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）：类固醇激素对于动物，昆虫和植物的生长，发育和稳态至关重要。在植物中，一类称为黄铜固醇（BRS）的多羟基化类固醇在整个植物王国中具有广泛的分布和独特的生长促进活动。在过去的十年中，我们已经分析了BRS的生物合成和细胞功能，并鉴定了膜钙化受体BRI1及其许多下游信号传导成分。 BRI1是一种类似的受体激酶，由大型细胞外配体结合结构域组成，其中包含24个富含亮氨酸的重复序列，跨膜段和一个细胞质激酶结构域。先前的研究表明，BRI1的细胞外结构域结合了类固醇激素，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BKI1的解离。这增加了BRI1对其共受体Bak1的亲和力，Bak1是一种具有5个LRR的受体激酶。然后，受体的激酶结构域与受体受体之间的广泛跨磷酸化事件导致完全激活的信号传导复合物。在这里，我们建议将结构生物学/生物化学与遗传学/细胞生物学结合起来，以剖析受体的分子机制。我们要分析其配体识别和激活的模式，与共受体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，该建议具有以下特定目的：（1）确定在BRI1的细胞外域中LRR模块的背景下，由94个氨基酸类固醇结合结构域确定了类固醇激素识别的详细机制； （2）开发一种用于受体激活的机械模型，并在体外和体内测试该模型； （3）在BRI1激酶活性的负调控中，阐明了BKI1和6相关蛋白的分子作用模式和特异性。我们的长期目标是开发一个经过验证的机械模型，该模型准确地描述了BR信号通路的刺激和调节。 BRI1是对多细胞真核生物中类固醇激素提出的任何细胞表面受体的研究最多，并且最了解的。因此，此处描述的实验将为我们对类固醇激素感知和来自细胞表面受体的信号传导的新范式的机械理解提供信息。拟南芥作为遗传模型的强度允许鉴定受体及其受体受体的数十个突变等位基因（既有功能和功能丧失），并阐明了从受体到核基因表达变化的整个信号传导途径。因此，我们对解释结构研究进行了充分的精力。 Bri1是植物中最大的受体激酶家族的创始成员，并与哺乳动物的先天免疫受体共享重要同源性。因此，我们的研究将提供对其他植物信号通路的机械洞察力，以及它们与研究精良的哺乳动物系统的进化关系。 公共卫生相关性：尽管它们与十亿年前的共同祖先有分歧，但动植物都利用类固醇作为激素来调节控制生长，发育和稳态的基因表达。类固醇的合成和周转途径非常保守，但是类固醇受体似乎在动植物中独立发展，这可能是由于动物和植物的独特捕食者/猎物关系所致。因此，本提案中描述的实验不仅将为我们对类固醇激素感知的新范式的理解提供理解，而且还可能影响我们关于由于饮食而导致人类信号传导途径演变的思想。