Structure and Function of CB2 Cannabinoid Receptor

描述（由申请人提供）：本研究提案的长期目标是确定CB 2大麻素受体功能的分子基础。为实现这一目标，将采取以下步骤。在目标1和2中，将进行相互作用诱变、分子建模和新化合物测试研究。这种结合的方法促进了我们的理解，并应继续揭示大麻素配体如何被其受体识别，受体亚型选择性的起源以及这些受体如何被激活/失活的新见解。大麻素受体与其他研究充分的G蛋白偶联受体（GPCR）（如视紫红质和生物胺受体）中的许多保守序列基序相同。然而，在大麻素受体中，存在于许多其他GPCR中的几个螺旋弯曲残基和基序以及关键的二硫键缺失。目的3：利用取代半胱氨酸可及性方法，对CB 2大麻素受体两个跨膜结构域的配体结合缝隙暴露进行定位。这种方法应阐明大麻素受体的这些transmembrane域的序列分歧的结构和功能的后果。 在目标4中，CB 2受体的二硫键形成将直接通过最先进的质谱方法来确定。研究CB 2受体独特的二硫键或不存在二硫键将提供重要的螺旋折叠信息，这将对该受体的配体结合裂缝和激活机制具有重要意义。最后，在目标5中，将致力于过量表达和纯化大量功能性CB 2受体。这项具有挑战性的任务，如果成功完成，将产生重大影响，为未来的高分辨率生物物理研究提供急需的纯受体蛋白。总之，本研究有助于我们从分子水平更详细地了解CB 2受体的结构和功能。CB 2主要分布在免疫系统中。它对大麻的免疫调节作用非常重要。从长远来看，这项研究还应该帮助我们开发更好的大麻素模拟物，用于治疗免疫系统疾病，如炎症和自身免疫性疾病。专门针对CB 2的药物应该没有大麻的副作用。

DESCRIPTION (provided by applicant): The long-term goal of this research proposal is to identify the molecular basis underlying the functions of the CB2 cannabinoid receptor. To achieve this purpose, the following steps will be taken. In Aim 1 and 2, the interactive mutagenesis, molecular modeling and new compound testing studies will be carried out. This combined approach has advanced our understanding, and should continue to reveal new insights on how cannabinoid ligands are recognized by their receptors, the origin of receptor subtype-selectivity, and how these receptors are activated/inactivated. Cannabinoid receptors share many of the conserved sequence motifs seen in other well-studied G protein-coupled receptors (GPCRs), such as rhodopsin and biogenic amine receptors. However, several helix-bending residues and motifs, and a key disulfide bridge present in many other GPCRs are missing in cannabinoid receptors. In Aim 3, Ligand binding crevice exposure of two transmembrane domains of CB2 cannabinoid receptor will be mapped using substituted-cysteine accessibility method. This approach should elucidate structural and functional consequences of the sequence divergence in these trasmembrane domains of cannabinoid receptors. In Aim 4, Disulfide bond formation of CB2 receptor will be determined directly by the state-of-art methodologies of mass spectrometry. Investigating the unique disulfide bond or absence of a disulfide bond should provide important helix folding information of CB2 receptor, and this will have important implications regarding ligand binding crevice and activation mechanisms of this receptor. Finally, in Aim 5, effort will be devoted to over-express and purify large amounts of functional CB2 receptor. This challenging task, if successfully completed, should have a significant impact by providing the badly needed pure receptor proteins for future high-resolution biophysical studies. Overall, This study should help us to understand in more molecular detail the structure and function of CB2 receptor. CB2 is primarily distributed in the immune system. It is very important for the immune-modulatory effects of marijuana. In the long run, this study should also help us to develop better cannabinoid mimetics for the treatment of immune system illnesses, such as inflammation and autoimmune diseases. The drugs that specifically targeted at CB2 should be devoid of psvchoactive side effects of marijuana.