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
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
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DOI：
10.1124/mol.107.034645
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发表时间：
2007-06-01
影响因子：
3.6
通讯作者：
Abood, Mary E.
中科院分区：
文献类型：
--
作者：
Kapur, Ankur;Hurst, Dow P.;Abood, Mary E.
结构上不同性质的配体能够在CB 1大麻素受体结合，表明受体上存在多个结合位点。建模研究表明Ser2.60（173）和Ser7.39（383）可能是CB 1激动剂的相互作用位点。为了测试这些残基对于受体识别的重要性，在人胚肾293细胞中稳定表达的重组人CB 1受体被用于研究在放射性配体结合和鸟苷5 '-3-O-（硫代）三磷酸功能测定中突变Ser2.60（至S2.60A）或Ser7.39（至S7.39A）的结果。S7.39A突变体导致[3 H]（-）-3-[2-羟基4-（1，1-二甲基庚基）苯基]-4-[3-羟基丙基]环己烷1-α（CP 55，940）高亲和力结合完全消除。然而，S7.39A处的[3 H]（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]（1-萘基）甲酮（WIN 55，212-2）结合特性与野生型（WT）受体的结合特性相当。在S7.39A突变体处，（-）-11 β-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（HU 210）的结合亲和力急剧降低（50至100倍）。同样，HU 210和AM4056介导的S7.39A受体活化的EC 50增加> 200倍。相比之下，与WT人CB 1受体相比，在S2.60A突变体中，WIN 55，212-2，CP 55，940，HU 210和AM4056的结合亲和力和效力没有改变。这些结果清楚地表明，Ser7.39而不是Ser2.60在介导CP 55、940、HU 210和AM 4056在人CB 1受体上的配体特异性相互作用中起关键作用。我们的模型研究预测，G-x（1）构象的Ser7.39可以诱导TMH 7的螺旋弯曲，为CP 55，940结合提供对接空间; S7.39 A突变可以改变这种结合空间，排除CP 55，940结合。
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-hydroxyl4-(1,1-dimethylheptyl) phenyl]-4-[3-hydroxylpropyl] cyclohexan1-alpha (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 beta-hydroxy-3-(1', 1'-dimethylheptyl) hexahydro-cannabinol (AM4056) and (-)-11-hydroxydimethylheptyl-Delta(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 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-x(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.