Xu Wei, Sanz Arantxa, Pardo Leonardo, Liu-Chen Lee-Yuan
Department of Pharmacology, Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Biochemistry. 2008 Oct 7;47(40):10576-86. doi: 10.1021/bi800381v. Epub 2008 Sep 9.
We previously demonstrated that D3.49(164)Y or T6.34(279)K mutation in the rat mu opioid receptor (MOPR) resulted in agonist-independent activation. Here, we identified the cysteine(s) within the transmembrane domains (TMs) of the D3.49(164)Y mutant that became accessible in the binding-site crevice by use of methanethiosulfonate ethylammonium (MTSEA) and inferred conformational changes associated with receptor activation. While the C7.38(321)S mutant was insensitive to MTSEA, the D3.49(164)Y/C7.38(321)S mutant showed similar sensitivity as the D3.49(164)Y, suggesting that, in the D3.49(164)Y mutant, C7.38(321) becomes inaccessible while other cysteines are accessible in the binding-site crevice. Each of the other seven cysteines in the TMs was mutated to serine on the background of D3.49(164)Y/C7.38(321)S, and the resulting triple mutants were evaluated for [3H]diprenorphine and [d-Ala2,NMe-Phe4,Gly5-ol]-enkephalin (DAMGO) binding and effect of MTSEA on [3H]diprenorphine binding. The D3.49(164)Y/C7.38(321)S mutant and the triple mutants, except the C6.47(292)S triple mutant, retained similar affinities for [3H]diprenorphine and DAMGO as the D3.49(164)Y mutant. The second-order rate constants for MTSEA reactions showed that C3.44(159)S, C4.48(190)S, C5.41(235)S, and C7.47(330)S significantly reduced sensitivity to MTSEA, compared with the D3.49(164)Y/C7.38(321)S. These results suggest that the four cysteines may be rotated and/or tilted to become accessible. While the D3.49(164)Y/C7.38(321)S was similarly sensitive to MTSEA as the D3.49(164)Y mutant, the T6.34(279)K/C7.38(321)S was much less sensitive to MTSEA than the T6.34(279)K mutant, suggesting that the two constitutively active mutants assume different conformations and/or possess different dynamic properties. Molecular models of the MOPR monomer and homodimer, using the crystal structures of rhodopsin, the beta2-adrenergic receptor, and the ligand-free opsin, which contains several features characteristic of the active state, were employed to analyze these experimental results in a structural context.
我们之前证明，大鼠μ阿片受体（MOPR）中的D3.49(164)Y或T6.34(279)K突变导致了非激动剂依赖性激活。在此，我们通过使用甲硫代磺酸乙酯铵（MTSEA）确定了D3.49(164)Y突变体跨膜结构域（TMs）中可在结合位点裂隙中暴露的半胱氨酸，并推断了与受体激活相关的构象变化。虽然C7.38(321)S突变体对MTSEA不敏感，但D3.49(164)Y/C7.38(321)S突变体表现出与D3.49(164)Y相似的敏感性，这表明在D3.49(164)Y突变体中，C7.38(321)变得不可暴露，而其他半胱氨酸可在结合位点裂隙中暴露。在D3.49(164)Y/C7.38(321)S背景下，TMs中的其他七个半胱氨酸中的每一个都突变为丝氨酸，并对所得的三重突变体进行了[3H]二丙诺啡和[d-Ala2,NMe-Phe4,Gly5-ol]-脑啡肽（DAMGO）结合以及MTSEA对[3H]二丙诺啡结合的影响的评估。D3.49(164)Y/C7.38(321)S突变体和除C6.47(292)S三重突变体之外的三重突变体对[3H]二丙诺啡和DAMGO的亲和力与D3.49(164)Y突变体相似。MTSEA反应的二级速率常数表明，与D3.49(1,64)Y/C7.38(321)S相比，C3.44(159)S、C4.48(190)S、C5.41(235)S和C7.47(330)S对MTSEA的敏感性显著降低。这些结果表明，这四个半胱氨酸可能会旋转和/或倾斜以变得可暴露。虽然D3.49(164)Y/C7.38(321)S对MTSEA的敏感性与D3.49(164)Y突变体相似，但T6.34(279)K/C7.38(321)S对MTSEA的敏感性比T6.34(279)K突变体低得多，这表明这两个组成型活性突变体具有不同的构象和/或不同的动力学性质。利用视紫红质、β2-肾上腺素能受体和无配体视蛋白的晶体结构构建了MOPR单体和同二聚体的分子模型，这些晶体结构包含了几个活性状态的特征，用于在结构背景下分析这些实验结果。
