Conformation of the Muscarinic Acetylcholine Receptor

我们利用杆状病毒- sf9细胞建立了m2毒蕈碱受体突变体的大规模表达系统，并利用Co^<2+>-螯合柱建立了m2受体的简单纯化系统。我们制作了不同种类的m2受体突变体，并检查了它们的表达水平、稳定性和与G蛋白相互作用的能力。我们选择了一个突变体(1)Asn被Asp取代以避免糖基化，(2)删除第三个胞内环的中心部分以避免蛋白水解，(3)在羧基端添加6个组氨酸标签，以便使用Co^<2+>-柱进行纯化。我们每个月可以获得大约1mg纯化的m2受体突变体。该受体用于二维结晶和测量与m2受体结合的甲基胆碱（S (+)- o-乙酰- β -甲基胆碱）的转移核过hauser效应。我们可以观察到一个假定的受体的一维阵列，但尚未成功地获得二维晶体。我们可以得到证据表明，甲胆碱与m2受体结合的C_< α >-C_< β >键呈反式构象，而游离的甲胆碱键呈间扭式构象。在这个项目的过程中，我们发现(1)毒蕈碱受体的五种亚型（m1-m5）可以在杆状病毒- sf9系统中表达，这使我们能够检测它们的配体结合特性和活性形式的溶解能力；(2)大多数竞争配体结合具有相似亲和力的五种亚型，而变构配体，包括新发现的α -lapachone，以亚型特异性的方式结合。(3)在m2受体的羧基端存在棕榈酰残基，去棕榈酰化减弱了m2受体激活G蛋白的能力。

We have developed a large-scale expression system of a m2 muscarinic receptor mutant using the baculovirus-Sf9 cells and a simple purification system of the m2 receptor using Co^<2+>-chelating column. We have made different kinds of m2 receptor mutants and checked for their expression level, stability and ability to interact with G proteins. We chose a mutant with (1) substitution of Asn by Asp in order to avoid glycosylation, (2) deletion of the central part of the third intracellular loop to avoid proteolysis, and (3) addition of six histidine tag at the carboxy terminus in order to be used for purification with Co^<2+>-column. We could obtain approximately 1 mg of the purified m2 receptor mutant every month. The receptor was used for two-dimensional crystallization and for measurements of Transfer Nuclear Overhauser Effect of methacholine (S (+)-O-acetyl-beta-methylcholine) bound to the m2 receptor. We could observe a putative one-dimensional array of the receptor but have not yet succeeded in obtaining two-dimensional crystal. We could obtain evidence that the C_<alpha>-C_<beta> bond of methacholine bound to the m2 receptor takes the trans conformation, whereas the bond of free methacholine does the gauche conformation.In the course of this project, we have found that (1) the five subtypes of muscarinic receptors (m1-m5) could be expressed in the baculovirus-Sf9 system, enabling us to examine their ligand binding properties and capability to be solubilized in active forms, (2) most of competitive ligands bind to five subtypes with similar affinities, whereas allosteric ligands, including a newly found alpha-lapachone, bind in a subtype-specific manner, (3) a palmitoyl residue is present in the carboxyl terminus of the m2 receptor, and depalmitoylation attenuates the ability of the m2 receptor to activate G proteins.