Mechanistic Basis of Calcium Sensing Receptor Signaling

钙传感受体信号传导的机制基础

• 批准号: 10596176
• 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/10596176
• 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; Heterotrimeric GTP-Binding Proteins; Homeostasis; Human; Human body; Hyperactivity; Hypercalcemia; Hyperparathyroidism; Hypocalcemia; Intestines; Kidney; Kidney Calculi; Lead; Length; Ligand Binding; Ligands; Link; Lipids; Maps; Medical; Membrane; Metabotropic Glutamate Receptors; Minerals; Molecular Conformation; Monitor; Morbidity - disease rate; Muscle Cramp; Mutagenesis; Mutation; N-terminal; Neonatal; Osteoporosis; PTH gene; Parathyroid gland; Patients; Persons; 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; autosome; calcium metabolism; cinacalcet; conformational conversion; design; dimer; drug action; drug discovery; experimental study; extracellular; fluorophore; gain of function; improved; insight; interest; kidney dysfunction; loss of function mutation; member; mortality; nanodisk; nervous system disorder; new therapeutic target; novel therapeutics; pharmacologic; 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：钙敏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， 治疗慢性肾脏病患者发生的甲状旁腺疾病的突出药物靶点 疾病（CKD）。对于出现甲状旁腺功能亢进的肾功能不全患者， 作为CaSR的正向变构调节剂（PAM）是有利的治疗剂。PAM，如西那卡塞， evocalcet和etelcalcetide是获批的CKD治疗药物;然而，由于其 不良副作用通过阐明受体激活的动态结构机制， 小分子调节剂和G蛋白激活的特异性，我们试图详细了解CaSR 信号传导机制，并且能够合理设计调节受体功能的改进的治疗剂。 CaSR是G蛋白偶联受体（GPCR）的C家族成员，其还包括促代谢性CaSR。 谷氨酸受体（mGlus）和代谢型γ氨基丁酸受体（GABAB）。像其他 作为该家族的成员，CaSR作为具有N-末端胞外结构域（ECD）的专性同源二聚体发挥功能， 负责配体结合，连接到七跨膜（7 TM）结构域。我们最近确定 活性和非活性CaSR同源二聚体的近全长人CaSR同源二聚体的冷冻电镜（cryoEM）结构 非活性状态，揭示了在Ca 2+结合时ECD重排如何诱导7 TM的活化， 变构调节剂如何与受体结合。我们的结果说明了一个本质的不对称性，在活跃状态 其中每个CaSR原聚体由以两种不同构象结合到每个7 TM的PAM分子稳定 导致仅激活一个跨膜区，使其启动G蛋白偶联。在这里我们建议 为了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，以及具有不同药理学兴趣的变构调节剂的详细作用。 具体来说，我们寻求应用：基于结构的诱变与细胞信号转导分析相结合，监测细胞的增殖。 别构调节剂的作用; CaSR单独和与别构调节剂复合的冷冻电镜结构研究 调节剂和不同的G蛋白在一个接近天然的脂质环境;和单分子荧光 共振能量转移（smFRET）补充双电子-电子（DEER）光谱，以揭示 受体和G蛋白激活的动力学以及不同别构配体对其的调节。 总的来说，拟议的结构、细胞、生物化学和生物物理实验旨在提供一个完整的 CaSR跨膜信号传导的机制框架，并将指导未来新的 针对该受体的药物。