m_5 AChR-G_(11)融合蛋白筛选m_5受体配体的研究  

m_5AChR-G_(11) fusion protein as a tool for screening m_5 ligand

作　　者：王海波[1] 秦兴军[2] 郭政东[3] 杨君兰[4] 

机构地区：[1]中国医科大学基础医学院病理生理学教研室,辽宁沈阳110001 [2]中国医科大学附属第一医院口腔颌面外科 [3]中国医科大学基础医学院药理学教研室 [4]辽宁省疾病预防控制中心

出　　处：《中国医科大学学报》2005年第5期396-398,共3页Journal of China Medical University

基　　金：国家自然科学基金资助项目(30171077);辽宁省自然科学基金资助项目(002039)

摘　　要：目的:以m5AChR-G11融合蛋白为工具,筛选m5受体亚型特异性配体,并且探讨m5AChR-G11融合蛋白中两组分相互作用的机制。方法:采用杆状病毒Sf 9细胞表达系统,制备m5AChR-G11融合蛋白;通过[3H]QNB放射配体结合实验和[35S]GTPγS替代结合实验,检测m5AChR-G11融合蛋白的功能。结果:m5AChR-G11融合蛋白的表达水平为(60.39±1.95)pmol.mg-1蛋白。不同配体存在时使融合蛋白中G11与GDP的亲和力发生变化,ace-tylcholine,M cN-A-343,AF-102B,R-(+)-hyoscyam ine,trop icam ide,alcuron ium和atrop ine存在时以及无配体时,GDP的IC50值分别是134.90,46.77,38.02,10.00,13.80,18.62,5.89,23.40μmol/L。结论:杆状病毒Sf 9细胞表达系统表达的m5AChR-G11融合蛋白具备m受体配体结合特性和两组分间的偶联功能。乙酰胆碱是m5受体的完全激动剂,M cN-A-343和AF-102B是部分激动剂,R-(+)-hyoscyam ine,trop icam ide,alcuron ium和阿托品是拮抗剂。Objective： To prepare m5AChR-G11 fusion protein as a tool to screen muscarinic ligands specific for m5AChR, and to explore the mechanism of the effects between 2 partners in m5AChR-G11 fusion protein. Methods： m5AChR-G11, fused DNA was generated and then expressed in Baculovirus-Sf 9 cells. [^3H] QNB and [^35S] GTPγS binding experiments were performed to study the function of m5AChR-G11 fusion protein and to screen the ligands specific for m53ChRs. Results： The expression level of m5AChR-G11 was （60.39± 1.95 ） pmol·mg^-1 protein. The affinity of GDP to G11 partner changed in the presence of different muscarinic ligands. The IC50 values of GDP in the presence of acetylcholine （ACh）, McN-A-343, AF-102B, R-（ ＋ ）-hyoscyamine, tropicamide, alcuronium, and atropine were 134. 90, 46. 77, 38.02, 10.00, 13.80, 18.62, and 5.89 μmol·L^-1, respectively, The IC50 value in the absence of muscarinic ligand was 23.44μmol·L^-1. Conclusion：The m5AChR-G11 fusion protein has the pharmacological specificity of m5 receptor and the efficient signaling of 2 partners. ACh is a full agonist of m5AChRs. Mcn-A-343 and AF-102B are partial agonists of m5 AChRs. R-（ ＋ ）-hyoscyamine, tropicamide, alcuronium, and atropine are antagonists of m5AChRs.

关 键 词：融合蛋白 毒蕈碱乙酰胆碱受体 G蛋白偶联受体 兴奋剂 拮抗剂 

分 类 号：Q51[生物学—生物化学]