Institute of General Zoology and Endocrinology, Ulm University, D-89069 Ulm, Germany.
Insect Mol Biol. 2011 Aug;20(4):417-28. doi: 10.1111/j.1365-2583.2011.01077.x. Epub 2011 May 18.
Although it has no ligand, helix 12 in the ligand binding domain of Ultraspiracle (USP) is locked in an antagonistic position. To investigate whether this position is of functional importance, we enhanced the flexibility of helix 12 by mutating two amino acids (259, located in L1-3 and F491 in helix 12). Mutated USP reduces the stability of USP and all isoforms of the ecdysone receptor (EcR) and impairs nuclear localization and DNA binding of EcR/USP(L259A/F491/A), resulting in lower levels of basal transcriptional activity. Although the affinity of the ligand ponasterone A to EcR/USP(L259/F491) is moderately diminished, hormone-induced stimulation of transcriptional activity is normal. Potentiation of the ecdysone response by juvenile hormone (JH) is selectively increased in mutated heterodimers with EcR-B1, demonstrating that the antagonistic position impairs functional interaction of the EcR complex with JHIII.
虽然 Ultraspiracle（USP）的配体结合域中的螺旋 12 没有配体，但它被锁定在拮抗位置。为了研究这个位置是否具有功能重要性，我们通过突变两个氨基酸（位于 L1-3 的 259 和螺旋 12 中的 F491）增强了螺旋 12 的灵活性。突变的 USP 降低了 USP 和所有蜕皮激素受体（EcR）同工型的稳定性，并损害了 EcR/USP（L259A/F491/A）的核定位和 DNA 结合，导致基础转录活性降低。尽管 ponasterone A 与 EcR/USP（L259/F491）的亲和力适度降低，但激素诱导的转录活性刺激正常。在与 EcR-B1 的突变异二聚体中，保幼激素（JH）对蜕皮激素反应的增强作用被选择性增加，表明拮抗位置损害了 EcR 复合物与 JHIII 的功能相互作用。
