title: Subtype-specific differences in subcellular localization and chlorethylclonidine inactivation of alpha1-adrenoceptors.
abstract: Chlorethylclonidine (CEC) inactivation has been used as one criterion to subclassify the alpha1-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) alpha1-ARs, we have determined the relationship between CEC sensitivity and the cellular localization of alpha1-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 alpha1B-AR-expressing cells, but low signals in alpha1A-AR-expressing cells. Further examination with confocal microscopy showed that fluorescent signals densely localized intra-cellularly in alpha1A-AR-expressing cells, while most of alpha1B-AR localized on the cell surface. Furthermore, radioligand binding studies with [125I]HEAT showed that CEC (10 microM) treatment of intact cells inactivated approximately 30-40% of alpha1A-AR and >90% of alpha1B-AR, while the CEC treatment of membrane preparations resulted in >80% decrease in the alpha1A-AR density and >90% of alpha1B-AR density, respectively. The results showed that the hydrophilic alkylating agent CEC inactivated only alpha1-AR on the cell surface irrespective of its subtype, and that the subtype-specific sorting is a major determinant for CEC inactivation of alpha1-AR. Subtype-specific cellular localization suggests a new class of functional properties that may explain the signal and functional diversity of homologous alpha1-AR (as well as other G protein-coupled receptors) subtypes.
authors.affiliation: Department of Molecular, Cell Pharmacology, National Children's Medical Research Center, Tokyo, Japan. gtsujimoto@nch.go.jp
authors.forename: G
authors.initials: G
authors.lastname: Tsujimoto
authors.forename: A
authors.initials: A
authors.lastname: Hirasawa
authors.forename: T
authors.initials: T
authors.lastname: Sugawara
authors.forename: T
authors.initials: T
authors.lastname: Awaji
mesh_terms: D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology*; D000818:Animals; D019556:COS Cells; D003000:Clonidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000939:Epitopes; D019204:GTP-Binding Proteins / Q000378:metabolism; D007457:Iodine Radioisotopes; D010627:Phenethylamines / Q000378:metabolism; D018340:Receptors, Adrenergic, alpha-1 / Q000145:classification* / Q000187:drug effects* / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D013379:Substrate Specificity; D044024:Tetralones; D014162:Transfection
chemical_list: D000317:Adrenergic alpha-Antagonists; D000939:Epitopes; D007457:Iodine Radioisotopes; D010627:Phenethylamines; D018340:Receptors, Adrenergic, alpha-1; D044024:Tetralones; C051731:chlorethylclonidine; C048008:2-(beta-(3-iodo-4-hydroxyphenyl)ethylaminomethyl)tetralone; D019204:GTP-Binding Proteins; D003000:Clonidine