G蛋白偶联受体激酶4对大鼠血管平滑肌细胞AT_1受体的调节作用

G protein-coupled receptor kinase 4 regulates expression and phosphorylation status of AT_1 receptor in rat vascular smooth muscle cells

目的应用siRNA干扰抑制大鼠胸主动脉血管平滑肌细胞G蛋白偶联受体激酶4(G protein-coupled recep-tor kinase 4,GRK4)的表达,探讨GRK4对血管紧张素Ⅱ1型(angiotensinⅡtype 1,AT1)受体的调节作用。方法免疫组化检测大鼠回结肠动脉血管平滑肌组织GRK4蛋白表达;以大鼠胸主动脉平滑肌细胞株(A10细胞株)为研究对象,免疫印迹检测GRK4、AT1受体蛋白表达变化,免疫共沉淀检测GRK4和AT1受体的相互作用和AT1受体磷酸化改变。结果大鼠动脉平滑肌组织GRK4表达良好;siRNA干扰后,GRK4蛋白表达明显下降(P<0.05);AT1蛋白表达降低(P<0.05),AT1受体磷酸化明显增强(P<0.05);GRK4和AT1受体存在共连接,抑制GRK4表达后增加GRK4与AT1受体之间的共连接。结论 GRK4能够调控大鼠胸主动脉平滑肌细胞AT1受体蛋白表达及其磷酸化状态,该调节作用可能与两者的共连接有关。

Objective To determine the effect of G protein-coupled receptor kinase 4（GRK4）on angiotensin Ⅱ type 1（AT1） receptor in rat vascular smooth muscle cells by siRNA against GRK4.Methods S-P immunohistochemistry was used to detect the expression of GRK4 in SD rat ileocolic artery sample.Western blotting were employed to detect the expression of GRK4 and AT1 receptor in A10 cells,an embryonic thoracic aortic smooth muscle cell line from normotensive Berlin-Druckrey IX,with or without RNA interference.The phosphorylation of AT1 receptor and the interaction between GRK4 and AT1 receptor were determined by co-immunoprecipitation.Results GRK4 protein was expressed in rat aortic smooth muscle cells.GRK4 siRNA interference inhibited GRK4 protein expression and AT1 receptor expression in A10 cells（P0.05）,while the AT1 receptor phosphorylation was enhanced（P0.05）.There was co-immunoprecipitation between GRK4 and AT1 receptor,which was increased after GRK4 siRNA interference.Conclusion GRK4 regulates AT1 receptor expression and phosphorylation,which may be related with the linkage between GRK4 and AT1 receptor.