Structural Basis of PTH Receptor Function

项目摘要 该项目的目标是确定甲状旁腺激素(PTH)的结构机制。 受体(PTHR)信号对其功能不同的配体：PTH、PTH相关肽(PTHrP)和 长效PTH类似物(LA-PTH)。PTHR是一种主要的G蛋白偶联受体(GPCR)，调节 钙离子在血液和骨转换中的动态平衡，是治疗骨质疏松症最有效的靶点。它 也是第一个在甲状旁腺素受体内化后维持cAMP产生的gpr之一。 内体中的复合体。新近认识到的特征是PTHR对小鼠和 灵长类是由LA-PTH维持的，它也延长了内体cAMP的产生，正在改变我们的思维 关于PTHR如何调节其生理活动。这些发现中隐含的是有效的治疗 选择性靶向PTHR介导的内体cAMP可能更容易发生低钙血症 发信号。区分PTH及其类似物信号选择性的机制尚不清楚 这是进一步朝着开发基于甲状旁腺素的疗法的新方向前进的障碍，这些疗法具有 改善了治疗骨骼和矿物质疾病的疗效。因此，我们提出一项研究计划，以 克服这一障碍。因此，该项目的目标是确定PTHR的结构基础 PTH、PTHrP和LA-PTH对G蛋白的作用和激活。 为了发现PTHR信号的结构基础，我们提出了两个具体的目标。第一个目标是解决 甲状旁腺激素和LA-甲状旁腺激素通过稳定唯一cAMP促进长内体cAMP产生的假设 PTHR的结构排列和磷酸化模式不同于 PTHrP，它从质膜上诱导短暂的cAMP反应。为此，我们将使用 基于定量质谱学(MS)的蛋白质组学技术，如氢-氚交换 与MS(HDXMS)联用，以确定PTHR激活的结构变化和结构决定因素 与不同的配体结合，并鉴定PTHR-G蛋白复合体中的结合界面。第二 AIM将补充我们对PTHR功能选择性的X射线结晶学的理解 PTHR与PTH、PTHrP和LA-PTH结合。我们已经获得了全X-射线衍射晶体(4？) 与LA-PTH结合并与RNA聚合酶II形成复合体的长度PTHR。这里我们使用空腔 在RNA聚合酶II晶体中形成的“晶体海绵”，它创造了有利的结晶 PTHR的环境。我们将初步优化结晶条件，以解析其晶体结构。 PTHR与RNA聚合酶II以较高的分辨率(2~3？)结合在LA-PTH上。然后我们将重点关注 解析PTHR的PTH和PTHrP结合状态。这些研究将为PTHR如何 激活G蛋白及区分PTH、PTHrP和LA-PTH作用的结构机制。

Project Summary The goal of this project is to determine structural mechanisms by which for the parathyroid hormone (PTH) receptor (PTHR) signals in response to its functionally distinct ligands: PTH, PTH-related peptide (PTHrP) and the long-acting PTH analog (LA-PTH). The PTHR is a major G protein-coupled receptor (GPCR) that regulates Ca2+ homeostasis in blood and bone turnover, and is the most effective therapeutic target for osteoporosis. It also is one of the first GPCR found to sustain cAMP production after internalization of the PTH–receptor complex in endosomes. The recently recognized feature that the calcemic action of PTHR in mice and primates is sustained by LA-PTH, which also prolongs endosomal cAMP production, is changing our thinking about how PTHR mediates its physiological actions. Implicit in these findings is that efficient treatment of hypocalcemia might be more approachable with selective targeting of PTHR-mediated endosomal cAMP signaling. The mechanism that differentiating the signaling selectivity of PTH and its analogs are not known and this is an obstacle to further move toward new directions to develop PTH-based therapies that have improved efficacies for treating bone and mineral diseases. We therefore propose a research program to overcome this obstacle. The goal of this project is thus to determine the structural basis by which PTHR function and activate G proteins in response to PTH, PTHrP and LA-PTH. Two specific aims are proposed to discover structural basis of PTHR signaling. The first aim addresses the hypothesis that PTH and LA-PTH promote long endosomal cAMP production by stabilizing unique structural arrangements and phosphorylation pattern in the PTHR that are different from those stabilized by PTHrP, which induces a short cAMP response from the plasma membrane. To this end, we will use quantitative mass spectrometry (MS) based-proteomics technologies, such as Hydrogen-Deuterium exchange coupled to MS (HDXMS) to determine structural changes and structural determinants for PTHR activation upon binding to the distinct ligands, and identify the binding interface in PTHR–G protein complexes. The second aim will complement our understanding of the functional selectivity of PTHR by X-ray crystallography of PTHR and PTHR bound to PTH, PTHrP and LA-PTH. We have obtained X-ray diffractable crystals (4 Å) of the full length PTHR bound to LA-PTH and in complex with the RNA polymerase II. Here we are using the cavity formed in the RNA polymerase II crystal as a “crystal sponge” that creates a favorable crystallization environment for PTHR. We will initially optimize the crystallization conditions to resolve crystal structure of the PTHR bound to LA-PTH in complex with RNA polymerase II at higher resolution (2-3 Å). We will then focus of resolving PTH and PTHrP-bound states of PTHR. These studies will provide new insights into how PTHR activate G proteins and the structural mechanism differentiating the action of PTH, PTHrP and LA-PTH.