)-11β-hydroxy-3-(1′,1′-dimethylheptyl) hexahydrocannabinol (AM4056), and (–)-11-hydroxyldimethylheptyl-Δ8-tetrahydrocannabinol (HU210) for the stimulation of GTPγS binding, without affecting their binding affinities. Likewise, the I2.62T mutant selectively altered agonist potency without altering agonist affinity. It was surprising to note that the double mutant (I2.62T-D2.63N) displayed a drastic and synergistic increase (by ∼50-fold) in the EC50 for agonist-mediated activation. The profound loss of function in the I2.62T-D2.63N double mutant suggests that, although these residues are not obligatory for agonist recognition, they play a synergistic and crucial role in modulating signal transduction. The American Society for Pharmacology and Experimental Therapeutics ER -