Structural Mechanisms of parathyroid hormone receptor signaling through Gs

项目摘要 甲状旁腺激素（PTH）1型受体（PTHR）是骨转换和钙的关键调节因子 体内平衡PTHR是一种G蛋白偶联受体（GPCR），可激活多种G蛋白，包括 刺激性Gs，其随后激活腺苷酸环化酶并诱导cAMP的产生。 以前的研究表明，通过Gs的PTHR信号是骨愈合的主要介质。 因此，了解PTHR-Gs信号通路对于设计治疗骨和矿物离子损伤的新药至关重要 疾病，如骨质疏松症和低钙血症。 两种FDA批准的骨质疏松症治疗药物，PTH（1 - 34）（teriparbal或PTH）和改良的PTHrP（1 - 34） （abaloparterine或ABL），触发Gs/cAMP信号传导的不同模式。两种配体均刺激瞬时cAMP 在质膜上生产。此外，PTH刺激内体中cAMP的产生延长。 合成长效PTH（LA-PTH）比PTH更能促进内体Gs信号传导， 小鼠和灵长类动物的血钙水平。因此，LA-PTH是一种有前途的治疗低钙血症的药物。 虽然以前已经研究了PTH、LA-PTH和ABL的细胞效应，但分子机制 通过这三种配体中的每一种的偏置信号传导是未知的。在研究中，我们将 从两个方面研究PTHR-Gs信号通路的分子细节。在目标1中，我们将确定原子 通过冷冻电子显微镜观察配体结合的PTHR-Gs复合物的结构。这些结构将揭示 PTHR-Gs的PTH-、LA-PTH-和ABL-特异性状态。在目标2中，我们将确定 配体选择性PTHR信号传导。我们将使用氢氘交换（HDX）和化学交联 (CX)偶联到质谱法（MS），以在PTH、LA-1存在下鉴定PTHR-Gs复合物界面。 此外，HDXMS将揭示PTHR-Gs相互作用的结构动力学。数据 将用于设计PTHR突变体，其信号传导将在活细胞中进行测试。 从这两个目标收集的结构和功能数据将深入了解不同的PTHR-G 瞬时和长期cAMP生产所必需的构象。这些数据将用于今后的工作 设计治疗骨质疏松症和其他骨骼疾病的新型疗法。因此，拟议的研究支持 国家关节炎、肌肉骨骼和皮肤病研究所（NIAMS）的使命。

Project Abstract The parathyroid hormone (PTH) type 1 receptor (PTHR) is a key regulator of bone turnover and calcium homeostasis. PTHR is a G protein-coupled receptor (GPCR) that activates multiple G proteins including stimulatory Gs, which subsequently activates adenylate cyclases and induces the production of cAMP. Previous research has suggested that PTHR signaling through Gs is the major mediator of bone anabolism. Therefore, understanding PTHR-Gs signaling is critical for designing novel drugs to treat bone and mineral-ion diseases, such as osteoporosis and hypocalcemia. Two FDA-approved therapeutics for osteoporosis, PTH(1-34) (teriparatide or PTH) and modified PTHrP(1–34) (abaloparatide or ABL), trigger distinct modes of Gs/cAMP signaling. Both ligands stimulate transient cAMP production at the plasma membrane. In addition, PTH stimulates prolonged cAMP production in endosomes. Synthetic long-acting PTH (LA-PTH) promotes endosomal Gs signaling more than does PTH and increases blood calcium levels in mice and primates. Therefore, LA-PTH is a promising therapeutic for hypocalcemia. While the cellular effects of PTH, LA-PTH, and ABL have been previously studied, the molecular mechanisms of biased signaling through each of these three ligands are not known. In the proposed research, we will investigate the molecular details of PTHR-Gs signaling through two aims. In Aim 1, we will determine atomic structures of ligand-bound PTHR-Gs complexes via cryo-electron microscopy. These structures will reveal PTH-, LA-PTH-, and ABL-specific states of PTHR-Gs. In Aim 2, we will identify structural determinants of ligand-selective PTHR signaling. We will use hydrogen-deuterium exchange (HDX) and chemical crosslinking (CX) coupled to mass spectrometry (MS) to identify PTHR-Gs complex interfaces in the presence of PTH, LA- PTH, and ABL. Furthermore, HDXMS will reveal the structural dynamics of PTHR-Gs interactions. The data from HDXMS and CXMS will be used to design PTHR mutants, whose signaling will be tested in live cells. Structural and functional data gathered from both aims will give insight into the distinct PTHR-Gs conformations necessary for transient and prolonged cAMP production. These data will be used in future work to design novel therapies for osteoporosis and other bone diseases. As such, the proposed research supports the mission of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS).