Molecular Mechanisms of Steroid Hormone Perception at the Cell Surface

描述(由申请人提供)：类固醇激素对动物、昆虫和植物的生长、发育和动态平衡是必不可少的。在植物中，有一类多羟基类固醇化合物，称为油菜素类固醇(BRs)，广泛分布于整个植物界，具有独特的促生长活性。在过去的十年里，我们分析了BRs的生物合成和细胞功能，并鉴定了膜定位的受体BRI1及其许多下游信号成分。BRI1是一种Toll样受体蛋白，由一个含有24个富含亮氨酸重复序列的胞外配体结合域、一个跨膜片段和一个胞浆激酶域组成。以往的研究表明，BRI1的胞外区与类固醇激素结合，然后引起受体的构象变化，进而导致胞浆激酶域的自动磷酸化和激酶抑制蛋白BKI1的解离。这增加了BRI1对其辅助受体BAK1的亲和力，BAK1是一种具有5个LRRs的受体激酶。受体和辅受体的激酶域之间广泛的交叉磷酸化事件随后导致完全激活的信号复合体。在这里，我们建议结合结构生物学/生物化学和遗传学/细胞生物学来剖析受体的分子机制。我们想要分析它的配体识别和激活模式，它与辅助受体BAK1的相互作用，以及新型抑制剂BKI1对其激酶活性的调节。因此，本研究有以下具体的目的：(1)结合BRI1胞外区的LRR模块，确定94个氨基酸的类固醇结合结构域识别类固醇激素的详细机制；(2)建立受体激活的机制模型，并在体内外验证该模型；(3)阐明BKI1和6个相关蛋白在负调控BRI1‘S激酶活性中的分子作用方式和特异性。我们的长期目标是开发一个有效的机制模型，准确地描述BR信号通路的刺激和调节。BRI1是多细胞真核生物中研究最多、了解最多的类固醇激素细胞表面受体。因此，这里描述的实验将使我们从机制上理解类固醇激素感知和细胞表面受体信号传递的新范例。拟南芥作为一种遗传模型的强大功能使人们能够在受体及其辅助受体中识别数十个突变等位基因(包括功能获得和功能丧失)，并阐明了从受体到核基因表达变化的整个信号通路。因此，我们做好了解释结构研究的准备。BRI1是植物中最大的受体激酶家族的创始成员，与哺乳动物的天然免疫受体有显著的同源性。因此，我们的研究将提供对其他植物信号通路的机械性洞察，以及它们与研究得很好的哺乳动物系统的进化关系。

DESCRIPTION (provided by applicant): Steroid hormones are essential for growth, development, and homeostasis of animals, insects, and plants. In plants, one class of polyhydroxylated steroids, called brassinosteroids (BRs), has wide distribution throughout the plant kingdom and unique growth promoting activities. During the past decade, we have analyzed the biosynthesis and cellular functions of BRs, and identified the membrane-localized receptor, BRI1, as well as many of its downstream signaling components. BRI1 is a Toll-like receptor kinase comprised of a large extracellular ligand-binding domain containing 24 leucine-rich repeats, a trans-membrane segment and a cytoplasmic kinase domain. Previous studies indicate that the extracellular domain of BRI1 binds the steroid hormone, which then induces a conformational change in the receptor that in turn leads to auto- phosphorylation of the cytoplasmic kinase domain and the dissociation of the kinase inhibitor protein BKI1. This increases the affinity of BRI1 for its co-receptor, BAK1, a receptor kinase with 5 LRRs. Extensive cross- phosphorylation events between the kinase domains of the receptor and the co-receptor then lead to a fully activated signaling complex. Here, we propose combining structural biology/biochemistry with genetics/cell biology to dissect the molecular mechanism of the receptor. We want to analyze its mode of ligand recognition and activation, its interaction with the co-receptor BAK1, and the regulation of its kinase activity by the novel inhibitor, BKI1. Thus, this proposal has the following specific aims: (1) Determine the detailed mechanism of steroid hormone recognition by the 94 amino acid steroid-binding domain in context of the LRR modules in the extracellular domain of BRI1; (2) Develop a mechanistic model for receptor activation and test this model in vitro and in vivo; (3) Elucidate the molecular mode of action and specificity of BKI1 and 6 related proteins in the negative regulation of BRI1's kinase activity. Our long-term goal is to develop a validated mechanistic model that accurately describes the stimulation and regulation of the BR signaling pathway. BRI1 is the best studied and most understood of any cell surface receptor proposed for steroid hormones in multicellular eukaryotes. The experiments described here will thus inform our mechanistic understanding of a new paradigm for steroid hormone perception and signaling from a cell surface receptor. The strength of Arabidopsis as a genetic model has allowed the identification of dozens of mutant alleles (both gain- and loss-of-function) in both the receptor and its co-receptor, as well as elucidated an entire signaling pathway from the receptor to changes in nuclear gene expression. Thus, we are well-poised to interpret the structural studies. BRI1 is a founding member of the largest family of receptor kinases in plants, and shares significant homology with mammalian innate immunity receptors. As such, our studies will provide mechanistic insight into other plant signaling pathways, and their evolutionary relationship to well-studied mammalian systems.