EBP50 Regulation of PTH Receptor in Bone

EBP50 骨中 PTH 受体的调节

• 批准号: 8232049
• 负责人: Peter A Friedman
• 金额: $ 32.95万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8232049
• 关键词: Accounting; Affect; Affinity; Allografting; Amino Acids; Award; Binding; Binding Proteins; Biochemical; Biological; Biology; Bone Development; Bone Diseases; Bone Marrow Transplantation; Cell model; Cells; Cyclic AMP-Dependent Protein Kinases; Data; Dimerization; Drosophila genus; Engineering; Equilibrium; Exhibits; Fluorescence Resonance Energy Transfer; Functional disorder; Goals; Hormones; Human; Indium; Ions; Kidney; Knockout Mice; Lead; Life; Light; Marrow; Mass Spectrum Analysis; Measurement; Measures; Mediating; Metabolic Bone Diseases; Metabolism; Methodology; Microscopy; Minerals; Modeling; Molecular; Molecular Conformation; Molecular Models; Molecular Target; Mus; Mutation; Osteoblasts; Osteomalacia; Osteopenia; Osteoporosis; Outcome; Parathyroid Hormone Receptor; Parathyroid gland; Patients; Phenotype; Phosphorylation; Pilot Projects; Post-Translational Protein Processing; Regulation; Resistance; Rest; Role; Scaffolding Protein; Serine; Signal Transduction; Site; Source; Spectrum Analysis; Stem cells; Structure; Surface Plasmon Resonance; Testing; Time; Transplantation; Urine; Ursidae Family; Wild Type Mouse; Work; adapter protein; base; bone; bone cell; bone mass; bone turnover; ezrin; improved; inorganic phosphate; molecular modeling; mutant; novel; osteosarcoma; prevent; protein protein interaction; public health relevance; receptor; research study; scaffold; skeletal; small molecule; therapeutic target; tool; wasting

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the cellular mechanisms by which the 50-kDa ezrin-bind protein (EBP50) regulates parathyroid hormone receptor (PTHR)-mediated signaling and function in bone. Mice with targeted deletion of EBP50 exhibit a bone phenotype, as do patients with EBP50 mutations. Although it is thought that the bone disorder arises as a secondary consequence of renal dysfunction, our preliminary data identify direct effects of EBP50 on bone. This suggested a novel mechanism by which mutations interfere with EBP50 function and, by extension, that EBP50 is dynamically regulated by PTH in open and closed conformations. The unifying idea of the present proposal is that novel structural determinants in EBP50 and their posttranslational modification dictate EBP50 function on PTHR activity in bone. Three specific aims are developed to test this idea. In Aim 1 we will characterize EBP50 conformations and dimerization to test the hypothesis that the described mutations lock EBP50 in a closed configuration that interferes with PTHR function. These experiments will use molecular biological maneuvers to examine static interactions, molecular modeling to predict the effect of amino acid mutation on binding affinity, and biophysical measurements of fluorescence resonance energy transfer microscopy to acquire dynamic interactions in living cells and in real time, and surface plasmon resonance to quantify protein-protein interactions. Aim 2 will define post- translational modifications of EBP50 that determine its function. This will be accomplished by testing the hypothesis that PTH-induced phosphorylation of EBP50 induces the closed configuration. We will apply mass spectrometry to identify site-specific EBP50 phosphorylation, and molecular biological tools with phospho-mimics and phospho-resistant EBP50 derivatives to determine their structural conformation and their actions on bone cells. Aim 3 will delineate the direct effects of EBP50 on bone to test the hypothesis that EBP50 regulates bone development and turnover. Several approaches will be applied including allograft transplantation to determine if the bone phenotype of EBP50-null mice can be rescued by transplanting marrow stem cells from wild-type mice. Other experiments will involve transfecting bone cell models with mutant EBP50 or EBP50 harboring phospho-mimics or phospho- resistant forms of EBP50 to determine how these influence PTHR action. These studies will quantitatively examine the relations between EBP50 structure and function and characterize a novel mechanism to explain the regulation and origin of EBP50 effects on bone. The findings will generate new information that is relevant to understanding bone turnover. The outcomes will help define potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases. PUBLIC HEALTH RELEVANCE: The proposed studies will test how the adapter protein EBP50 regulates parathyroid hormone action on bone. Patients with EBP50 mutations and mice lacking EBP50 have decreased bone mineral (osteomalacia). Our preliminary studies show that this results from direct effects of EBP50 in bone and not indirectly from loss of phosphate in the urine, as thought. The actions and mechanism by which EBP50 affects bone is not understood. The planned experiments will fill this gap. The outcome of our studies is highly relevant to understanding bone biology and the factors that cause osteomalacia, osteopenia, osteoporosis, and other related bone disease. The results will help define potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases.

描述（由申请人提供）：该项目的长期目标是阐明 50-kDa ezrin 结合蛋白 (EBP50) 调节骨中甲状旁腺激素受体 (PTHR) 介导的信号传导和功能的细胞机制。靶向缺失 EBP50 的小鼠表现出骨表型，EBP50 突变的患者也是如此。尽管人们认为骨骼疾病是肾功能障碍的继发后果，但我们的初步数据确定了 EBP50 对骨骼的直接影响。这表明突变干扰 EBP50 功能的新机制，并且推而广之，EBP50 在开放和闭合构象下受到 PTH 的动态调节。本提案的统一思想是 EBP50 中的新结构决定因素及其翻译后修饰决定了 EBP50 对骨中 PTHR 活性的功能。制定了三个具体目标来测试这个想法。在目标 1 中，我们将表征 EBP50 构象和二聚化，以测试以下假设：所描述的突变将 EBP50 锁定在干扰 PTHR 功能的封闭构型中。这些实验将使用分子生物学操作来检查静态相互作用，使用分子建模来预测氨基酸突变对结合亲和力的影响，并使用荧光共振能量转移显微镜的生物物理测量来实时获取活细胞中的动态相互作用，以及表面等离子体共振来量化蛋白质-蛋白质相互作用。目标 2 将定义 EBP50 的翻译后修饰，以确定其功能。这将通过测试 PTH 诱导的 EBP50 磷酸化诱导闭合构型的假设来实现。我们将应用质谱法来识别位点特异性 EBP50 磷酸化，并使用磷酸模拟物和磷酸抗性 EBP50 衍生物的分子生物学工具来确定它们的结构构象及其对骨细胞的作用。目标 3 将描述 EBP50 对骨骼的直接影响，以检验 EBP50 调节骨骼发育和更新的假设。将应用多种方法，包括同种异体移植，以确定是否可以通过移植野生型小鼠的骨髓干细胞来挽救 EBP50 缺失小鼠的骨表型。其他实验将涉及用突变型 EBP50 或含有 EBP50 磷酸模拟物或磷酸抗性形式的 EBP50 转染骨细胞模型，以确定它们如何影响 PTHR 作用。这些研究将定量研究 EBP50 结构和功能之间的关系，并表征一种新机制来解释 EBP50 对骨影响的调节和起源。这些发现将产生与了解骨转换相关的新信息。研究结果将有助于确定潜在的治疗目标，以改善骨质疏松症和其他代谢性骨疾病的治疗。 公共健康相关性：拟议的研究将测试衔接蛋白 EBP50 如何调节甲状旁腺激素对骨骼的作用。具有 EBP50 突变的患者和缺乏 EBP50 的小鼠骨矿物质减少（骨软化症）。我们的初步研究表明，这是由 EBP50 对骨骼的直接影响造成的，而不是像人们认为的那样，由尿液中磷酸盐的损失间接造成的。 EBP50 影响骨骼的作用和机制尚不清楚。计划中的实验将填补这一空白。我们的研究结果与了解骨生物学以及导致骨软化、骨质减少、骨质疏松和其他相关骨病的因素高度相关。研究结果将有助于确定潜在的治疗目标，以改善骨质疏松症和其他代谢性骨疾病的治疗。