m_1AChR-G_(11)和m_4AChR-G_(16)融合蛋白的建立、表达及几种配体的作用  

Preparation of m_1AChR-G_(11)fusion protein and m_4AChR-G_(16)fusion protein and effects of various ligands on them

作　　者：王海波[1] 郭政东[2] 张海鹏[1] 王娟[1] 温华[3] 李新鸣[4] 

机构地区：[1]中国医科大学病理生理学教研室,辽宁沈阳110001 [2]中国医科大学药理学教研室,辽宁沈阳110001 [3]中国医科大学附属第一医院呼吸内科,辽宁沈阳110001 [4]中国医科大学病原生物学教研室,辽宁沈阳110001

出　　处：《中国病理生理杂志》2003年第12期1590-1595,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目 (No .30 1710 77);辽宁省自然科学基金资助项目(No.0 0 2 0 39)

摘　　要：目的 :通过Baculovirus-Sf9cells系统制备m1AChR -G11融合蛋白和m4AChR -G16融合蛋白 ,检测几种配体对m1AChR与G11及m4AChR与G16间相互作用的影响 ,并以m1AChR -G11和m4AChR -G16融合蛋白为工具 ,筛选m1和m4受体的特异性配体。方法 :两步PCR建立m1AChR -G11和m4AChR -G16融合DNA ,并在Sf9细胞内表达。通过 [3 H]QNB和 [3 5S]GTPγS结合实验检测m1AChR -G11融合蛋白和m4AChR -G16融合蛋白的药理学特性 ;通过 [3 5S]GTPγS替代结合实验研究几种配体包括acetylcholine(ACh) ,pilocarpine(Pilo)。 4 -hydroxy - 2 -butynyl- 1-trimethylam monium -m -chloro -carbanilatechloride (McN -A - 343) ,tetrandrine ,pirenzepine (PZ) ,alcuronium ,atropine ,R - (+) -hyoscyamine和gallamine对m1AChR -G11融合蛋白和m4AChR -G16融合蛋白作用的方式 ,并以这两种融合蛋白为模型筛选m1和m4受体的特异性配体。结果 :m1AChR -G11融合蛋白和m4AChR -G16融合蛋白表达水平分别为 (45 4±2 6 )nmol/gprotein和 (47 0± 1 6 )nmol/gprotein。不同配体使融合蛋白m1AChR -G11和m4AChR -G16中G11和G16与GDP的亲和力发生变化。结论 :杆状病毒 -Sf9细胞系统表达的m1AChR -G11融合蛋白和m4AChR -G16融合蛋白具备m受体配体结?AIM: To prepare m 1AChR-G 11 and m 4AChR-G 16 fusion protein in Baculovirus-Sf9 cell system and detect the effects of various muscarinic ligands on the interaction between m 1AChR and G 11 and m 4AChR and G 16 ,and screen different kinds of ligands specific for m 1 and m 4. METHODS: To prepare fused DNA of m 1AChR-G 11 and m 4AChR-G 16 in two PCR, then expressed in Sf9 cells and detect the pharmacological function of m 1AChR-G 11 fusion protein and m 4AChR-G 16 fusion protein by [ 3H]QNB and [ 35 S]GTPγS binding experiments; To expore the way of the activation of m 1AChR-G 11 and m 4AChR-G 16 fusion protein by various ligands includingcetylcholine (ACh), Pilocarpine (Pilo), 4-hydroxy-2-butynyl-1-trimethylammonium-m-chloro-carbanilatechloride (McN-A-343),tetrandrine, pirenzepine (PZ), alcuronium, atropine,R-(+)-hyoscyamine and gallamine by displacement by GDP on [ 35 S]GTPγS binding experiments. RESULTS: The expression levels of m 1AChR-G 11 and m 4AChR-G 16 fusion protein were (45 39±2 62) nmol·g -1 protein, (47 04±1 58) nmol·g -1 protein. The affinity of GDP to G 11 and G 16 partner changed in the presence of different muscarinic ligands. CONCLUSION: The m 1AChR-G 11 and m 4AChR-G 16 showed the pharmacological specificity to m 1 and m 4 receptor and the efficient signaling of the two partners. Ligands of m 1AChR and m 4AchR mediated different signal transduction by changing the affinity of G 11 /G 16 and GDP. So m1AChR-G 11 fusion protein and m 4AChR-G 16 fusion protein can be taken as a tool to screen ligands specific for m 1AChR and m 4AChR.

关 键 词：受体 毒蕈碱 信号转导 配体 G蛋白 

分 类 号：R363[医药卫生—病理学]