Development of Purification Methods of G Protein- Linked Receptors

本项目的目的是开发一种纯化G蛋白连接受体的通用方法。策略和初步计划如下：(1)纯化大量的毒菌碱受体（mAChRs）作为模型系统，(2)研究重组系统中mAChRs和G蛋白的相互作用，(3)溶解mAChRs和G蛋白的复合物，(4)使用G蛋白或抗G蛋白作为配体的亲和层析分离受体，并使用GTP的受体兴奋剂从这些凝胶中特异性洗脱受体。杆状病毒-昆虫细胞（Sf9）系统的引入和亲和柱的改进使我们能够以较高的数量和重复性纯化mAChRs （m2）。对重组体系中mAChR （m2）与G蛋白（Gi, Go, Gn）相互作用的详细研究表明，与无颗粒核苷酸的G蛋白（含或不含Mg^<2+>）复合物的mAChR对受体具有更高的亲和力，而Mg^<2+>存在的复合物对受体具有更高的亲和力，被认为是mAChR作用于G蛋白的中间体。G蛋白对mAChR激酶对毒蕈碱受体激动剂依赖性磷酸化的抑制作用也表明了该复合物的形成。研究发现，与G蛋白孵育的心房膜中的单月桂酸钠可以溶解mAChR和G蛋白的复合物，并在相同的洗涤剂溶液中形成。我们一直在尝试将单月桂酸钠溶解后的machr或mAChR-G蛋白复合物与以G蛋白或抗G蛋白为配体的琼脂糖凝胶结合，并从凝胶中特异性洗脱，但尚未找到满意的条件。

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.