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 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.
Footnotes
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A.A.C. holds a Biotechnology and Biological Sciences Research Council research studentship.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.088914.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; PKA, protein kinase A; GRK, G protein-coupled receptor kinase; MAP, mitogen-activated protein; β-gal, β-galactosidase; CGP 12177, (-)-4-(3-tert-utylamino-2-hydroxypropoxy)-benzimidazol-2-one; DMEM, Dulbecco's modified Eagle's medium; DMEM F12, Dulbecco's modified Eagle's medium Ham's F-12; β-AR, β2-adrenoceptor; FCS, fetal calf serum; CHO, Chinese hamster ovary; HBH, Hanks' balanced salt solution; PBS, phosphate-buffered saline; ICI 118551, (-)-1-(2,3-[dihydro-7-methyl-1H-inden-4-yl]oxy)-3-([1-methylethyl]-amino)-2-butanol; CGP 20712A, 2-hydroxy-5-(2-[{hydroxyl-3-(4-[1-methyl-4-trifluoromethyl-2-imidazolyl]phenoxy)-propyl}amino]ethoxy)benamide.
- Received May 2, 2005.
- Accepted July 26, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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