TY - JOUR T1 - Subtype-Specific Differences in Subcellular Localization of α<sub>1</sub>-Adrenoceptors: Chlorethylclonidine Preferentially Alkylates the Accessible Cell Surface α<sub>1</sub>-Adrenoceptors Irrespective of the Subtype JF - Molecular Pharmacology JO - Mol Pharmacol SP - 764 LP - 770 DO - 10.1124/mol.52.5.764 VL - 52 IS - 5 AU - Akira Hirasawa AU - Tatsuo Sugawara AU - Takeo Awaji AU - Keiko Tsumaya AU - Hiroshi Ito AU - Gozoh Tsujimoto Y1 - 1997/11/01 UR - http://molpharm.aspetjournals.org/content/52/5/764.abstract N2 - Selective inactivation of α1B-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 (α1A and α1B) 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 α1B-AR cells, but very few GFP-expressing α1A-AR cells, were detected by the anti-amino terminus antibodies. The immunocytochemical and GFP-fluorescence confocal micrographs showed that α1A-ARs predominantly localize intracellularly, whereas α1B-ARs localize on the cell surface. Furthermore, CEC (10 μm) treatment of intact cells resulted in an inactivation of approximately 42% of α1A-ARs and 93% of α1B-ARs, whereas treatment of the membrane preparations resulted in an inactivation of approximately 83% of α1A-ARs and 88% of α1B-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. ER -