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：
8010759
负责人：
JOHN A.A. LADIAS
金额：
$ 22.44万
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 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8010759
关键词：
Active Sites Address 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 Medicine Methods Molecular Molecular Biology Molecular Conformation Molecular Sieve Chromatography Muramidase Osteoporosis Pathology Physiology Play Proteins Purinergic P1 Receptors 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）：本提案重点关注人大麻素受体 hCB2 的晶体结构测定，hCB2 是一种 G 蛋白偶联受体 (GPCR)，在调节大麻素和内源性大麻素在人体生理学和病理学中的作用中发挥核心作用。对 GPCR 激活和随后信号传输至同源 G 蛋白的分子机制的准确结构理解是现代分子生物学和医学面临的重大挑战。该研究旨在通过关注活性和非活性状态下 hCB2 受体的晶体学分析来填补 GPCR 信号传导结构基础上的知识空白。具体目标是： 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。为了增加hCB2受体的稳定性和结晶性，其大部分胞内环3将被T4溶菌酶(T4L)取代，并且受体的C端胞质区域将被删除。优化的 hCB2-T4L 融合蛋白将在昆虫细胞中表达，并使用亲和层析、离子交换层析和尺寸排阻层析进行纯化。纯化的受体将与合成的激动剂或拮抗剂共价复合，并在脂质中间相中结晶。将使用同步加速器辐射收集所获得晶体的完整衍射数据集，并使用尖端的高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。所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开发。由于 hCB2 受体在介导内源性大麻素信号传导中发挥核心作用，因此将开发针对许多疾病的新疗法，包括炎症和神经性疼痛、肝纤维化、动脉粥样硬化和骨质疏松症。公共健康相关性：人类大麻素受体 hCB2 在介导人类生理学和病理学中的（内）大麻素信号传导方面发挥着核心作用。拟议的研究重点是利用 X 射线晶体学在原子水平上阐明 hCB2 功能的机制。对 hCB2 三维结构的准确了解将推进（内）大麻素和 GPCR 领域的研究，并将有效促进严重人类疾病（包括滥用药物和艾滋病毒）创新疗法的开发。