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

Abstract

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 α₁-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 α₁-AR subtypes (α_1A and α_1B) and CEC sensitivity. Constructing α₁-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 α₁-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 α₁-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 α₁-AR. Subtype-specific subcellular localization suggests an additional class of functional properties that provide new insight into drug action.