Structural Basis for Cannabinoid Receptor 2 Signaling

描述（由申请人提供）：该提案侧重于人类大麻素受体hCB 2的晶体结构测定，hCB 2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类生理学和病理学中的作用方面发挥核心作用。对GPCR激活和随后的信号传递到同源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。拟议的研究旨在填补知识空白的GPCR信号的结构基础上，通过专注于在活性和非活性状态的hCB 2受体的晶体学分析。具体目的是：1）确定活性状态下hCB 2受体的晶体结构。2)阐明hCB 2受体失活的结构基础。为了增加hCB 2受体的稳定性和可结晶性，其大部分胞内环3将被T4溶菌酶（T4L）取代，并且受体的C-末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，并将在介晶中间相中结晶。将使用同步辐射收集所获得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。所获得的结构知识将极大地促进hCB 2选择性激动剂、部分激动剂和拮抗剂的开发。由于hCB 2受体在介导内源性大麻素信号传导中的核心作用，将开发用于许多医学病症的新治疗方法，包括炎性和神经性疼痛、肝纤维化、动脉粥样硬化和骨质疏松症。 公共卫生相关性：人大麻素受体hCB 2在介导人体生理学和病理学中的（内）大麻素信号传导中起着核心作用。拟议的研究重点是使用X射线晶体学在原子水平上阐明hCB2功能的机制。对hCB 2三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HIV）的创新疗法的开发。

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.