Purification of Cannabinoid Receptors Type 1 and 2

描述(申请人提供)：获得膜蛋白的原子分辨结构，如G蛋白偶联受体(GPCRs)，由于难以纯化足够数量的膜蛋白而受到阻碍。唯一可用的GPCR晶体结构是牛视紫质，它可以从牛视网膜中大量分离出来。我们提出了一种新的方法来产生来源来纯化大量的受体，这可以解决GPCR结晶的瓶颈。视紫红质的共价结合的视网膜起到反向激动剂的作用，使受体牢固地保持在非常稳定的非活动状态。蛋白质的稳定性和均一性是形成高分辨率衍射的高度有序晶体的关键因素。大麻素受体(CBR)是最接近视紫红质的受体，因为当它们与反向激动剂结合时，它们表现出非常稳定的非活性状态，在这种情况下与G蛋白有关，使它们成为具有吸引力的GPCR结晶和结构阐明的靶标。在第一阶段，我们将测试我们用于CB1和CB2受体受体纯化的新来源的可行性，这是第二阶段应用于CB1和CB2受体结晶和结构鉴定的先决条件，单独使用并与处于反向激动剂结合状态的G蛋白复合。

DESCRIPTION (provided by applicant): Obtaining atomic resolution structures of membrane proteins such as G-protein coupled receptors (GPCRs) has been hampered by the difficulty to purify sufficient amounts of membrane protein. The only GPCR crystal structure available is bovine rhodopsin, which can be isolated in large amounts from bovine retina. We propose a novel approach to generate a source to purify large amounts of receptor, which could solve the bottleneck for GPCR crystallization. The covalently bound retinal of rhodopsin acts as an inverse agonist that maintains firmly the receptor in a very stable inactive state. Stability and homogeneity of the protein are key factors for the formation of highly ordered crystals that diffract at high resolution. The cannabinoid receptors (CBR) are the closest to rhodopsin in that when bound to the inverse agonist they exhibit a very stable inactive state, in this case associated with the G-protein, making them attractive GPCR targets for crystallization and structural elucidation. In Phase I we will test the feasibility of our novel source for receptor purification for CB1 and CB2 receptors, a prerequisite to a Phase II application for crystallization and structural elucidation of the CB1 and CB2 receptors, alone and complexed with G-protein in the inverse agonist bound state.