Abstract
BackgroundThe cellular localization of the α1D-adrenergic receptor (α1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the α1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled α1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity.ResultsThe adenovirally expressed α1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the α1D-AR was also demonstrated with immunocytochemistry using an α1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the α1A-α1B- and α1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other α1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the α1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the α1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The α1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the α1D-AR heterodimerizes with the β2-AR and that desensitization of the β2-AR results in α1D-AR desensitization. In the rat aorta, desensitization of the β2-AR had no effect on contractile responses mediated by the α1D-AR.ConclusionOur results suggest that the dimerization of the α1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the α1D-AR.
Highlights
The cellular localization of the α1D-adrenergic receptor (α1D-Adrenergic receptor (AR)) is controversial
Cellular localization An adenoviral vector was constructed to drive the efficient expression of a Green fluorescent protein (GFP)-labeled α1D-adrenergic receptor (α1D-AR)
Infection of aortic smooth muscle cells with virus expressing the α1D-AR/ GFP resulted in approximately 80% receptor transfectional efficiency demonstrating that adenovirus can be useful in cells that have been traditionally difficult to transfect with the α1-ARs
Summary
Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Results from heterologous expression systems have demonstrated that the α1B-AR is localized on the cell surface, as expected for a GPCR, while the α1A-AR is localized on the cell and in intracellular compartments [10,11]. We have shown that the α1D-AR is localized intracellularly [11,12] These results of nonconical cellular locatization are consistent with emerging data that show specific GPCR families can be localized to intracellular sites and on the nuclear membrane [13]
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