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Vol. 56, Issue 6, 1182-1191, December 1999
2-Adrenoceptor-Green
Fluorescent Protein Fusion Proteins
Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow, Scotland, United Kingdom (A.J.M.,
G.M.); and Biological Chemistry Unit, Glaxo-Wellcome Research and
Development, Stevenage, England, United Kingdom (N.B., S.R.)
Fusion proteins were generated by attachment of green fluorescent
protein (GFP) to the C-terminal tail of either the wild-type human
2-adrenoceptor or a form with enhanced constitutive
activity. Sustained treatment of HEK293 cells stably expressing the
constitutively active mutant (CAM) 2-adrenoceptor-GFP
with the inverse agonist betaxolol resulted in a marked up-regulation
of the fusion protein that could be monitored by both fluorescence and
immunoblotting of membrane fractions. This was not observed for the
wild-type 2-adrenoceptor-GFP. Addition of the agonist
isoprenaline to CAM 2-adrenoceptor-GFP expressing cells
previously treated with betaxolol resulted in rapid internalization of
the receptor into punctate intracellular vesicles in a manner similar
to wild-type 2-adrenoceptor-GFP. A range of
"-blockers" replicated the up-regulation of the CAM 2-adrenoceptor-GFP, although pharmacological specificity
was maintained, as it was not produced by 
1- and
2-adrenoceptor-selective antagonists/inverse agonists.
Parallel intact cell binding studies with
[3H]dihydroalprenolol confirmed up-regulation of the CAM
2-adrenoceptor-GFP by betaxolol but failed to predict
the optically monitored up-regulation produced by high concentrations
of alprenolol. The cellular distribution of the up-regulated CAM
2-adrenoceptor-GFP was not identical after
sustained treatment of the cells with different -blockers. Inverse
agonists, able to reduce basal intracellular cAMP levels, such as
betaxolol and ICI118551, resulted in both increased plasma membrane
receptor and increased diffuse intracellular staining. In contrast,
treatment with labetolol and alprenolol resulted in a
significant fraction of the intracellular receptor displaying a
punctate distribution pattern. These ligands displayed substantial agonism to stimulate intracellular cAMP levels via the CAM
2-adrenoceptor-GFP.
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