Abstract

β-Arrestin is an adaptor protein that has been shown to couple G protein-coupled receptors (GPCRs) to clathrin-coated pits and target them for subsequent internalization. More recently, β-arrestin 2 has also been shown to be involved in the activation of mitogen-activated protein kinase cascades by G protein-coupled receptors independently of G protein activation. Direct interactions between proteins can be monitored using enzyme complementation between two inactive deletion mutants of β-galactosidase (β-gal; Δα and Δω). In the present study, we have used fusion proteins of the human β₂-adrenoceptor (C-terminal β-gal Δα) and β-arrestin 2 (β-gal Δω) to study directly the pharmacology of this interaction in C2C12 cells expressing the β₂-adrenoceptor-β-gal Δα fusion protein at low physiological levels (38.2 ± 2.7 fmol · mg protein^-1). Isoprenaline, noradrenaline, and adrenaline (-log EC₅₀ = 5.9, 5.5, and 5.7, respectively) stimulated an association between the β₂-adrenoceptor and β-arrestin 2 at much higher concentrations than required for activation of cAMP accumulation (-log EC₅₀ = 7.6, 6.3, and 7.7, respectively). This was sensitive to inhibition by the β₂-adrenoceptor antagonists propranolol, timolol, and ICI 118551. Both salbutamol and terbutaline behaved as partial agonists of β-gal complementation. Furthermore, the long-acting β₂-agonist salmeterol (-log K_D for inhibition of [³H]CGP12177 binding = 8.7) behaved as an antagonist of isoprenaline-stimulated β₂-adrenoceptor-arrestin 2 interactions (-log K_D = 8.0), whereas acting as a full agonist of cAMP accumulation in the same cells (-log EC₅₀ = 9.2). These data suggest that salmeterol can discriminate between receptor-G_S protein and receptor-arrestin 2 complexes (in terms of efficacy and affinity) in a way that is favorable for its long duration of action.