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：钙传感受体信号的机理基础 钙传感受体（CASR）是人类钙代谢的主要调节剂，代表 慢性肾脏患者发展的甲状旁腺疾病的杰出药物靶标 疾病（CKD）。对于患有甲状旁腺功能亢进症的肾功能不全的患者，钙化药物 充当CASR的阳性变构调节剂（PAM）是受欢迎的治疗剂。 pam，例如cinacalcet， Evocalcet和Etelcalcetide被批准用于CKD；但是，由于他们的临床使用受到限制 不良副作用。通过阐明受体激活的动态结构机制，其调节 小分子调节剂以及G蛋白激活的特异性，我们试图详细了解CASR 信号传导机制，并使改进的治疗剂调节受体功能的合理设计。 CASR是G蛋白偶联受体（GPCR）的家族成员，其中还包括代谢型 谷氨酸受体（MGLUS）和代谢性γ氨基丁酸受体（GABAB）。像其他 CASR的成员是具有N末端细胞外域（ECD）的强制同型二聚体（ECD） 负责配体结合，与七跨膜（7TM）结构域相连。我们最近确定了 活性和活性和 不活跃的状态，揭示了Ca2+结合上的ECD重排如何诱导7TM和 变构调节剂如何使受体接合。我们的结果说明了活跃状态下必不可少的不对称性 其中每个CASR原始物通过与每个7tm结合的PAM分子以两个不同的构象稳定 导致仅一个跨膜区域的激活，将其用于G蛋白偶联。我们在这里提出 为了扩展这些研究以表征CASR的G蛋白激活的机制和特异性， 它的动态以及具有独特药理兴趣的变构调节剂的详细作用。 具体而言，我们寻求应用：基于结构的诱变以及监测的细胞信号传导测定法 变构调节剂的影响； CASR的冷冻结构研究单独且具有变构的复杂性 调节剂和在接近天然脂质环境中的不同G蛋白；和单分子荧光 共振能量转移（SMFRET）与双电子电子（鹿）光谱相辅相成 受体和G蛋白激活的动力学以及不同的变构配体的调节。 总体而言，提出的结构，细胞，生化和生物物理实验旨在提供完整的 CASR的跨膜信号的机理框架，将指导新颖的未来发展 针对该受体的药物。