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Molecular Pharmacology, Volume 52, Issue 5, 764-770
1-Adrenoceptors: Chlorethylclonidine Preferentially
Alkylates the Accessible Cell Surface 1-Adrenoceptors
Irrespective of the Subtype
Department of Molecular, Cell Pharmacology, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo, 154 Japan (A.H., T.S., T.A., G.T.), and Department of Biological Sciences, Tokyo Institute of Technology, Midori-ku, Yokohama, 226 Japan (K.T., H.I.)
Selective inactivation of 
1B-adrenoceptor (AR) by the
site-directed alkylating agent chlorethylclonidine (CEC) has been used as one of major pharmacological criteria to subclassify
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 between the subcellular localization of
1-AR subtypes (1A and 1B)
and CEC sensitivity. Constructing 1-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 1-ARs expressed in COS-7 cells. Flow
cytometry analysis showed that most populations of GFP-expressing 1B-AR cells, but very few GFP-expressing
1A-AR cells, were detected by the anti-amino terminus
antibodies. The immunocytochemical and GFP-fluorescence confocal
micrographs showed that 1A-ARs predominantly localize
intracellularly, whereas 1B-ARs localize on the cell
surface. Furthermore, CEC (10 µM) treatment of intact cells resulted in an inactivation of approximately 42% of
1A-ARs and 93% of 1B-ARs, whereas
treatment of the membrane preparations resulted in an inactivation of
approximately 83% of 1A-ARs and 88% of
1B-ARs, respectively. Together, the results showed that a hydrophilic alkylating agent CEC preferentially inactivates 1-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
1-AR. Subtype-specific subcellular localization suggests
an additional class of functional properties that provide new insight into drug action.
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