RT Journal Article SR Electronic T1 Visualizing Differences in Ligand Regulation of Wild-Type and Constitutively Active Mutant β2-Adrenoceptor-Green Fluorescent Protein Fusion Proteins JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1182 OP 1191 DO 10.1124/mol.56.6.1182 VO 56 IS 6 A1 McLean, Alison J. A1 Bevan, Nicola A1 Rees, Stephen A1 Milligan, Graeme YR 1999 UL http://molpharm.aspetjournals.org/content/56/6/1182.abstract AB Fusion proteins were generated by attachment of green fluorescent protein (GFP) to the C-terminal tail of either the wild-type human β2-adrenoceptor or a form with enhanced constitutive activity. Sustained treatment of HEK293 cells stably expressing the constitutively active mutant (CAM) β2-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 β2-adrenoceptor-GFP. Addition of the agonist isoprenaline to CAM β2-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 β2-adrenoceptor-GFP. A range of “β-blockers” replicated the up-regulation of the CAM β2-adrenoceptor-GFP, although pharmacological specificity was maintained, as it was not produced by α1- and α2-adrenoceptor-selective antagonists/inverse agonists. Parallel intact cell binding studies with [3H]dihydroalprenolol confirmed up-regulation of the CAM β2-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 β2-adrenoceptor-GFP was not identical after sustained treatment of the cells with different β-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 β2-adrenoceptor-GFP.