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)是骨转换和钙离子的关键调节因子 动态平衡。PTHR是一种G蛋白偶联受体，可激活多种G蛋白，包括 刺激性Gs，随后激活腺苷环化酶并诱导cAMP的产生。 以往的研究表明，PTHR通过Gs信号是骨合成代谢的主要调节因子。 因此，了解PTHR-Gs信号转导机制对于设计治疗骨和矿物质离子的新药至关重要。 骨质疏松症和低钙血症等疾病。 FDA批准的两种治疗骨质疏松症的药物，PTH(1-34)(Teriparatide或PTH)和改良的PTHrP(1-34) (abaloparatide或ABL)，触发Gs/cAMP信号的不同模式。两种配体都能刺激一过性cAMP 在质膜上生产。此外，甲状旁腺激素还能刺激内吞体内长时间产生cAMP。 合成长效甲状旁腺素(LA-PTH)比PTH更能促进内体Gs信号转导 老鼠和灵长类动物的血钙水平。因此，LA-PTH是一种很有前途的治疗低血钙症的药物。 虽然甲状旁腺激素、LA-甲状旁腺激素和ABL的细胞作用已有研究，但其分子机制 通过这三种配体中的每一种都有多少偏向的信号尚不清楚。在拟议的研究中，我们将 通过两个AIMS研究PTHR-Gs信号转导的分子细节。在目标1中，我们将确定原子 用冷冻电子显微镜研究配体结合的PTHR-Gs络合物的结构。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，我们将确定以下结构性决定因素 配体选择性PTHR信号转导。我们将使用氢-氚交换(HDX)和化学交联 (CX)与质谱仪(MS)联用，以在PTH、LA-G存在的情况下鉴定PTHR-Gs复合界面 Pth和ABL.此外，HDXMS将揭示PTHR-Gs相互作用的结构动力学。数据 HDXMS和CXMS将被用于设计PTHR突变体，其信号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使我们深入了解不同的PTHR-Gs 短暂和长期产生cAMP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病的新疗法。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).