@article {Carter839, author = {Alison A. Carter and Stephen J. Hill}, title = {Characterization of Isoprenaline- and Salmeterol-Stimulated Interactions between β2-Adrenoceptors and β-Arrestin 2 Using β-Galactosidase Complementation in C2C12 Cells}, volume = {315}, number = {2}, pages = {839--848}, year = {2005}, doi = {10.1124/jpet.105.088914}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, 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 {\textpm} 2.7 fmol {\textperiodcentered} 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 KD for inhibition of [3H]CGP12177 binding = 8.7) behaved as an antagonist of isoprenaline-stimulated β2-adrenoceptor-arrestin 2 interactions (-log KD = 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-GS protein and receptor-arrestin 2 complexes (in terms of efficacy and affinity) in a way that is favorable for its long duration of action. The American Society for Pharmacology and Experimental Therapeutics}, issn = {0022-3565}, URL = {https://jpet.aspetjournals.org/content/315/2/839}, eprint = {https://jpet.aspetjournals.org/content/315/2/839.full.pdf}, journal = {Journal of Pharmacology and Experimental Therapeutics} }