Subtype-specific differences in subcellular localization and chlorethylclonidine inacttvation of αl-adrenoceptors

Abstract

Chlorethylclonidine (CEC) inactivation has been used as one criterion to subclassify the αl-adrenoceptors (AR); however, the extent of CEC inactivation can vary depending on the CEC treatment. By constructing the FLAG-tagged (N-terminus) and green fluorescent protein (GFP)-fused (C-terminus) α1-ARs, we have determined the relationship between CEC sensitivity and the cellular localization of α1-AR subtypes using COS-7 cells. In GFP-expressing cells, flow cytometry analysis with anti-FLAG N-terminus antibody detected strong fluorescent signals in most of α1 B-AR-expressing cells, but low signals in α1A-AR-expressing cells. Further examination with confocal microscopy showed that fluorescent signals densely localized intra-cellularly in α1 A-AR-expressing cells, while most of α1B-AR localized on the cell surface. Furthermore, radioligand binding studies with [ 125I]HEAT showed that CEC (10 μM) treatment of intact cells inactivated approximately 30–40% of α1A-AR and > 90% of α1B-AR, while the CEC treatment of membrane preparations resulted in > 80% decrease in the α1A-AR density and > 90% of α1B-AR density, respectively. The results showed that the hydrophilic alkylating agent CEC inactivated only α1-AR on the cell surface irrespective of its subtype, and that the subtype-specific sorting is a major determinant for CEC inactivation of α1-AR. Subtype-specific cellular localization suggests a new class of functional properties that may explain the signal and functional diversity of homologous α1-AR (as well as other G protein-coupled receptors) subtypes.