SUBTYPE-SPECIFIC DIFFERENCES IN SUBCELLULAR-LOCALIZATION AND CHLORETHYLCLONIDINE INACTIVATION OF ALPHA-1-ADRENOCEPTORS

Chlorethylclonidine (CEC) inactivation has been used as one criterion to subclassify the alpha 1-adrenoceptors (AR); however, the extent of CEC inactivation can vary depending on the CEC treatment. By construct ing the FLAG-tagged (N-terminus) and green fluorescent protein (GFP)-f used (C-terminus) alpha 1-ARs, we have determined the relationship bet ween CEC sensitivity and the cellular localization of alpha 1-AR subty pes using COS-7 cells. In GFP-expressing cells, flow cytometry analysi s with anti-FLAG N-terminus antibody detected strong fluorescent signa ls in most of alpha 1B-AR-expressing cells, but low signals in alpha 1 A-AR-expressing cells. Further examination with confocal microscopy sh owed that fluorescent signals densely localized intra-cellularly in al pha 1A-AR-expressing cells, while most of alpha 1B-AR localized on the cell surface. Furthermore, radioligand binding studies with [I-125]HE AT showed that CEC (10 mu M) treatment of intact cells inactivated app roximately 30-40% of alpha 1A-AR and >90% of alpha 1B-AR, while the CE C treatment of membrane preparations resulted in >80% decrease in the alpha 1A-AR density and >90% of alpha 1B-AR density, respectively. The results showed that the hydrophilic alkylating agent CEC inactivated only alpha 1-AR on the cell surface irrespective of its subtype, and t hat the subtype-specific sorting is a major determinant for CEC inacti vation of alpha 1-AR. Subtype-specific cellular localization suggests a new class of functional properties that may explain the signal and f unctional diversity of homologous alpha 1-AR (as well as other G prote in-coupled receptors) subtypes.