Abstract

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.