An intramolecular folding sensor for imaging estrogen receptor-ligand interactions
An intramolecular folding sensor for imaging estrogen receptor-ligand interactions
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DOI：
10.1073/pnas.0607385103
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发表时间：
2006-10-24
影响因子：
11.1
通讯作者：
Gambhir, Sanjiv S.
中科院分区：
文献类型：
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作者：
Paulmurugan, Ramasanny;Gambhir, Sanjiv S.
高通量分析各种激素和药物与雌激素受体（ER）之间相互作用的策略对于加速了解雌激素受体生物学和药理学至关重要。通过仔细分析人类内质网（hER）配体结合域（hER- lbd）在不同配体复合物中的晶体结构，我们假设hER- lbd分子内折叠模式可以用来区分内质网激动剂、选择性内质网调节剂和纯抗雌激素。因此，我们构建并验证了编码各种hER-LBD融合蛋白的分子内折叠传感器，这些融合蛋白可以在适当配体存在的情况下导致Renilla/firefly荧光素酶报告互补分裂。一种对循环雌二醇亲和力低的hER-LBD突变体也被确定用于活体成像。稳定表达表达野生型和突变型hER-LBD的分子内折叠传感器的细胞被用于成像配体诱导的活体小鼠分子内折叠。这是第一个hER- lbd分子内折叠传感器，适用于高通量定量分析hER与激素和药物之间的相互作用，使用细胞裂解物、完整细胞和小活体的分子成像。所开发的策略也可以扩展到研究和成像其他重要的蛋白质分子内折叠系统。
Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla/firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.