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| 不同配体对m_5乙酰胆碱受体-G蛋白亚单位G_(11)融合蛋白表达和功能的影响 | |
| 引用本文： | 郭政东,王海波,李智,刘洪瑞,郭政礼,赵立斌. 不同配体对m_5乙酰胆碱受体-G蛋白亚单位G_(11)融合蛋白表达和功能的影响[J]. 中国药理学与毒理学杂志, 2004, 18(1): 11-16 |
| 作者姓名： | 郭政东 王海波 李智 刘洪瑞 郭政礼 赵立斌 |
| 作者单位： | 1. 中国医科大学药理学教研室,辽宁,沈阳,110001 2. 中国医科大学病理生理学教研室,辽宁,沈阳,110001 3. 沈阳市中心医院药剂科,辽宁,沈阳,110024 |
| 基金项目： | 国家自然科学基金，辽宁省自然科学基金 |
| 摘 要： | 目的 制备毒蕈碱乙酰胆碱受体m5亚型与G 蛋白亚单位G1 1 (m5AChR G1 1 )融合蛋白 ,探索m5AChR与G1 1 之间的耦联功能、相互作用及其影响因素。方法 两步PCR法建立m5AChR与G1 1 融合cDNAs,并在Sf9细胞内表达。 [3 H]QNB和 [3 5S]GTPγS结合实验检测m5AChR与G1 1 融合蛋白的功能。结果m5AChR G1 1 融合蛋白的表达水平为 (60 .4± 2 .0 )nmol·g-1 膜蛋白。不同配体存在使融合蛋白中G1 1 与GDP的亲和力发生变化。乙酰胆碱(ACh)、卡巴胆碱、毛果芸香碱、异丙铵、异丙嗪及阿托品存在时 ,GDP的IC50值分别为 1 35,63 ,1 82 ,4.1 ,8.9和 5.9μmol·L-1 ,无配体存在时 ,GDP的IC50 值为 2 3.4μmol·L-1 。结论 杆状病毒Sf9细胞系统表达的m5AChR G1 1 融合蛋白具备m受体配体结合特性和组分间耦联的功能。m5AChR G1 1 融合蛋白对GDP亲和力取决于m受体配体的性质 ,分析GDP的亲和力的变化有利于筛选和鉴别m5受体亚型特异性配体 |
| 关 键 词： | 受体,毒蕈碱 G蛋白类 融合蛋白 胆碱能激动剂 胆碱能拮抗剂 |
| 收稿时间： | 2003-03-20 |
Effects of various muscarinic ligands on the expression and function of m5 muscarinic acetylcholine receptor-G11 fusion protein |
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| GUO Zheng-Dong, WANG Hai-Bo, LI Zhi, LIU Hong-Rui, GUO Zheng-Li, ZHAO Li-Bin. Effects of various muscarinic ligands on the expression and function of m5 muscarinic acetylcholine receptor-G11 fusion protein[J]. Chinese Journal of Pharmacology and Toxicology, 2004, 18(1): 11-16 | |
| Authors: | GUO Zheng-Dong WANG Hai-Bo LI Zhi LIU Hong-Rui GUO Zheng-Li ZHAO Li-Bin |
| Affiliation: | (1. Department of Pharmacology, 2. Department of Pathophysiology, China Medical University, Shenyang 110001, China; 3. Deptartment of Pharmacy, Central Hospital of Shenyang, Shenyang 110024, China) |
| Abstract: | AIM To test the couping function, the interreaction and the influence factors between m5 muscarinic acetylcholine receptor(m5AChR) and G11 protein by generating m5AChR and G11 fusion protein(m5AchR-G11) in baculovirus-Sf9 cells system. METHODS m5AchR-G11 Fused DNA was generated in a two- step PCR and then expressed in Sf9 cells to produce fusion protein. ［3H］QNB and ［γ-35S］GTP binding experiments were used to study the function of m5AchR-G11 fusion protein. RESULTS The expression level of m5AchR-G11 was (60.4±2.0)nmol•g-1 protein. The affinity of GDP to G11 partner changed in the presence of different muscarinic ligands. IC50 values of GDP in the presence of acetylcholine(ACh), carbachol, pilocarpine, isopropamide, promethazine and atropine were 135, 63, 182, 4.1, 8.9 and 5.9 μmol•L-1, respectively, and that in the absence of muscarinic ligand was 23.4 μmol•L-1. CONCLUSION The m5AchR-G11 fusion protein expressed in baculovirus-sf9 cells system has pharmacological specificity for m5 receptor and the efficient coupling function between the two partners. Affinity of GDP to ligand-bound fusion protein represents the species of muscarinic ligands. This is helpful to screen and detect new specific ligands of the muscarinic receptors subtype. |
| Keywords: | receptors muscarinic G proteins fusion proteins cholinergic agonists cholinergic antagonists |
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