激动剂作用下α_1-肾上腺素受体亚型在HEK293A细胞中的分布变化

Redistribution of three α_1-adrenergic receptor subtypes in the stably transfected HEK 293A cells upon agonist stimulation

为了明确α1-肾上腺素受体(α1-adrenergicreceptor,α1-AR)三种亚型在人胚胎肾(humanembryonickidney,HEK)293A细胞株中的分布特点,及其在激动剂作用下在细胞内的定位改变,本研究采用放射配体结合实验、实时荧光共聚焦成像和Westernblot方法检测α1-AR三种亚型在细胞中的定位及蛋白质表达的变化。结果发现:(1)α1-AR三种亚型在HEK293A细胞株转染效率相同,均达90%以上。三株细胞的粗制膜上α1B-AR表达量最高,α1D-AR最低,α1A-AR居中,但三者的解离常数(Kd)相等;(2)在无激动剂作用时,α1A-AR均匀地分布在HEK293A细胞的胞膜和胞浆,α1B-AR主要位于胞膜,而α1D-AR则主要分布在胞浆中;(3)用α1-AR激动剂苯肾上腺素(phenylephrine,PE)刺激细胞1h后,α1A-和α1B-AR在胞膜上分布明显减少,而在胞浆中分布增加,其中α1B-AR变化更为显著,α1D-AR的分布在PE作用下无明显变化。以上结果提示,在激动剂作用下,α1-AR三种亚型在HEK293A细胞中的定位特点和分布变化各有不同。

To investigate the subcellular distribution of three α1-adrenergic receptor subtypes and their intemalization and trafficking upon agonist stimulation in human embryonic kidney （HEK） 293A cell line, saturation radioligand binding assay, laser confocal imaging,and Western blot were applied to examine the distribution and changes in localization of three α1-AR subtypes in transfected HEK 293A cells prior to and after treatment with phenylephrine. The results are as follows： （1） The transfection efficiency was over 90% and was equal among three α1-AR subtypes, α1B-AR expression in cell membrane was the highest, and α1D-AR was the lowest, as determined by ^125I-BE2254 binding assay, however, KdS were not significantly different among the three receptor subtypes. （2） Without agonist stimulation, α1A-AR was detected not only on the cell surface but also in the cytosol, α1B-AR was predominantly located on the cell surface, whereas α1D-AR was mostly detected in the cytosol. （3） After 1 h of stimulation with phenylephrine, as observed using confocal microscope, less α1A- and α1B-AR were detected on the cell surface but more in the cytosol. The change was more remarkable in α1B-AR than that in α1A-AR, whereas no change of distribution was detected in α1D-AR in response to phenylephrine. However, when examined by Westem blot, no change in distribution was detected in α1A- and α1D-AR, only α1B-AR showed the same change as that shown in confocal imaging. It is suggested that the characteristics of localization and changes of distribution are different among three α1-AR subtypes in HEK293A cells upon phenylephrine stimulation.