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：钙敏感受体信号转导的机制基础 钙敏感受体(CaSR)是人体钙代谢的主要调节器，代表着 治疗慢性肾脏病患者甲状旁腺疾病的杰出药物靶点 疾病(CKD)。对于患有甲状旁腺功能亢进症的肾功能不全患者，拟钙药物 作为CASR的正性变构调节剂(PAM)是目前治疗的热点。PAMs，如Cinacalcet， Evocalcet和etelcalcetie是被批准的治疗慢性肾脏病的药物；然而，由于它们的临床应用受到限制。 不良副作用。通过阐明受体激活的动态结构机制，它的调节 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 信号转导机制，并使合理设计改进的治疗药物调节受体功能。 CaSR是G蛋白偶联受体(GPCRs)的C家族成员，也包括代谢性受体 谷氨酸受体(MGlus)和代谢性γ-氨基丁酸受体(GABAB)。像其他人一样 作为这个家族的成员，CaSR作为具有N端胞外域(ECD)的专性同源二聚体发挥作用。 负责配体结合，连接到七跨膜(7TM)结构域。我们最近确定了 人近全长CaSR同源二聚体在活性和非激活状态下的冷冻电子显微镜结构 非活性状态，揭示了钙结合时ECD重排是如何诱导7TM激活的，以及 变构调节剂如何与受体结合。我们的结果说明了有源态的本质不对称性。 其中每个CaSR原构体由以两种不同构象结合到每个7TM上的PAM分子稳定 导致只有一个跨膜区被激活，为G蛋白偶联做准备。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， 它的动态，以及具有不同药理意义的变构调节剂的详细作用。 具体地说，我们试图应用：基于结构的突变与细胞信号分析相结合，监测 变构调节剂的作用；CaSR单独的和与变构的络合物的低温电子显微镜结构研究 在接近天然脂质环境中的调节剂和不同的G蛋白；以及单分子荧光 共振能量转移(SmFRET)与双电子-电子(DER)光谱互补揭示 受体和G蛋白激活的动态以及不同变构配体对其的调节。 总的来说，拟议的结构、细胞、生化和生物物理实验旨在提供完整的 CASR跨膜信号转导的机制框架，并将指导未来的发展 针对这种受体的药物。