Development of Purification Methods of G Protein- Linked Receptors

本项目的目的是建立一种通用的纯化G蛋白连接受体的方法。战略和初步时间表如下：（1）大量毒蕈碱受体（mAChRs）的纯化，其将用作模型系统，（2）mAChrS和G蛋白在重构系统中的相互作用的研究，（3）mAChRs和G蛋白的复合物的增溶，（4）通过使用G蛋白或抗G蛋白作为配体的亲和层析分离受体，并通过GTP激动剂从这些凝胶中特异性洗脱受体。杆状病毒-昆虫细胞（Sf 9）系统的引入和亲和柱的改进使我们能够以较高的量和重复性纯化mAChRs（m2）。对mAChR（m2）与G蛋白（Gi，Go，Gn）在重组体系中相互作用的研究表明，mAChR与G蛋白复合物中的mAChR与Mg^<2+>有或无Mg^<2+>，但在Mg^<2+>存在下的复合物对激动剂有较高的亲和力，被认为是mAChR作用于G蛋白的中间体。G蛋白对mAChR激酶激动剂依赖性毒蕈碱受体磷酸化的抑制作用也表明了复合物的形成。发现mAChR和G蛋白的复合物用来自心房膜的单月桂酸钠溶解，所述心房膜已与G蛋白孵育并在相同的洗涤剂溶液中形成。我们一直试图将用单月桂酸钠增溶的mAChR或mAChR-G蛋白复合物结合到以G蛋白或抗G蛋白为配体的琼脂糖凝胶上，并从这些凝胶中特异性地将它们分离，但我们尚未找到令人满意的条件。

The purpose of the present project is to develop a general method to purify G protein-linked receptors. The strategy and initial schedule are as follows ; (1) purification of a large quantity of muscarinic receptors (mAChRs) which will be used as a model system, (2) studies of the interaction of mAChrS and G proteins in the reconstitution system, (3) solubilization of the complex of mAChRs and G proteins, (4) isolation of receptors by the use of an affinity chromatography with G proteins or anti-G proteins as ligands and a specific elution of receptors from these gels by agonists of GTP. An introduction of baculovirus-insect cell (Sf9) system and the improvement of the affinity column enablied us to purify mAChRs (m2) in a relatively high quantity and reproducebly. Detailed studies on the interaction of mAChRs (m2) and G proteins (Gi, Go, Gn) in reconstitution system have indicatied that mAChRs from the complex with the granine nucleotide-free G proteins with or without Mg^<2+> but the complex in the presence of Mg^<2+> has higher affinities for agonists and is considered to be an intermediate for the action of mAChR on G proteins. Inhibitory effects of G proteins on the agonist-dependent phosphorylation of muscarinic receptors by mAChR kinase also indicated the formation of the complex. The complex of mAChR and G proteins was found to be solubilized with sodium monolaurate from atrial membranes which had been incubated with G proteins and to be formed in the same detergent solution. We have been attempting to bind mAChRs or mAChR-G protein complex, which were solubilized with sodium monolaurate, to agarose gels with G proteins or anti-G proteins as ligands and to elute them from these gels specifically, but we have not yet found the satisfactory conditions.