Structural Basis for Cannabinoid Receptor 2 Signaling

描述（由申请人提供）：本提案侧重于确定人类大麻素受体hCB2的晶体结构，hCB2是一种G蛋白偶联受体（GPCR），在介导大麻素和内源性大麻素在人体生理和病理中的作用中起核心作用。对GPCR激活和随后信号传递到同源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的主要挑战。本研究旨在通过对hCB2受体在活性和非活性状态下的晶体学分析，填补关于GPCR信号传导结构基础的知识空白。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。为了增加hCB2受体的稳定性和结晶性，其胞内环3大部分将被T4溶菌酶（T4L）取代，受体的c端细胞质区将被删除。优化后的hCB2-T4L融合蛋白将在昆虫细胞中表达，并通过亲和层析、离子交换层析和大小隔离层析进行纯化。纯化后的受体将与合成的激动剂或拮抗剂共价络合，并在脂质中间期结晶。所获得晶体的完整衍射数据集将使用同步辐射收集，结构将使用尖端的大分子晶体学方法确定。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的核心作用，新的治疗方法将被开发用于许多疾病，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。

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.