A proteomic microarray approach for exploring ligand-initiated nuclear hormone receptor pharmacology, receptor selectivity, and heterodimer functionality
A proteomic microarray approach for exploring ligand-initiated nuclear hormone receptor pharmacology, receptor selectivity, and heterodimer functionality
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DOI：
10.1074/mcp.m400192-mcp200
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发表时间：
2005-03-01
影响因子：
7
通讯作者：
Katzenellenbogen, JA
中科院分区：
文献类型：
--
作者：
Kim, SH;Tamrazi, A;Katzenellenbogen, JA
核激素受体（NHRs）是多器官系统发育和稳态的主要调节因子。这些蛋白质是配体调节的转录因子，其响应于其同源激素或激素类似物的循环水平的变化来调节基因表达。当NHR结合配体时，它们采用不同的构象，以反映配体的激动剂与拮抗剂特性的方式使辅调节蛋白结合或不结合。使用雌激素受体配体结合域作为NHR家族的代表性成员，我们显示功能蛋白质微阵列的发展，并使用它们来探索辅激活剂的招聘和NHR同源和异源二聚体的功能。这些NHR蛋白质微阵列可以正向模式（共激活物募集到打印的NHR）或反向模式（NHR募集到打印的共激活物）制造。从这些微阵列，我们可以预测各种NHR配体的效力和药理学特性，通过其辅激活剂招聘的性质。另外，不同的辅激活蛋白可以在功能上分类，并且可以定量它们对NHR的亲和力。NHR选择性拮抗剂配体和小分子共激活剂模拟物破坏共激活剂-NHR复合物。这种新的蛋白质组学方法也被用来评估辅激活蛋白的募集，以探索异源二聚体的功能。发现雌激素受体的异源二聚体仅在两个单体与激动剂配体结合时才能招募共激活剂，这一观察结果提供了对作用于包含两种NHR亚型的组织的激素的复杂生物学的深入了解。我们可以将这种NHR蛋白质组学方法扩展到多结构域全长NHR构建体的分析，并且可以同时监测不同类别的NHR的激活状态，其中具有不同NHR选择性和药理学的内源性或合成配体的混合物。
Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist versus antagonist character of the ligand. Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo- and heterodimer functionality. These NHR protein microarrays can be fabricated in either a forward mode ( coactivator recruited to printed NHR) or a reversed mode ( NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally different coactivator proteins can be functionally classified and their affinity for NHRs can be quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore heterodimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology.