血管紧张素Ⅱ 1型受体牵张激活位点的筛选  

Screening the stress sensitive sites of angiotensin Ⅱ type I receptor

作　　者：牛玉宏[1] 邹云增[1] 王翔飞[1] 徐丹令[1] 梁艳艳[1] 马桢[1] 

机构地区：[1]复旦大学附属中山医院心血管病研究所,上海200032

出　　处：《中国病理生理杂志》2009年第3期427-431,共5页Chinese Journal of Pathophysiology

基　　金：国家杰出青年科学基金资助项目(No.30525018);上海市优秀学科带头人资助项目(No.05XD14003);国家自然科学基金资助项目(No.30670840);教育部博士点基金资助项目(No.20060246079);国家重点基础发展规划项目(No.2007CB512003)

摘　　要：目的:寻找血管紧张素Ⅱ1型受体(angiotensinⅡ type 1 receptor,AT1受体)上的机械负荷感受位点。方法:制备AT1受体不同位点的突变体,转染既不表达血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)也不表达AT1的COS7细胞,Western blotting法检测细胞牵张后细胞外信号调节激酶(ERKs)的磷酸化水平。结果:构建了C76A、K199Q、H256A、Q257A、C289A、C296A、K199Q/H256A、K199Q/Q257A8种突变体。COS7细胞转染AT1受体以前,AngⅡ和牵张刺激都不能引起细胞内ERKs磷酸化升高;而转染野生型AT1后,AngⅡ和牵张刺激引起细胞内ERKs磷酸化明显升高,各突变体中,Q257A和C289A转染细胞后细胞对牵张刺激的反应受到明显抑制,提示AT1的牵张感受位点在Q257A和C289A。结论:结果提示AT1受体上位于257位的谷氨酰胺和289位的胱氨酸在机械牵张引起的AT1受体激活中起了重要作用。AIM： To find out the stress sensitive sites in angiotensin Ⅱ type 1 receptor （AT1 receptor）. METHODS ： AT1 receptor mutants were constructed by PCR from wild AT1 receptor. The plasmid with wild AT1 receptor or its mutants was transfected into COS7 cells that express neither angiotensin Ⅱ （Ang Ⅱ ） nor AT1 receptor. Western blotting was used to detect the phosphorylated extracellular signal - regulated kinase （ERKs）. RESULTS ： Eight mutants of AT1 were constructed （C76A, K199Q, H256A, Q257A, C289A, C296A, K199Q/H256A, K199Q/Q257A）. Before transfected with AT1, neither Ang Ⅱ nor stretch increased the activation of ERKs. After transfected with AT1, pretreatment with Ang Ⅱ for 8 min or stretch for 10 min activated ERKs obviously. However, the activation of ERKs was significantly suppressed after the COS7 cells were transfected with C289A and Q257A. The result indicated that C289A and Q257A were the stretch sensitive region. CONCLUSION ： The results suggest that Cys 289 and Gin 257 play important roles in the activation of AT1 receptor induced by mechanical stresses.

关 键 词：受体 血管紧张素 牵张感受器 COS7细胞 

分 类 号：R363[医药卫生—病理学]