贩卖雄激素受体突变体对绿色荧光蛋白融合:一个新的调查部分雄激素不敏感综合征。
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Georget V, Terouanne B, Lumbroso年代,尼古拉斯JC,苏丹C
贩卖雄激素受体突变体对绿色荧光蛋白融合:一个新的调查部分雄激素不敏感综合征。
中国性金属底座。1998年10月,83 (10):3597 - 603。
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9768671 (在PubMed]
- 文摘
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天然突变的雄激素受体(AR),检测患者的雄激素不敏感综合征(AIS),目前在体外实验中分析了。不幸的是,这些化验的演示并不总是允许的体外活性受体之间的直接关系和表型的严重程度(尤其是突变患者中发现部分AIS)。我们最近研究贩卖野生型的基于“增大化现实”技术,融合绿色荧光蛋白(GFP)的活细胞。在目前的研究中,我们应用此方法对AR突变体的分析找出它是否可能是一个互补的AIS的调查方法。GFP-AR突变体融合蛋白的建设后,androgen-binding特征、核移植能力,和转录活动进行评估。核移植是量化的各种浓度的双氢睾酮(DHT)。我们研究了两个突变体与部分AIS相关:G743V R840C。androgen-binding特征的突变体都受到影响,与正常相比AR。尽管亲和力是相似,离解速率GFP-AR-G743V GFP-AR-R840C的两倍。在转录分析,这两个突变体活性只有在高浓度的雄激素。核贩运的突变体是由两个参数评价:1)核转移的速度; and 2) the maximal amount of receptors imported into the nucleus. At 10(-6) mol/L DHT, the GFP-AR mutants entered into the nucleus in a fashion similar to that of GFP-AR-wt. At 10(-7) mol/L DHT, the rate and maximal degree of nuclear import were both reduced, even more, for GFP-AR-G743V. The difference between mutants was more pronounced at 10(-9) mol/L DHT, because GFP-AR-G743V entered into the nucleus with even slower kinetics. Though the androgen-binding affinity and transcriptional activity assays did not reveal major differences between mutants, the dissociation rate and the trafficking capacity measurements permitted the activity of the mutants to be differentiated. We observed that the nuclear transfer capacities of these mutants are in correlation with the severity of the phenotype. The GFP-AR model provides an opportunity both to observe the dynamics of the hormone/receptor complex in living cells and to study the impact of the ligand-binding domain mutation, as opposed to certain in vitro techniques. Because the nuclear import capacity correlates well with the degree of androgen insensitivity, the GFP-AR is a useful complementary tool to understanding the phenotype/genotype relationship of AR function in patients with AIS.