EBP50 Regulation of PTH Receptor in Bone

描述(申请人提供)：这个项目的长期目标是阐明50 kDa Ezrin结合蛋白(EBP50)调节甲状旁腺激素受体(PTHR)介导的信号和骨骼功能的细胞机制。EBP50靶向缺失的小鼠表现出骨表型，EBP50突变的患者也是如此。虽然认为骨病是肾功能不全的继发性后果，但我们的初步数据证实了EBP50对骨骼的直接影响。这提示了一种新的机制，即突变干扰EBP50的功能，进而，EBP50在开放和闭合构象中受到PTH的动态调节。本方案的统一思想是EBP50中新的结构决定因素及其翻译后修饰决定了EBP50对骨骼中PTHR活性的作用。为了测试这一想法，我们制定了三个具体目标。在目标1中，我们将表征EBP50的构象和二聚化，以检验所描述的突变将EBP50锁定在干扰PTHR功能的封闭构型中的假设。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，以及使用表面等离子体共振来量化蛋白质与蛋白质的相互作用。AIM 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EBP50磷酸化导致闭合构型的假设来实现。我们将应用质谱学来鉴定特定部位的EBP50磷酸化，以及分子生物学工具与磷酸模拟物和耐磷EBP50衍生物，以确定它们的结构构象及其对骨细胞的作用。目的3描述EBP50对骨骼的直接影响，以验证EBP50调节骨骼发育和转换的假说。将采用几种方法，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EBP50缺失小鼠的骨表型。其他实验将涉及将突变的EBP50或EBP50突变的EBP50导入骨细胞模型，以确定这些因素如何影响PTHR的作用。这些研究将定量研究EBP50的结构和功能之间的关系，并描述一种新的机制来解释EBP50对骨骼的影响的调节和来源。这些发现将产生与了解骨骼转换相关的新信息。研究结果将有助于确定改善骨质疏松症和其他代谢性骨骼疾病治疗的潜在治疗目标。 公共卫生相关性：拟议的研究将测试接头蛋白EBP50如何调节甲状旁腺激素对骨骼的作用。EBP50突变的患者和缺乏EBP50的小鼠的骨矿物质减少(骨软化症)。我们的初步研究表明，这是由于EBP50在骨骼中的直接作用，而不是像人们认为的那样，间接导致尿磷丢失。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和导致骨软化、骨量减少、骨质疏松症和其他相关骨骼疾病的因素高度相关。这一结果将有助于确定改善骨质疏松症和其他代谢性骨骼疾病治疗的潜在治疗目标。

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.