[Redistribution of three alpha1-adrenergic receptor subtypes in the stably transfected HEK 293A cells upon agonist stimulation.].
To investigate the subcellular distribution of three alpha(1)-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 alpha(1)-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 alpha(1)-AR subtypes. alpha(1B ) -AR expression in cell membrane was the highest, and alpha(1D ) -AR was the lowest, as determined by (125)I-BE2254 binding assay, however, K(d)s were not significantly different among the three receptor subtypes. (2) Without agonist stimulation, alpha(1A ) -AR was detected not only on the cell surface but also in the cytosol, alpha(1B ) -AR was predominantly located on the cell surface, whereas alpha(1D ) -AR was mostly detected in the cytosol. (3) After 1 h of stimulation with phenylephrine, as observed using confocal microscope, less alpha(1A)- and alpha(1B ) -AR were detected on the cell surface but more in the cytosol. The change was more remarkable in alpha(1B)-AR than that in alpha(1A)-AR, whereas no change of distribution was detected in alpha(1D)-AR in response to phenylephrine. However, when examined by Western blot, no change in distribution was detected in alpha(1A)- and alpha(1D)-AR, only alpha(1B)-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 alpha(1)-AR subtypes in HEK293A cells upon phenylephrine stimulation.