Conformation of the Muscarinic Acetylcholine Receptor

我们已经建立了一个利用杆状病毒-Sf 9细胞的m2受体突变体的大规模表达系统和一个利用Co^<2+>-螯合柱的m2受体的简单纯化系统。我们已经制备了不同类型的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。我们选择了一种突变体，其具有（1）用Asp取代Asn以避免糖基化，（2）缺失第三胞内环的中心部分以避免蛋白水解，和（3）在羧基末端添加六个组氨酸标签以用于用Co^<2+>-柱纯化。我们每个月可以获得大约1 mg纯化的m2受体突变体。该受体用于二维结晶和测定与m2受体结合的乙酰甲胆碱（S（+）-O-乙酰基-β-甲基胆碱）的转移核Overhauser效应。我们可以观察到一个假定的一维阵列的受体，但尚未成功地获得二维晶体。本<alpha><beta>研究发现：（1）M_2受体的5种亚型（m_1-m_5）在杆状病毒-Sf_9系统中均能表达，并能检测它们的配体结合特性和增溶活性;（2）大多数竞争性配体以相似的亲和力与5种亚型结合，而变构配体（包括新发现的α-拉帕醌）以亚型特异性方式结合。（3）在m2受体的羧基端存在棕榈酰残基，脱棕榈酰化减弱了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.