Subtype-Specific Differences in Subcellular Localization of α1-Adrenoceptors: Chlorethylclonidine Preferentially Alkylates the Accessible Cell Surface α1-Adrenoceptors Irrespective of the Subtype

Selective inactivation of α 1B -adrenoceptor (AR) by the site-directed alkylating agent chlorethylclonidine (CEC) has been used as one of major pharmacological criteria to subclassify α 1 -AR; however, the mechanism for the differential CEC sensitivity of the two subtypes is uncertain, and the extent of CEC inactivation varies depending on the treatment employed. In this study, we examined the correlation between the subcellular localization of α 1 -AR subtypes (α 1A and α 1B ) and CEC sensitivity. Constructing α 1 -AR tagged with the FLAG epitope at the amino terminus and/or green fluorescent protein (GFP) at the carboxyl terminus, we examined the subcellular distribution of α 1 -ARs expressed in COS-7 cells. Flow cytometry analysis showed that most populations of GFP-expressing α 1B -AR cells, but very few GFP-expressing α 1A -AR cells, were detected by the anti-amino terminus antibodies. The immunocytochemical and GFP-fluorescence confocal micrographs showed that α 1A -ARs predominantly localize intracellularly, whereas α 1B -ARs localize on the cell surface. Furthermore, CEC (10 μm) treatment of intact cells resulted in an inactivation of approximately 42% of α 1A -ARs and 93% of α 1B -ARs, whereas treatment of the membrane preparations resulted in an inactivation of approximately 83% of α 1A -ARs and 88% of α 1B -ARs, respectively. Together, the results showed that a hydrophilic alkylating agent CEC preferentially inactivates α 1 -AR on the cell surface irrespective of its subtype, and that the subtype-specific subcellular localization rather than the receptor structure is a major determinant for CEC inactivation of α 1 -AR. Subtype-specific subcellular localization suggests an additional class of functional properties that provide new insight into drug action.