Structural Basis for Cannabinoid Receptor 2 Signaling

大麻素受体 2 信号传导的结构基础

基本信息

批准号：
8109386
负责人：
JOHN A.A. LADIAS
金额：
$ 19.98万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-15 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8109386
关键词：
Active Sites Address Adenosine A2A Receptor Adopted Adrenergic Receptor Affinity Agonist Atherosclerosis Award Basic Science Binding Biomedical Research C-terminal Cannabinoids Cells Chimeric Proteins Complex Crystal Formation Crystallization Crystallography Data Set Development Disease Drug Addiction Endocannabinoids Engineering Equilibrium Experimental Designs Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Goals HIV Health Heterogeneity Human In Vitro Insecta Ion Exchange Knowledge Laboratories Ligands Liver Fibrosis Masks Mediating Medical Methods Molecular Molecular Biology Molecular Conformation Molecular Medicine Molecular Sieve Chromatography Muramidase Osteoporosis Pathology Physiology Play Proteins Receptor Activation Receptor Inhibition Research Role Signal Transduction Structure Tail Therapeutic X-Ray Crystallography base cannabinoid receptor covalent bond drug discovery drug of abuse flexibility human disease in vivo inflammatory neuropathic pain innovation meetings novel strategies public health relevance receptor synchrotron radiation three dimensional structure transmission process

项目摘要

DESCRIPTION (provided by applicant): This proposal focuses on the crystal structure determination of the human cannabinoid receptor hCB2, a G protein-coupled receptor (GPCR) that plays a central role in mediating the effects of cannabinoids and endocannabinoids in human physiology and pathology. An accurate structural understanding of the molecular mechanisms underlying GPCR activation and subsequent signal transmission to the cognate G proteins represents a major challenge for modern molecular biology and medicine. The proposed research aims at filling the knowledge gap on the structural basis of GPCR signaling by focusing on the crystallographic analysis of the hCB2 receptor in the active and inactive states. The specific aims are: 1) To determine the crystal structure of the hCB2 receptor in the active state. 2) To elucidate the structural basis for hCB2 receptor inactivation. To increase the stability and crystallizability of the hCB2 receptor, most of its intracellular loop 3 will be replaced with the T4 lysozyme (T4L) and the C-terminal cytoplasmic region of the receptor will be deleted. The optimized hCB2-T4L fusion protein will be expressed in insect cells and purified using affinity, ion exchange, and size exclusion chromatography. The purified receptor will be covalently complexed with a synthetic agonist or antagonist, and will be crystallized in lipidic mesophases. Complete diffraction data sets of the obtained crystals will be collected using synchrotron radiation and the structures will be determined using cutting-edge macromolecular crystallography methods. The successful completion of these studies will have a major impact on biomedical research and human health. The obtained structural knowledge will greatly facilitate the development of hCB2-selective agonists, partial agonists, and antagonists. Because of the central role of the hCB2 receptor in mediating endocannabinoid signaling, new treatments will be developed for a number of medical conditions, including inflammatory and neuropathic pain, hepatic fibrosis, atherosclerosis, and osteoporosis. PUBLIC HEALTH RELEVANCE: The human cannabinoid receptor hCB2 plays a central role in mediating the (endo)cannabinoid signaling in human physiology and pathology. The proposed research focuses on the elucidation of the mechanisms underlying hCB2 function at the atomic level using X-ray crystallography. Accurate knowledge of the three- dimensional structure of hCB2 will advance the research in the (endo)cannabinoid and GPCR fields, and will effectively facilitate the development of innovative therapeutics for serious human diseases, including drugs of abuse and HIV.

描述（由申请人提供）：该提案的重点是人类大麻素受体HCB2的晶体结构测定，该受体HCB2是G蛋白偶联受体（GPCR），在介导大麻素和内源性诺碱在人体生理学和病理学中的影响中起着核心作用。对GPCR激活和随后向同源G蛋白传播的分子机制的准确理解是对现代分子生物学和医学的主要挑战。拟议的研究旨在通过重点关注主动状态和非活动状态中HCB2受体的晶体学分析来填补GPCR信号传导的结构基础的知识差距。具体目的是：1）确定活性状态下HCB2受体的晶体结构。 2）阐明HCB2受体失活的结构基础。为了提高HCB2受体的稳定性和结晶性，其大部分细胞内环3将被T4溶菌酶（T4L）取代，并将删除受体的C末端细胞质区域。优化的HCB2-T4L融合蛋白将在昆虫细胞中表达，并使用亲和力，离子交换和大小排除色谱法纯化。纯化的受体将与合成的激动剂或拮抗剂共价络合，并将在脂质中间酶中结晶。将使用同步加速器辐射收集获得的晶体的完整衍射数据集，并使用尖端的大分子晶体学方法确定结构。这些研究的成功完成将对生物医学研究和人类健康产生重大影响。获得的结构知识将极大地促进HCB2选择性激动剂，部分激动剂和拮抗剂的发展。由于HCB2受体在介导内源性大麻素信号传导中的核心作用，因此将针对多种医学疾病（包括炎症性和神经性疼痛，肝纤维化，动脉粥样硬化和骨质疏松症）开发新的治疗方法。公共卫生相关性：人类大麻素受体HCB2在介导（ENDO）大麻素信号传导中在人类生理和病理学中起着核心作用。提出的研究重点是使用X射线晶体学在原子水平上阐明HCB2功能的机制。对HCB2的三维结构的准确知识将推进（内部）大麻素和GPCR领域的研究，并将有效地促进针对严重人类疾病的创新治疗剂的开发，包括虐待和HIV药物。