Structural Basis of PTH Receptor Function

PTH 受体功能的结构基础

• 批准号: 9895802
• 负责人: Jean-Pierre Vilardaga
• 金额: $ 42.93万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895802
• 关键词: Address; Affinity; Amino Acids; Animals; Behavior; Binding; Biological; Biomedical Research; Blood; Calcium; Cell Culture Techniques; Cell membrane; Chemicals; Complement; Complex; Coupled; Crystallization; Crystallography; Cyclic AMP; Deuterium; Development; Disease; Early Endosome; Endosomes; Environment; Foundations; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Goals; Health; Homeostasis; Human; Hydrogen; Hypocalcemia result; Hypoparathyroidism; Ions; Length; Ligand Binding; Ligands; Mass Spectrum Analysis; Mediating; Medical; Minerals; Molecular; Molecular Conformation; Mus; Mutagenesis; Osteoporosis; PTH gene; Parathyroid Hormone Receptor; Patients; Pattern; Peptides; Pharmacology; Phosphorylation; Physiological; Porifera; Primates; Process; Production; Property; Proteomics; RNA Polymerase II; Receptor Activation; Receptor Signaling; Research; Resolution; Roentgen Rays; Side; Signal Transduction; Stable Isotope Labeling; Structure; Technology; Testing; Thinking; Treatment Efficacy; X-Ray Crystallography; analog; base; bone; bone turnover; calcium phosphate; clinically relevant; crosslink; design; hormone analog; improved; insight; mutant; parathyroid hormone-related protein; programs; receptor; receptor binding; receptor function; receptor internalization; response; small molecule; therapeutic target

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.

项目摘要 该项目的目的是确定甲状旁腺（PTH）的结构机制 受体（PTHR）信号响应其功能不同的配体：PTH，PTH相关肽（PTHRP）和 长效PTH类似物（LA-PTH）。 PTHR是一种调节的主要G蛋白偶联受体（GPCR） 血液和骨骼更新中的Ca2+稳态，是骨质疏松症的最有效治疗靶点。它 同样是PTH - 受体内在化后，首先发现cAMP生产的GPCR之一 内体中的复合物。最近公认的特征，PTHR在小鼠和 灵长类动物由LA-PTH持续，该LA-PTH也延长了内体阵营的生产，正在改变我们的思想 关于PTHR如何介导其身体行为。这些发现中隐含的是对 通过选择性靶向PTHR介导的内体cAPP，低钙血症可能更容易接近 信号。尚不清楚区分PTH及其类似物的信号传导选择性的机制 这是进一步发展新方向以开发具有PTH的疗法的障碍 改善治疗骨骼和矿物质疾病的效率。因此，我们建议一个研究计划 克服这个障碍。因此，该项目的目的是确定PTHR的结构基础 响应PTH，PTHRP和LA-PTH的功能并激活G蛋白。 提出了两个具体的目的，以发现PTHR信号的结构基础。第一个目的解决 PTH和LA-PTH通过稳定独特而促进长内体cAMP生产的假设 PTHR中的结构排列和磷酸化模式与稳定的结构排列和磷酸化模式不同 PTHRP，引起了质膜的短暂cAMP反应。为此，我们将使用 基于定量质谱（MS）的蛋白质组学技术，例如氢欧交换 耦合到MS（HDXM），以确定结构性变化和结构确定剂，以进行PTHR激活 与不同的配体结合，并鉴定PTHR -G蛋白复合物中的结合界面。第二个 AIM将通过PTHR的X射线晶体学完成我们对PTHR功能选择性的理解 PTHR绑定到PTH，PTHRP和LA-PTH。我们获得了完整的X射线衍射晶体（4Å） 长度PTHR与LA-PTH结合并与RNA聚合酶II复合体。在这里，我们正在使用腔 在RNA聚合酶II晶体中形成，作为“晶体赞助商”，产生有利的结晶 PTHR的环境。我们最初将优化结晶条件，以解决 PTHR与RNA聚合酶II在较高分辨率（2-3Å）的复合物中结合到LA-PTH。然后我们将集中精力 解决PTHR的PTH和PTHRP结合状态。这些研究将为PTHR提供新的见解 激活G蛋白和结构机制，从而区分了PTH，PTHRP和LA-PTH的作用。