SUBTYPE-SPECIFIC DIFFERENCES IN SUBCELLULAR-LOCALIZATION OF ALPHA(1)-ADRENOCEPTORS - CHLORETHYLCLONIDINE PREFERENTIALLY ALKYLATES THE ACCESSIBLE CELL-SURFACE ALPHA(1)-ADRENOCEPTORS IRRESPECTIVE OF THE SUBTYPE

Selective inactivation of alpha(1B)-adrenoceptor (AR) by the site-dire cted alkylating agent chlorethylclonidine (CEC) has been used as one o f major pharmacological criteria to subclassify alpha(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 bet ween the subcellular localization of alpha(1)-AR subtypes (alpha(1A) a nd alpha(1B)) and CEC sensitivity. Constructing alpha(1)-AR tagged wit h the FLAG epitope at the amino terminus and/or green fluorescent prot ein (GFP) at the carboxyl terminus, we examined the subcellular distri bution of alpha(1)-ARs expressed in COS-7 cells. Flow cytometry analys is showed that most populations of GFP-expressing alpha(1B)-AR cells, but very few GFP-expressing alpha(1A)-AR cells, were detected by the a nti-amino terminus antibodies. The immunocytochemical and GFP-fluoresc ence confocal micrographs showed that alpha(1A)-ARs predominantly loca lize intracellularly, whereas alpha(1B)-ARs localize on the cell surfa ce. Furthermore, CEC (10 mu M) treatment of intact cells resulted in a n inactivation of approximately 42% of alpha(1A)-ARs and 93% of alpha( 1B)-ARs, whereas treatment of the membrane preparations resulted in an inactivation of approximately 83% of alpha(1A)-ARs and 88% of alpha(1 B)-ARs, respectively. Together, the results showed that a hydrophilic alkylating agent CEC preferentially inactivates alpha(1)-AR on the cel l surface irrespective of its subtype, and that the subtype-specific s ubcellular localization rather than the receptor structure is a major determinant for CEC inactivation of alpha(1)-AR. Subtype-specific subc ellular localization suggests an additional class of functional proper ties that provide new insight into drug action.