与绿色荧光蛋白融合的雄激素受体突变体的转运:部分雄激素不敏感综合征的新研究。
文章的细节
-
引用
复制到剪贴板 -
乔治五世，特鲁安B，伦布罗索S，尼古拉斯JC，苏丹C
与绿色荧光蛋白融合的雄激素受体突变体的转运:部分雄激素不敏感综合征的新研究。
中华内分泌杂志。1998年10月;83(10):3597-603。
- PubMed ID
-
9768671 (在PubMed］
- 摘要
-
在雄激素不敏感综合征(AIS)患者中检测到的雄激素受体(AR)自然发生的突变，目前正在通过体外试验进行分析。不幸的是，这些检测并不总是能够证明受体的体外活性和表型的严重程度之间的直接关系(特别是在部分AIS患者中检测到的突变)。我们最近研究了野生型AR在活细胞中与绿色荧光蛋白(GFP)融合的转运。在本研究中，我们将该方法应用于AR突变体的分析，看看是否可以作为研究AIS的一种补充方法。构建GFP-AR突变融合蛋白后，评估其雄激素结合特性、核转移能力和转录活性。在不同浓度的双氢睾酮(DHT)存在下定量核转移。我们研究了两个与部分AIS相关的突变体:G743V和R840C。与正常的AR相比，这两个突变体的雄激素结合特性都受到了影响。尽管亲和力相似，GFP-AR-G743V的解离率是GFP-AR-R840C的两倍。在转录实验中，两种突变体只有在高浓度雄激素时才有活性。用两个参数评价突变体的核转移:1)核转移率; 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.