RhoGDI2与β2肾上腺素能受体减敏的关系及机制研究

Study on the relationship between RhoGDI2 and β2-adrenergic receptor desensitization and its mechanism

目的探讨Rho鸟苷酸解离抑制因子2（RhoGDI2）对气道平滑肌细胞B。。肾上腺素能受体（口zAR）减敏的关系。方法培养人气道平滑肌细胞，以RhoGDI2高表达质粒转染后，用Western blot法检测RhoGDI2的蛋白表达量，用酶联免疫吸附试验（ELISA）检测细胞内腺苷-3’，5’-环化一磷酸（cAMP）的含量，并探究两者关系。检测转染后细胞膜表面B2AR蛋白表达量随时间的变化，及转染后G蛋白耦联受体激酶2（GRK2）、蛋白激酶A（PKA）磷酸化位点蛋白表达情况，再加入GRK2及PKA抑制剂后，用ELISA检测细胞内cAMP的变化。结果RhoGDI2高表达组与阴性对照组及空白组相比，高表达组平滑肌细胞内cAMP含量降低，差异有统计学意义，转染后24h、36h、48h、60h、72h气道平滑肌细胞膜表面8zAR蛋白表达量随转染时间的延长呈下降趋势；RhoGDI2高表达组的BzAR的355，356丝氨酸位点磷酸化水平高于345，346位丝氨酸位点磷酸化水平。RhoGDI2转染后加GRK2抑制剂组、RhoGDI2转染后加PKA抑制剂组与空白对照组相比，气道平滑肌细胞内cAMP含量降低，其中，RhoGDI2转染后加PKA抑制剂组与空白对照组相比差异有统计学意义，RhoGDI2转染后加GRK2抑制剂组与空白对照组相比差异无统计学意义。结论①RhoGDI2高表达可引起人气道平滑肌细胞β2AR的减敏。②RhoGDI2引起人气道平滑肌细胞膜表面β2AR的减敏是通过GRK2磷酸化β2AR起作用。

Objective To investigate the effect and mechanism of Rho GDP dissociation inhibitor 2 （RhoGDI2） affecting β2-adrenergic receptor （β2AR） desensitization in airway smooth muscle cells. Methods The expression of RhoGDI2 was detected by Western blot and the content of cyclic adenosine monophosphate （cAMP） in the cells was detected by enzyme linked immunosorbent assay （ELISA）. The expression of cAMP in the airway smooth muscle cells and RhoGDI2 was detected by Western blot. Western blot was used to detect the change of β2AR after transfection. After treatment of G protein- coupled receptor kinases 2（GRK2） and protein kinase A （PKA）, the ELISA method was used to detect cAMP in intracellular. Results The expression of cAMP in airway smooth muscle cells was significantly lower in RhoGDI2 high expression group than that in control group and blank group. The expression of β2 AR on the surface of airway smooth muscle cells gradually reduced at 24 h, 36 h, 48 h, 60 h and 72 h after transfecting. The phosphorylation level of the 355,356 serine site of the ~z receptor was higher than that of the 345,346 serine site. After RhoGDI2 transfection, GRK2 inhibitor group and PKA inhibitor group comparing with the blank control group, the cAMP expression of airway smooth muscle cell decreased. However, PKA inhibitor group compared with the blank control group was statisticallysignificant, GRK2 inhibitor group compared with the blank control group was no statistically significant difference. Conclusions @The overexpression of RhoGDI2 affects the desensitization of β2AR in human airway smooth muscle cells.②The mechanism is that the desensitization of β2 AR is caused by GRK2 phosphorylation.