Mechanistic Basis of Calcium Sensing Receptor Signaling

钙传感受体信号传导的机制基础

• 批准号: 10467554
• 负责人: Georgios Skiniotis
• 金额: $ 60.27万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467554
• 关键词: Address; Adopted; Affect; Bartter Disease; Binding; Biochemical; Biological Assay; Biophysics; Blood; Bone Resorption; Calcitonin; Calcium; Calcium Signaling; Calcium-Sensing Receptors; Cell Membrane Proteins; Cell membrane; Cell surface; Cells; Chemosensitization; Child; Chronic Kidney Failure; Clinical; Clinical Treatment; Clinical Trials; Complement; Complex; Coupled; Couples; Coupling; Cryoelectron Microscopy; Cysteine; Defect; Development; Disease; Disease of parathyroid glands; Drug Modulation; Electrons; Engineering; Environment; Extracellular Domain; Family; Family member; Future; G-Protein-Coupled Receptors; GABA Receptor; GTP-Binding Proteins; Genetic; Health; Homeostasis; Human; Human body; Hyperactivity; Hypercalcemia; Hyperparathyroidism; Hypocalcemia; Intestines; Kidney; Kidney Calculi; Lead; Length; Ligand Binding; Ligands; Light; Link; Lipids; Maps; Medical; Metabotropic Glutamate Receptors; Minerals; Molecular Conformation; Monitor; Morbidity - disease rate; Muscle Cramp; Mutagenesis; Mutation; N-terminal; Neonatal; Osteoporosis; PTH gene; Parathyroid gland; Patients; Persons; Pharmaceutical Preparations; Pharmacology; Preparation; Proteins; Protomer; Receptor Activation; Receptor Signaling; Regulation; Role; Seizures; Side; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Spectrum Analysis; Structure; System; Tail; Therapeutic; Tissues; Transmembrane Domain; Work; absorption; base; calcium metabolism; cinacalcet; conformational conversion; design; dimer; drug discovery; experimental study; extracellular; fluorophore; gain of function; improved; insight; interest; loss of function mutation; member; mortality; nanodisk; nervous system disorder; new therapeutic target; novel therapeutics; positive allosteric modulator; protein activation; rational design; receptor; receptor function; recruit; sensor; side effect; single-molecule FRET; small molecule; targeted treatment

ABSTRACT: Mechanistic Basis of Calcium Sensing Receptor Signaling The calcium sensing receptor (CaSR) is the master regulator of calcium metabolism in human and represents an outstanding drug target for the treatment of parathyroid disorders that develop in patients with chronic kidney diseases (CKDs). For patients with renal disfunction that develop hyperparathyroidism, calcimimetic drugs that act as positive allosteric modulators (PAMs) of the CaSR are the favored therapeutics. PAMs, such as cinacalcet, evocalcet and etelcalcetide, are approved treatment for CKD; however, their clinical use is limited due to their adverse side effects. By elucidating the dynamic structural mechanisms of receptor activation, its modulation by small-molecule modulators, and the specificity of G protein activation, we seek to understand in detail the CaSR signaling mechanism and enable the rational design of improved therapeutics modulating receptor function. CaSR is a family C member of G protein-coupled receptors (GPCRs), which also include the metabotropic glutamate receptors (mGlus) and the metabotropic gamma aminobutyric acid receptor (GABAB). Like other members of this family, CaSR functions as an obligate homodimer with an N-terminal extracellular domain (ECD) responsible for ligand binding, linked to the seven-transmembrane (7TM) domain. We have recently determined cryo-electron microscopy (cryoEM) structures of the near-full-length human CaSR homodimer in active and inactive states, revealing how ECD rearrangement upon Ca2+ binding induces the activation of the 7TMs and how allosteric modulators engage the receptor. Our results illustrate an essential asymmetry in the active state where each CaSR protomer is stabilized by a PAM molecule bound to each 7TM in two distinct conformations leading to the activation of only one transmembrane region, priming it for G protein coupling. Here we propose to extend these studies in order to characterize the mechanism and specificity of G protein activation by CaSR, its dynamics, as well as the detailed action of allosteric modulators with distinct pharmacological interest. Specifically, we seek to apply: structure-based mutagenesis coupled with cell signaling assays that monitor the effects of allosteric modulators; cryoEM structural studies of CaSR alone and in complex with allosteric modulators and distinct G proteins in a near native lipid environment; and single-molecule fluorescence resonance energy transfer (smFRET) complemented by double electron-electron (DEER) spectroscopy to reveal the dynamics of receptor and G protein activation as well as its modulation by different allosteric ligands. Collectively, the proposed structural, cellular, biochemical and biophysical experiments aim to provide a full mechanistic framework for transmembrane signaling by CaSR and will guide the future development of novel drugs targeting this receptor.

摘要：钙传感受体信号传导的机制基础 钙敏感受体（CaSR）是人体钙代谢的主要调节因子，代表 治疗慢性肾病患者发生的甲状旁腺疾病的杰出药物靶点 疾病（CKD）。对于出现甲状旁腺功能亢进的肾功能障碍患者，可使用拟钙药物 作为 CaSR 的正变构调节剂 (PAM) 是受欢迎的治疗方法。 PAM，例如西那卡塞， evocalcet 和 etelcalcetide 被批准用于治疗 CKD；然而，它们的临床应用由于其 不良副作用。通过阐明受体激活的动态结构机制，其调节通过 小分子调节剂和 G 蛋白激活的特异性，我们寻求详细了解 CaSR 信号机制并能够合理设计改进的调节受体功能的疗法。 CaSR 是 G 蛋白偶联受体 (GPCR) 的 C 家族成员，该受体还包括代谢型受体 谷氨酸受体（mGlus）和代谢型γ氨基丁酸受体（GABAB）。和其他人一样 作为该家族的成员，CaSR 作为具有 N 末端细胞外结构域 (ECD) 的专性同二聚体发挥作用 负责配体结合，与七跨膜 (7TM) 结构域相连。我们最近确定 活性和近全长人 CaSR 同二聚体的冷冻电子显微镜 (cryoEM) 结构 非活性状态，揭示了 Ca2+ 结合后 ECD 重排如何诱导 7TM 的激活和 变构调节剂如何与受体结合。我们的结果说明了活跃状态下的本质不对称性 其中每个 CaSR 原聚体均由以两种不同构象与每个 7TM 结合的 PAM 分子稳定 导致仅激活一个跨膜区域，从而启动 G 蛋白偶联。在这里我们建议 扩展这些研究，以表征 CaSR 激活 G 蛋白的机制和特异性， 其动力学，以及具有独特药理学意义的变构调节剂的详细作用。 具体来说，我们寻求应用：基于结构的诱变与监测细胞信号转导分析相结合。 变构调节剂的作用； CaSR 单独和与变构复合物的冷冻电镜结构研究 接近天然脂质环境中的调节剂和独特的 G 蛋白；和单分子荧光 共振能量转移 (smFRET) 辅以双电子 - 电子 (DEER) 光谱来揭示 受体和 G 蛋白激活的动力学及其通过不同变构配体的调节。 总的来说，所提出的结构、细胞、生物化学和生物物理实验旨在提供完整的 CaSR 跨膜信号传导的机制框架，并将指导新型药物的未来发展 靶向该受体的药物。