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Redistribution of three α_(1)-adrenergic receptor subtypes in the stably transfected HEK 293A cells upon agonist stimulation

Wang Shuyi, Song Yao, Xu Ming, Hao Tianpao, Han Qide, Zhang Youyi

Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education. Beijing 100083, China

Abstract

To investigate the subcellular distribution of three α1-adrenergic receptor subtypes and their internalization 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 a1D-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 a1D-AR in response to phenylephrine. However, when examined by Western 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.

Key words: α_(1)-adrenergic receptor;Distribution;Localization;Cell line

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Citing This Article：

Wang Shuyi, Song Yao, Xu Ming, Hao Tianpao, Han Qide, Zhang Youyi. Redistribution of three α_(1)-adrenergic receptor subtypes in the stably transfected HEK 293A cells upon agonist stimulation. Acta Physiol Sin 2005; 57 (4): (in Chinese with English abstract).