Molecular Mechanisms of Steroid Hormone Perception at the Cell Surface

细胞表面类固醇激素感知的分子机制

• 批准号: 8186473
• 负责人: JOANNE CHORY
• 金额: $ 35.77万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8186473
• 关键词: 3-Dimensional; Affinity; Alleles; Amino Acids; Anabolism; Animals; Arabidopsis; Binding; Binding Sites; Biochemical; Biochemistry; Bioinformatics; Biological Assay; C-terminal; Cell Death; Cell Surface Receptors; Cell physiology; Cell surface; Cells; Cellular biology; Complex; Crystallography; Data; Development; Diet; Dissociation; Eukaryota; Event; Evolution; Extracellular Domain; Family; Family member; Gene Expression; Gene Family; Genetic; Genetic Models; Glycoproteins; Goals; Growth and Development function; Homeostasis; Hormone Receptor; Hormones; Human; In Vitro; Insecta; Lead; Leucine; Leucine-Rich Repeat; Ligand Binding; Ligand Binding Domain; Ligands; Maps; Membrane; Modeling; Molecular; N-terminal; Natural Immunity; Nuclear; Output; Pathway interactions; Peptides; Perception; Phosphorylation; Phosphotransferases; Plant Proteins; Plants; Plasma; Protein Family; Protein Kinase Inhibitors; Proteins; Publishing; Receptor Activation; Recombinants; Recruitment Activity; Regulation; Reporting; Resolution; Role; Signal Pathway; Signal Transduction; Specificity; Steroid Receptors; Steroids; System; Testing; Thinking; Toll-like receptors; extracellular; growth promoting activity; in vitro Model; in vivo; inhibitor/antagonist; insight; kinase inhibitor; loss of function; member; mutant; novel; protein kinase inhibitor; receptor; research study; small molecule; steroid hormone; structural biology; trafficking

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. PUBLIC HEALTH RELEVANCE: Despite their divergence from a common ancestor over 1 billion years ago, both plants and animals utilize steroids as hormones to regulate gene expression that controls growth, development, and homeostasis. The pathways of synthesis and turnover of steroids are remarkably conserved, yet steroid receptors appear to have evolved independently in plants and animals, which may be due to the unique predator/prey relationship of animals and plants. The experiments described in this proposal will thus not only inform our understanding of a new paradigm for steroid hormone perception, but they may also influence our thinking concerning the evolution of signaling pathways in humans as a result of our diet.

描述（由申请人提供）：类固醇激素对动物、昆虫和植物的生长、发育和体内平衡至关重要。在植物中，一类称为油菜素类固醇（brassinosteroids，BR）的多羟基化类固醇在整个植物界中具有广泛的分布和独特的生长促进活性。在过去的十年中，我们分析了BR的生物合成和细胞功能，并确定了膜定位受体BRI 1及其许多下游信号组分。BRI 1是Toll样受体激酶，由含有24个富含亮氨酸的重复序列的大的胞外配体结合结构域、跨膜区段和胞质激酶结构域组成。先前的研究表明，BRI 1的细胞外结构域结合类固醇激素，然后诱导受体的构象变化，进而导致细胞质激酶结构域的自磷酸化和激酶抑制剂蛋白BKI 1的解离。这增加了BRI 1对其共受体BAK 1的亲和力，BAK 1是一种具有5个LRR的受体激酶。受体和共受体的激酶结构域之间的广泛的交叉磷酸化事件然后导致完全活化的信号传导复合物。在这里，我们建议结合结构生物学/生物化学与遗传学/细胞生物学来剖析受体的分子机制。我们想分析它的配体识别和激活模式，它与辅助受体BAK 1的相互作用，以及新型抑制剂BKI 1对其激酶活性的调节。因此，本研究的目的是：（1）确定BRI 1胞外区LRR模块中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，并在体外和体内测试该模型;（3）阐明BKI 1及其6个相关蛋白在BRI 1激酶活性负调控中的分子作用方式和特异性。我们的长期目标是开发一个经过验证的机制模型，准确地描述BR信号通路的刺激和调节。BRI 1是多细胞真核生物中类固醇激素的细胞表面受体中研究最多和最了解的。因此，这里描述的实验将告知我们对来自细胞表面受体的类固醇激素感知和信号传导的新范例的机械理解。拟南芥作为遗传模型的优势已经允许在受体及其辅助受体中鉴定出数十种突变等位基因（功能获得和丧失），并阐明了从受体到核基因表达变化的整个信号通路。因此，我们准备好解释结构研究。BRI 1是植物中最大的受体激酶家族的创始成员，并且与哺乳动物先天免疫受体具有显著的同源性。因此，我们的研究将为其他植物信号通路及其与哺乳动物系统的进化关系提供机制性见解。 公共卫生相关性：尽管植物和动物在10亿年前与共同的祖先不同，但它们都利用类固醇作为激素来调节控制生长，发育和体内平衡的基因表达。类固醇的合成和周转途径非常保守，但类固醇受体似乎在植物和动物中独立进化，这可能是由于动物和植物独特的捕食者/猎物关系。因此，本提案中描述的实验不仅将告知我们对类固醇激素感知的新范式的理解，而且它们还可能影响我们对人类饮食导致的信号通路演变的思考。