Kapur Ankur, Samaniego Patrick, Thakur Ganesh A, Makriyannis Alexandros, Abood Mary E
California Pacific Medical Center Research Institute, San Francisco, California, USA.
J Pharmacol Exp Ther. 2008 Apr;325(1):341-8. doi: 10.1124/jpet.107.133256. Epub 2008 Jan 3.
Amino acid residues in the transmembrane domains of the CB(1) 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 (CB(1)) 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 CB(1) receptors, stably expressed in human embryonic kidney 293 cells, were assayed for ligand affinity and agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) 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-ylmethanone (WIN55212-2), (-)-11beta-hydroxy-3-(1',1'-dimethylheptyl) hexahydrocannabinol (AM4056), and (-)-11-hydroxyldimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210) for the stimulation of GTPgammaS 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 approximately 50-fold) in the EC(50) 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.
CB(1)受体跨膜结构域中的氨基酸残基对于配体识别和信号转导至关重要。我们使用定点诱变来确定跨膜螺旋II结构域中两个相邻新残基Ile2.62和Asp2.63的作用。我们通过将大麻素受体（CB(1)）功能中2.63位保守的带负电荷的天冬氨酸替换为天冬酰胺（D2.63N）和谷氨酸（D2.63E），研究了其作用。此外，还研究了突变体I2.62T单独以及与D2.63N组合（双突变体）对结构多样配体的亲和力和效价的影响。对稳定表达于人类胚胎肾293细胞中的重组人CB(1)受体进行配体亲和力和激动剂刺激的鸟苷5'-3-O-(硫代)三磷酸（GTPγS）结合检测。电荷保守突变体D2.63E的表现与野生型相似。电荷中和突变D2.63N减弱了(-)-3-[2-羟基-4-(1,1-二甲基庚基)苯基]-4-[3-羟丙基]环己醇（CP55940）、(R)-(-)-2,3-二氢-5-甲基-3-[(4-吗啉基)甲基]吡咯并[1,2,3-de]-1,4-苯并恶嗪-6-基甲酮（WIN55212-2）、(-)-11β-羟基-3-(1',1'-二甲基庚基)六氢大麻酚（AM4056）和(-)-11-羟基二甲基庚基-Δ(8)-四氢大麻酚（HU210）刺激GTPγS结合的效价，而不影响它们的结合亲和力。同样，I2.62T突变体选择性地改变了激动剂效价，而不改变激动剂亲和力。值得注意的是，双突变体（I2.62T-D2.63N）在激动剂介导的激活的半数有效浓度（EC(50)）上显示出急剧的协同增加（约50倍）。I2.62T-D2.63N双突变体中功能的严重丧失表明，尽管这些残基对于激动剂识别不是必需的，但它们在调节信号转导中起协同且关键的作用。
