EBP50 Regulation of PTH Receptor in Bone

描述（由申请人提供）：本项目的长期目标是阐明50-kDa ezrin结合蛋白（EBP 50）调节甲状旁腺激素受体（PTHR）介导的信号传导和骨功能的细胞机制。靶向缺失EBP 50的小鼠表现出骨表型，EBP 50突变的患者也是如此。尽管人们认为骨疾病是肾功能不全的继发性后果，但我们的初步数据确定了EBP 50对骨的直接影响。这表明突变干扰EBP 50功能的一种新机制，并且通过扩展，EBP 50在开放和闭合构象中受到PTH的动态调节。本建议的统一思想是，EBP 50及其翻译后修饰的新结构决定因素决定了EBP 50对骨中PTHR活性的功能。三个具体的目标来测试这个想法。在目标1中，我们将表征EBP 50构象和二聚化以测试所述突变将EBP 50锁定在干扰PTHR功能的闭合构型中的假设。这些实验将使用分子生物学演习来检查静态相互作用，分子建模来预测氨基酸突变对结合亲和力的影响，以及荧光共振能量转移显微镜的生物物理测量来获得活细胞和真实的时间中的动态相互作用，以及表面等离子体共振来量化蛋白质-蛋白质相互作用。目的2将定义决定其功能的EBP 50的翻译后修饰。这将通过检验PTH诱导的EBP 50磷酸化诱导闭合构型的假设来实现。我们将应用质谱法来鉴定位点特异性EBP 50磷酸化，并使用磷酸模拟物和磷酸抗性EBP 50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。目的3描述EBP 50对骨的直接作用，以验证EBP 50调节骨发育和转换的假说。将应用几种方法，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EBP 50缺失小鼠的骨表型。其他实验将涉及用突变型EBP 50或具有磷酸模拟物或磷酸抗性形式的EBP 50的EBP 50来检测骨细胞模型，以确定这些如何影响PTHR作用。这些研究将定量研究EBP 50结构和功能之间的关系，并描述一种新的机制来解释EBP 50对骨的作用的调节和起源。这些发现将产生与理解骨转换相关的新信息。这些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病的治疗。

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.